Neurogenetics: scientific and clinical advances

Author: David Robinson Lynch

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Neurogenetics: Scientific and Clinical Advances

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NEUROLOGICAL DISEASE AND THERAPY Advisory Board Louis R. Caplan, M.D. Professor of Neurology Harvard University School of Medicine Beth Israel Deaconess Medical Center Boston, Massachusetts

William C. Koller, M.D. Mount Sinai School of Medicine New York, New York

John C. Morris, M.D. Friedman Professor of Neurology Co-Director, Alzheimer’s Disease Research Center Washington University School of Medicine St. Louis, Missouri

Bruce Ransom, M.D., Ph.D. Warren Magnuson Professor Chair, Department of Neurology University of Washington School of Medicine Seattle, Washington

Kapil Sethi, M.D. Professor of Neurology Director, Movement Disorders Program Medical College of Georgia Augusta, Georgia

Mark Tuszynski, M.D., Ph.D. Associate Professor of Neurosciences Director, Center for Neural Repair University of California–San Diego La Jolla, California

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1. Handbook of Parkinson’s Disease, edited by William C. Koller 2. Medical Therapy of Acute Stroke, edited by Mark Fisher 3. Familial Alzheimer’s Disease: Molecular Genetics and Clinical Perspectives, edited by Gary D. Miner, Ralph W. Richter, John P. Blass,

Jimmie L. Valentine, and Linda A. Winters-Miner 4. Alzheimer’s Disease: Treatment and Long-Term Management,

edited by Jeffrey L. Cummings and Bruce L. Miller 5. Therapy of Parkinson’s Disease, edited by William C. Koller

and George Paulson 6. Handbook of Sleep Disorders, edited by Michael J. Thorpy 7. Epilepsy and Sudden Death, edited by Claire M. Lathers

and Paul L. Schraeder 8. Handbook of Multiple Sclerosis, edited by Stuart D. Cook 9. Memory Disorders: Research and Clinical Practice,

edited by Takehiko Yanagihara and Ronald C. Petersen 10. The Medical Treatment of Epilepsy, edited by Stanley R. Resor, Jr.,

and Henn Kutt 11. Cognitive Disorders: Pathophysiology and Treatment,

edited by Leon J. Thal, Walter H. Moos, and Elkan R. Gamzu 12. Handbook of Amyotrophic Lateral Sclerosis, edited by Richard Alan Smith 13. Handbook of Parkinson’s Disease: Second Edition, Revised and Expanded,

edited by William C. Koller 14. Handbook of Pediatric Epilepsy, edited by Jerome V. Murphy

and Fereydoun Dehkharghani 15. Handbook of Tourette’s Syndrome and Related Tic and Behavioral Disorders, edited by Roger Kurlan 16. Handbook of Cerebellar Diseases, edited by Richard Lechtenberg 17. Handbook of Cerebrovascular Diseases, edited by Harold P. Adams, Jr. 18. Parkinsonian Syndromes, edited by Matthew B. Stern

and William C. Koller 19. Handbook of Head and Spine Trauma, edited by Jonathan Greenberg 20. Brain Tumors: A Comprehensive Text, edited by Robert A. Morantz

and John W. Walsh 21. Monoamine Oxidase Inhibitors in Neurological Diseases,

edited by Abraham Lieberman, C. Warren Olanow, Moussa B. H. Youdim, and Keith Tipton 22. Handbook of Dementing Illnesses, edited by John C. Morris 23. Handbook of Myasthenia Gravis and Myasthenic Syndromes,

edited by Robert P. Lisak 24. Handbook of Neurorehabilitation, edited by David C. Good

and James R. Couch, Jr. 25. Therapy with Botulinum Toxin, edited by Joseph Jankovic

and Mark Hallett 26. Principles of Neurotoxicology, edited by Louis W. Chang 27. Handbook of Neurovirology, edited by Robert R. McKendall

and William G. Stroop 28. Handbook of Neuro-Urology, edited by David N. Rushton 29. Handbook of Neuroepidemiology, edited by Philip B. Gorelick

and Milton Alter

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30. Handbook of Tremor Disorders, edited by Leslie J. Findley

and William C. Koller 31. Neuro-Ophthalmological Disorders: Diagnostic Work-Up and Management, edited by Ronald J. Tusa and Steven A. Newman 32. Handbook of Olfaction and Gustation, edited by Richard L. Doty 33. Handbook of Neurological Speech and Language Disorders,

edited by Howard S. Kirshner 34. Therapy of Parkinson’s Disease: Second Edition, Revised and Expanded,

edited by William C. Koller and George Paulson 35. Evaluation and Management of Gait Disorders, edited by

Barney S. Spivack 36. Handbook of Neurotoxicology, edited by Louis W. Chang

and Robert S. Dyer 37. Neurological Complications of Cancer, edited by Ronald G. Wiley 38. Handbook of Autonomic Nervous System Dysfunction,

edited by Amos D. Korczyn 39. Handbook of Dystonia, edited by Joseph King Ching Tsui

and Donald B. Calne 40. Etiology of Parkinson’s Disease, edited by Jonas H. Ellenberg,

William C. Koller and J. William Langston 41. Practical Neurology of the Elderly, edited by Jacob I. Sage

and Margery H. Mark 42. Handbook of Muscle Disease, edited by Russell J. M. Lane 43. Handbook of Multiple Sclerosis: Second Edition, Revised and Expanded,

edited by Stuart D. Cook 44. Central Nervous System Infectious Diseases and Therapy,

edited by Karen L. Roos 45. Subarachnoid Hemorrhage: Clinical Management, edited by

Takehiko Yanagihara, David G. Piepgras, and John L. D. Atkinson 46. Neurology Practice Guidelines, edited by Richard Lechtenberg

and Henry S. Schutta 47. Spinal Cord Diseases: Diagnosis and Treatment, edited by

Gordon L. Engler, Jonathan Cole, and W. Louis Merton 48. Management of Acute Stroke, edited by Ashfaq Shuaib

and Larry B. Goldstein 49. Sleep Disorders and Neurological Disease, edited by Antonio Culebras 50. Handbook of Ataxia Disorders, edited by Thomas Klockgether 51. The Autonomic Nervous System in Health and Disease,

David S. Goldstein 52. Axonal Regeneration in the Central Nervous System, edited by

Nicholas A. Ingoglia and Marion Murray 53. Handbook of Multiple Sclerosis: Third Edition, edited by Stuart D. Cook 54. Long-Term Effects of Stroke, edited by Julien Bogousslavsky 55. Handbook of the Autonomic Nervous System in Health and Disease,

edited by C. Liana Bolis, Julio Licinio, and Stefano Govoni 56. Dopamine Receptors and Transporters: Function, Imaging, and Clinical Implication, Second Edition, edited by Anita Sidhu,

Marc Laruelle, and Philippe Vernier 57. Handbook of Olfaction and Gustation: Second Edition, Revised and Expanded, edited by Richard L. Doty

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58. Handbook of Stereotactic and Functional Neurosurgery, edited by

Michael Schulder 59. Handbook of Parkinson’s Disease: Third Edition, edited by Rajesh Pahwa,

Kelly E. Lyons, and William C. Koller 60. Clinical Neurovirology, edited by Avindra Nath and Joseph R. Berger 61. Neuromuscular Junction Disorders: Diagnosis and Treatment, Matthew N. Meriggioli, James F. Howard, Jr., and C. Michel Harper 62. Drug-Induced Movement Disorders, edited by Kapil D. Sethi 63. Therapy of Parkinson’s Disease: Third Edition, Revised and Expanded, edited by Rajesh Pahwa, Kelly E. Lyons, and William C. Koller 64. Epilepsy: Scientific Foundations of Clinical Practice, edited by Jong M. Rho, Raman Sankar, and José E. Cavazos 65. Handbook of Tourette’s Syndrome and Related Tic and Behavioral Disorders: Second Edition, edited by Roger Kurlan 66. Handbook of Cerebrovascular Diseases: Second Edition, Revised and Expanded, edited by Harold P. Adams, Jr. 67. Emerging Neurological Infections, edited by Christopher Power and Richard T. Johnson 68. Treatment of Pediatric Neurologic Disorders, edited by Harvey S. Singer, Eric H. Kossoff, Adam L. Hartman, and Thomas O. Crawford 69. Synaptic Plasticity : Basic Mechanisms to Clinical Applications, edited by Michel Baudry, Xiaoning Bi, and Steven S. Schreiber 70. Handbook of Essential Tremor and Other Tremor Disorders, edited by Kelly E. Lyons and Rajesh Pahwa 71. Handbook of Peripheral Neuropathy, edited by Mark B. Bromberg and A. Gordon Smith 72. Carotid Artery Stenosis: Current and Emerging Treatments, edited by Seemant Chaturvedi and Peter M. Rothwell 73. Gait Disorders: Evaluation and Management, edited by Jeffrey M. Hausdorff and Neil B. Alexander 74. Surgical Management of Movement Disorders (HBK), edited by Gordon H. Baltuch and Matthew B. Stern 75. Neurogenetics: Scientific and Clinical Advances, edited by David R. Lynch 76. Epilepsy Surgery: Principles and Controversies, edited by John W. Miller and Daniel L. Silbergeld 77. Clinician's Guide To Sleep Disorders, edited by Nathaniel F. Watson and Bradley Vaughn 78. Amyotrophic Lateral Sclerosis, edited by Hiroshi Mitsumoto, Serge Przedborski and Paul H. Gordon

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Neurogenetics: Scientific and Clinical Advances Edited by

David R. Lynch, M.D., Ph.D. Children’s Hospital Philadelphia, Pennsylvania, U.S.A.

Associate Editor

Jennifer M. Farmer, M.S., C.G.C. Genetic Counselor, Department of Pathology and Laboratory Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, U.S.A.

New York London

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Published in 2006 by Taylor & Francis Group 270 Madison Avenue New York, NY 10016 © 2006 by Taylor & Francis Group, LLC No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8247-2942-0 (Hardcover) International Standard Book Number-13: 978-0-8247-2942-4 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Catalog record is available from the Library of Congress

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Foreword Those working in the ﬁeld of neurogenetics view the advances of the last decade as a fantastic explosion of knowledge. But to others, particularly the practicing neurologist, it may seem less than fantastic. It can seem, well, downright daunting. Take Charcot-Marie-Tooth disease, for example. What once was broadly classiﬁed into two clinically distinguishable forms of neuropathy now represents more than 20 genetic conditions. Or take the hereditary ataxias: over 25 genetic causes already identiﬁed—and that’s just the dominant forms of disease! Diseases that, clinically, we thought we knew so well sometimes seem to change once the underlying genes are discovered. Friedreich’s ataxia, for example, was originally deﬁned as a childhood disorder, but the gene now tells us otherwise: it is a disease of adults, too, and can look altogether different in adults. In short, while the onslaught of advances in neurogenetics has helped to clarify disease mechanisms and improve diagnosis, it sometimes leaves non-geneticists wanting to throw in the towel. My advice to you: don’t throw in that towel just yet. First, read this book. David Lynch has assembled a remarkable group of experts who have written a wide-ranging collection of chapters on neurogenetics principles. These authors clearly and succinctly explain neurogenetics—both its general, underlying principles and speciﬁc heritable diseases—as we understand them in 2006. When I say read this book, I don’t mean read only the chapter about your favorite disease, then place it on the shelf. I mean sit down and read this book from page one. The opening chapters on neurogenetics in the clinic, genetic testing, genetic counseling, and ethical issues are a beautiful foray into the practical nuts and bolts of neurogenetics that physicians, including neurologists, increasingly must deal with. These introductions to fundamental aspects of neurogenetics are written in a compelling and straightforward manner that will leave readers, whether they are training scientists, resident physicians or even seasoned professors, with a clearer

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understanding of what it means for a disease to ‘‘look genetic’’ and how best to pursue the underlying genetic cause. If, on top of that, you ﬁnish your reading also knowing how to draw a pedigree (well described in Bennett’s genetic counseling chapter), that’s icing on the cake. Once these general principles are introduced in the ﬁrst few chapters, they are then applied by disease experts to a wide range of inherited neurological diseases. The 28 chapters comprising this book cover nearly all of the major areas of neurogenetics that an adult neurologist will see in the clinic, as well as a good many pediatric disorders. Neuromuscular and neurodegenerative disease are particularly well covered. This comes as no surprise since they have led the way in neurogenetics. But areas in which genetics are just beginning to make their full presence known—epilepsy and paroxysmal disorders, to name a few—are also well represented. Even someone who studies molecular mechanisms of genetic diseases for a living (like I do) will learn quite a bit of cutting edge information about a wide range of important diseases. A major strength of this book is that most chapters ground their discussion where it matters most—in the clinical realm. The disease experts typically begin by describing the clinical features of disease followed by a discussion of the underlying genetic basis and, when known, the molecular mechanism. This often then leads to a discussion of where treatment stands today and how recent insights from the underlying neurogenetics are leading to novel treatments. This last point is emphasized in certain chapters that are dedicated to describing such advancements, such as Pierson and Wolfe’s chapter on gene therapy. But even chapters that concentrate on particular diseases, for example, Leavitt’s and Raymond’s chapter on Huntington’s disease, compellingly describe how modern science is leading to novel therapies. Finally, perhaps we can stop lamenting the complexity of neurogenetic disorders. In many cases, knowing the genes and the underlying genetic mechanisms has actually simpliﬁed the view of the class of diseases. When experts write about these diseases in a clear and compelling manner, as they do here, classiﬁcation schemes that formerly seemed vexing begin to make sense. Provided you read the book, of course. Henry L. Paulson, MD, PhD University of Iowa Carver College of Medicine

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Preface In the past decade, no ﬁeld of neurology has changed more than that of neurogenetics. Once limited to the clinical description of rare, inherited neurologic disease, the introduction of molecular technologies and genomic medicine approaches have allowed for more precise diagnosis, better scientiﬁc understanding of the pathophysiology, and introduction of novel therapies for neurogenetic disease. Molecular technology has also broadened the ﬁeld to recognize genetic contributions and signiﬁcance in complex diseases, such as Parkinson’s disease, epilepsy, and Alzheimer’s disease, leading to advances in the understanding and treatment of these disorders as well. These advances demand greater knowledge of basic genetic principles, the ability to translate the utility of genetic advances into clinical practice, appreciation for ethical issues raised by genetic testing, and recognition of a growing group of conditions in neurogenetic disease, from clinical neurology practitioners, academic neurologists, and neuroscience researchers. The huge volume of information on neurogenetic disease now allows the clinician to answer more clinical and diagnostic questions than previously and potentially allows researchers to ask new questions directed at disease mechanisms. However, understanding the utility of these advances is not easy, and the limitations of genetic approaches are frequently misunderstood. In addition, new ethical dilemmas arise in situations such as predictive genetic testing, in which a genetic test can be used to inform individuals of whether they will eventually acquire a disease years before they may feel any symptoms. Paradigms for application of such testing to patients are frequently unfamiliar to even some of the best-informed clinicians. The present book should allow clinicians and researchers to be familiar with modern neurogenetic approaches and information, and apply that information to the questions that arise in practice and research. In this volume, we seek to present the most current perspectives on neurogenetic disease from both clinical and scientiﬁc perspectives. There

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are three sections of this book. In the ﬁrst, authors present overviews of modern approaches to genetic testing and counseling, and the dilemmas that arise with molecular technologies and genomic medicine. These chapters illustrate the type of information now available, how it can be used, and how it can be confounded in general ways using speciﬁc practical examples. In the second section of the book, each chapter reviews a classical genetic neurological disorder (or group of disorders) with an application of modern knowledge to clinical evaluation, management and treatment, and basic pathophysiology. In the ﬁnal section of the book, each chapter examines traditionally non-genetic neurological illness, and demonstrates the signiﬁcance and utility of genetic approaches and technology in the pathophysiological understanding and clinical management of these disorders. This approach should provide clinical neurologists, academicians, and researchers with the tools and the knowledge to answer modern questions in neurogenetic illnesses. David R. Lynch Jennifer M. Farmer

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Contents

Foreword Preface Contributors 1. Neurogenetics in the Clinic Thomas D. Bird Factors Suggesting a Neurogenetic Disorder Neurogenetic Diseases ‘‘Hiding’’ in Nonspeciﬁc Categories Importance of Family History Assessment of Sporadic Cases Genetic Counseling Genetic Testing Neurogenetics Information Resources An Integrated Clinical Neurogenetics Strategy References 2. Genetic Testing for Neurological Disorders Martha A. Nance Introduction Principles of Genetic Inheritance Genotype Changes and Their Signiﬁcance in Phenotypic Appearance Gene Mutations and Assays to Detect Them Clinical Use of Gene Tests

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Resources for the Clinician References 3. Genetic Counseling Robin L. Bennett Introduction Are Genetic Disorders Unique? Who Are Genetic Counselors? The Process of Genetic Counseling Genetic Family History—The Pedigree Pedigree Analysis and Risk Perception Summary References 4. Gene Therapy for Inherited Diseases of the Central Nervous System Tyler Mark Pierson and John H. Wolfe Introduction General Properties of CNS Gene Therapy Candidate Diseases for Gene Therapy in the CNS Animal Models of Genetic CNS Disorders Lysosomal Storage Disease: Animal Models as a Paradigm for CNS Gene Therapy Gene Delivery Vehicles Summary References 5. Ethical Dilemmas in Neurogenetics Wendy R. Uhlmann Testing Minors Testing a Child for Adoption Testing an Adult Child When the At-Risk Parent Refuses Testing Is There a ‘‘Duty to Warn’’ and Share Test Results with Other At-Risk Family Members? Testing Identical Twins Non-Disclosure of Test Results Testing Siblings Simultaneously Copyright 2006 by Taylor & Francis Group, LLC

Testing Individuals Who Work in Professions Where Neurological Deﬁcits Can Jeopardize the Safety of Others Testing Symptomatic Individuals Who Think They Are Asymptomatic Testing ‘‘Asymptomatic’’ Individuals Who have Recently Attempted Suicide Testing a Blood Sample from Someone Who Has Just Committed Suicide Testing a Pregnancy Giving Informed Consent for Individuals with Cognitive/Psychiatric Impairment Insurance Issues Anonymous Testing Legal Cases Resources Summary Recommended Reading References 6. Autosomal Dominant Charcot-Marie-Tooth Disease and Related Disorders Craig L. Bennett Scope of This Chapter CMT: Background and General Clinical Features Electrophysiological Classiﬁcation of CMT Major Forms of Demyelinating CMT1 Major Forms of Demyelinating CMT2 Prognosis in CMT Summary Electronic Databases References 7. Duchenne and Becker Muscular Dystrophies Leta S. Steffen and Louis M. Kunkel Introduction Clinical Symptoms Clinical Diagnosis Clinical Treatment History of the Dystrophin Discovery Dystrophin—Molecular Characterization Copyright 2006 by Taylor & Francis Group, LLC

Animal Models of DMD Current Research for DMD/BMD Therapies Conclusion References 8. The Congenital and Limb-Girdle Muscular Dystrophies Janbernd Kirschner and Carsten G. Bo¨nnemann Introduction Congenital Muscular Dystrophies Limb-Girdle Muscular Dystrophies References 9. Non-dystrophic Myotonias and Periodic Paralyses Arie Struyk and Stephen Cannon Introduction Chloride Channel Myotonias (Myotonia Congenita) Sodium Channel Myotonia and Periodic Paralysis Hypokalemic Periodic Paralysis Andersen’s Syndrome Conclusions References 10. The Myotonic Dystrophies—Effects of an RNA Mutation John W. Day and Laura P. W. Ranum Introduction Clinical Features of the Myotonic Dystrophies Genetics of the Myotonic Dystrophies Molecular Pathogenesis of the Myotonic Dystrophies Reports of a Third Form of Myotonic Dystrophy Summary References 11. Spinal Muscular Atrophy Stephen J. Kolb and J. Paul Taylor Introduction Clinical Features of Autosomal Recessive SMA Copyright 2006 by Taylor & Francis Group, LLC

Molecular Genetics of Autosomal Recessive SMA SMA Unrelated to Mutations in SMN1 Basic Research on Autosomal Recessive SMA: The SMN Complex Model Systems Therapeutics References 12. Hereditary Spastic Paraplegia Kleopas A. Kleopa Introduction Clinical and Pathological Features of HSP Genetics and Neurobiology of HSP Diagnostic Approach and Treatment in HSP Conclusion References 13. Mitochondrial Disorders Clotilde Lagier-Tourenne and Michio Hirano Introduction Principles of Mitochondrial Genetics Primary Mitochondrial DNA Mutations Nuclear DNA Mutations General Features of Mitochondrial Diseases Speciﬁc Mitochondrial Diseases MELAS References 14. Friedreich Ataxia Martin B. Delatycki, Michael C. Fahey, Louise Corben, and Andrew Churchyard Introduction Clinical Features Pathology Molecular Genetics Pathogenesis Management of Patients with FRDA Potential Pharmacological Therapies for FRDA Conclusion References Copyright 2006 by Taylor & Francis Group, LLC

15. Autosomal Dominant Ataxias Susan L. Perlman Introduction The Typical Dominant Ataxias SCA1 SCA2 SCA3 SCA4 SCA5 SCA6 SCA7 SCA8 SCA9 SCA10 SCA11 SCA12 SCA13 SCA14 SCA15 SCA16 SCA17 SCA18 SCA19 SCA20 SCA21 SCA22 SCA23 SCA24 SCA25 SCA26 SCA Due to FGF14 Conclusion References 16. Huntington’s Disease: Mechanisms of Pathogenesis and Development of New Therapies Blair R. Leavitt and Lynn A. Raymond Clinical, Pathologic, and Genetic Features of Huntington’s Disease Animal Models of HD Copyright 2006 by Taylor & Francis Group, LLC

Pathogenesis of HD Preclinical Trials in Mouse Models of HD Clinical Trials in HD Conclusions References 17. Wilson’s Disease George J. Brewer Introduction and Historical Overview Pathophysiology Genetics and Epidemiology Clinical Presentations Recognition, Screening, and Diagnosis Treatment with Anticopper Drugs Prognosis References 18. Neurogenetics of Dystonia and Paroxysmal Dyskinesias Jennifer Friedman and David G. Standaert Dystonia Genetics of Primary Dystonias Genetics of Dystonia Plus Syndromes Genetics of Paroxysmal Dystonias and Dyskinesias References 19. Inherited Epilepsies Yr Sigurdardottir and Annapurna Poduri Introduction Channelopathies Myoclonic Epilepsies Generalized Epilepsy Syndromes Localization-Related Epilepsy Syndromes Conclusion References 20. Leukodystrophies James Y. Garbern Introduction Disorders of Myelin Lipid Metabolism Copyright 2006 by Taylor & Francis Group, LLC

Closing Thoughts Resources References 21. Lysosomal Storage Disorders David A. Wenger and Stephanie Coppola Introduction General Concepts Clinical Features that Suggest a LSD Diagnosis of LSDS Treatment of LSDS References 22. The Tuberous Sclerosis Complex: Clinical Manifestations and Molecular Genetics John R. Pollard and Peter B. Crino Clinical Features of TSC Neurological Manifestations Neuropathology Genetics Signaling Pathway and Cell Biology Conclusion References 23. Neuroﬁbromatosis Amit Malhotra, James Dowling, and David H. Gutmann Introduction Neuroﬁbromatosis 1 Clinical Features of Neuroﬁbromatosis 2 References 24. Genetics of Parkinson’s Disease Sathya R. Sriram, Valina L. Dawson, and Ted M. Dawson Introduction Genes Linked to Familial PD Clinical Testing for Genetic Forms of PD Conclusion References

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25. Alzheimer’s Disease R. Scott Turner Introduction Epidemiology Clinical Criteria Pathologic Criteria Risk Factors Genetics of Familial AD Genetics of Sporadic (Late-Onset) Alzheimer’s Disease The Amyloid Hypothesis Genetic Testing Current Treatment Strategies Future Treatment Strategies Perspectives References 26. Tauopathies John C. van Swieten Introduction Tau Gene—Tau Protein Frontotemporal Dementia FTDP-17 Pathology of FTDP-17 Conclusions References 27. Amyotrophic Lateral Sclerosis Teepu Siddique and Lisa Dellefave ALS Background Approaches to Mendelian Inherited ALS Understanding ALS Pathophysiology Through Mendelian Inherited ALS Approaches to SALS as a Genetically Complex or Multifactorial Disorder Clinical Management Remaining Questions to Be Answered References

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28. Prion Diseases William S. Baek and James A. Mastrianni Introduction Historical Background Prion Protein (PrP) and the Prion Concept Pathogenesis of Prions Transmissible Properties of Prions Epidemiology Mutations of PRNP Cause Familial PrD PRNP Polymorphisms Prion-Like Protein Gene (Prnd ) The Phenotypes of PrD Diagnosis of Prion Disease Diagnostic Studies Treatment References About the Editor

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Contributors

William S. Baek Department of Neurology, University of Chicago Hospitals, Chicago, Illinois, U.S.A. Craig L. Bennett Division of Genetics and Developmental Medicine, Department of Pediatrics, University of Washington School of Medicine, Seattle, Washington, U.S.A. Robin L. Bennett University of Washington, Medical Genetics, Seattle, Washington, U.S.A. Thomas D. Bird Department of Neurology, University of Washington Geriatric Research Center, VA Medical Center, Seattle, Washington, U.S.A. Carsten G. Bo¨nnemann Division of Neurology, The Children’s Hospital of Philadelphia, Abramson Research Center, Philadelphia, Pennsylvania, U.S.A. George J. Brewer Department of Human Genetics and Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, U.S.A. Stephen Cannon Department of Neurology, University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A. Andrew Churchyard Monash Institute for Neurological Disease, Southern Health, Monash Medical Centre, Victoria, Australia

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Stephanie Coppola Department of Neurology, Jefferson Medical College, Philadelphia, Pennsylvania, U.S.A. Louise Corben Bruce Lefroy Centre for Genetic Health Research, Murdoch Childrens Research Institute, Royal Children’s Hospital, Victoria, Australia Peter B. Crino Department of Neurology and Epilepsy Center, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, U.S.A. Ted M. Dawson Departments of Neurology and Neuroscience and Program in Cellular and Molecular Medicine, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. Valina L. Dawson Departments of Neurology, Neuroscience, and Physiology and Program in Cellular and Molecular Medicine, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. John W. Day Department of Neurology, Institute of Human Genetics, University of Minnesota School of Medicine, Minneapolis, Minnesota, U.S.A. Martin B. Delatycki Bruce Lefroy Centre for Genetic Health Research, Murdoch Childrens Research Institute, Royal Children’s Hospital, Victoria, Australia Lisa Dellefave Davee Department of Neurology and Clinical Neuroscience, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, U.S.A. James Dowling The Division of Neurology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Michael C. Fahey Bruce Lefroy Centre for Genetic Health Research, Murdoch Childrens Research Institute, Royal Children’s Hospital, Victoria, Australia Jennifer Friedman Department of Neurology, Children’s Hospital and Health Center, San Diego, California, U.S.A. James Y. Garbern Department of Neurology, Wayne State University School of Medicine, Detroit, Michigan, U.S.A.

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David H. Gutmann Department of Neurology, Washington University School of Medicine and St. Louis Children’s Hospital, St. Louis, Missouri, U.S.A. Michio Hirano Department of Neurology, Columbia University College of Physicians and Surgeons, New York, New York, U.S.A. Janbernd Kirschner Division of Neuropediatrics and Muscle Disorders, University Children’s Hospital, Freiburg, Germany Kleopas A. Kleopa Nicosia, Cyprus

The Cyprus Institute of Neurology and Genetics,

Stephen J. Kolb Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A. Louis M. Kunkel Program in Genomics, Children’s Hospital Boston, Department of Genetics, Harvard Medical School, and Howard Hughes Medical Institute, Boston, Massachusetts, U.S.A. Clotilde Lagier-Tourenne Department of Neurology, Columbia University College of Physicians and Surgeons, New York, New York, U.S.A. Blair R. Leavitt Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics, and Division of Neurology, Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada Amit Malhotra Department of Neurology, Washington University School of Medicine and St. Louis Children’s Hospital, St. Louis, Missouri, U.S.A. James A. Mastrianni Department of Neurology, University of Chicago Hospitals, Chicago, Illinois, U.S.A. Martha A. Nance Department of Neurology, University of Minnesota, Minneapolis, Minnesota, U.S.A. Susan L. Perlman Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A. Tyler Mark Pierson Division of Neurology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

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Annapurna Poduri Division of Epilepsy and Neurophysiology, Children’s Hospital Boston, Boston, Massachusetts, U.S.A. John R. Pollard Department of Neurology and Epilepsy Center, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, U.S.A. Laura P. W. Ranum Department of Genetics, Cell Biology and Development, Institute of Human Genetics, University of Minnesota School of Medicine, Minneapolis, Minnesota, U.S.A. Lynn A. Raymond Department of Psychiatry, Division of Neurology, Department of Medicine, and Brain Research Centre, University of British Columbia, Vancouver, British Columbia, Canada Teepu Siddique Davee Department of Neurology and Clinical Neurosciences and Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, U.S.A. Yr Sigurdardottir Department of Pediatrics, University Hospital Iceland, Reykjavik, Iceland Sathya R. Sriram Institute for Cell Engineering and Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. David G. Standaert MassGeneral Institute for Neurodegenerative Disorders, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, U.S.A. Leta S. Steffen Program in Genomics, Children’s Hospital Boston, and Department of Genetics, Harvard Medical School, Boston, Massachusetts, U.S.A. Arie Struyk Department of Neurology, University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A. J. Paul Taylor Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A. R. Scott Turner Department of Neurology and Neurology Service and Neuroscience Program and VA Geriatric Research Education and Clinical Center, Institute of Gerontology, University of Michigan, Ann Arbor, Michigan, U.S.A.

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Wendy R. Uhlmann Department of Internal Medicine, Division of Molecular Medicine and Genetics, and Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, U.S.A. John C. van Swieten Department of Neurology, Erasmus Medical Center, Rotterdam, The Netherlands David A. Wenger Department of Neurology, Jefferson Medical College, Philadelphia, Pennsylvania, U.S.A. John H. Wolfe Division of Neurology, Children’s Hospital of Philadelphia and W. F. Goodman Center for Comparative Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

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About the Editor DAVID R. LYNCH is Associate Professor of Neurology and Pediatrics and Director of the Integrated Friedreich’s Ataxia Clinic, University of Pennsylvania, Philadelphia. Dr. Lynch received the B.S. degree from Yale University, New Haven, Connecticut, and the M.D. and Ph.D. degrees from The Johns Hopkins University School of Medicine, Baltimore, Maryland. He completed residency and fellowship training at the University of Pennsylvania, Philadelphia.

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1 Neurogenetics in the Clinic Thomas D. Bird Department of Neurology, University of Washington, Geriatric Research Center, VA Medical Center, Seattle, Washington, U.S.A.

The clinical practice of neurogenetics is complex, challenging, and rewarding. Several guidelines will be helpful. This chapter will brieﬂy focus on eight aspects of clinical neurogenetics, namely, (i) Factors suggesting the presence of a genetic disease, (ii) Neurogenetic diseases that may be ‘‘hiding’’ in nonspeciﬁc categories, (iii) The importance of family history, (iv) The assessment of sporadic cases, (v) Genetic counseling, (vi) Genetic testing, (vii) Available information resources, and (viii) An integrated clinical neurogenetic strategy. [A discussion of this approach has appeared previously in Ref. (1).]

FACTORS SUGGESTING A NEUROGENETIC DISORDER Table 1 highlights several of the clues indicating an increased probability that a patient may have a neurogenetic disorder. First and foremost is the presence of a positive family history. By nature, genetic diseases are usually inherited (although there are exceptions), and thus it is common to identify other affected family members. This is true of all patterns of inheritance including autosomal dominant, autosomal recessive, X-linked, and mitochondrial. Therefore, a positive family history of at least two similarly affected individuals is a compelling sign of a genetic disorder. However, there are two major exceptions to ﬁnding a positive family history. The ﬁrst is that isolated or sporadic cases without a positive family history can still be genetic. The various explanations for sporadic cases are discussed later in

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Table 1 Clues to the Presence of a Neurogenetic Disease Positive family history Similarity to a known genetic syndrome Chronic, progressive clinical course Consanguinity Increased frequency in a speciﬁc ethnic group

this chapter. The second exception is that not all familial disorders are genetic. In other words, family members sharing the same environment may be affected with the same nongenetic acquired disease. For example, common exposure to infectious or toxic agents may produce a positive family history of a disorder (such as neuropathy or ataxia), which is not genetic. Thus, although a positive family history is a strong sign of a genetic disease, all familial disorders are not always genetic and all genetic disorders are not always familial. Probably the next most important clue suggesting a genetic disorder is a constellation of signs and symptoms suggesting a known genetic syndrome. This is common sense, but often requires a detailed knowledge of, and memory for syndromic identiﬁcation. For example, atypical facial ‘‘acne’’ and a seizure disorder suggest tuberous sclerosis, progressive ataxia with loss of reﬂexes suggests Friedreich ataxia, white matter disease with hyperactive tendon reﬂexes in a male suggests adrenoleukodystrophy or Pelizaeus-Merzbacher disease, and weakness with cataracts suggests myotonic dystrophy. Other factors can be helpful in identifying a genetic disease but are less speciﬁc. For example, most (but not all) genetic diseases have a slow, subtle onset, and prolonged, chronic course. Many have onset in childhood, although it is not uncommon to have a later onset (especially in autosomal dominant triplet repeat conditions). Consanguinity (matings between blood relatives) is a clue to autosomal recessive disorders. In addition, certain diseases are more common in speciﬁc ethnic groups. Examples include Tay-Sachs disease in Eastern European Jews, Unverricht-Lundborg myoclonic epilepsy in Finns, dentatorubral-pallidoluysian atrophy (DRPLA) in Japanese, spinocerebellar ataxia (SCA) 3 in Portuguese and cerebral cavernous malformations or SCA10 in Mexicans.

NEUROGENETIC DISEASES ‘‘HIDING’’ IN NONSPECIFIC CATEGORIES Certain nonspeciﬁc diagnostic categories often harbor patients with neurogenetic diseases and can be viewed as ‘‘reservoirs’’ where neurogenetic patients may be ‘‘hiding.’’ Several of these categories are listed in Table 2. These categories are quite general and such diagnoses, when applied to

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Table 2 Diagnostic Reservoirs of Neurogenetic Diseases Cerebral palsy Mental retardation Epilepsy Movement disorders Ataxias Dementias Atypical multiple sclerosis Peripheral neuropathy

individual patients, often warrant additional investigation. This is particularly true of cerebral palsy, epilepsy, mental retardation, and multiple sclerosis (MS). Certainly, any patient with one of these diagnoses and with a positive family history of a similarly affected individual should initiate a further search for a more speciﬁc genetic cause. Patients referred to our clinic with a diagnosis of cerebral palsy actually have been discovered to have familial spastic paraplegia, ataxia telangiectasia, or Pelizaeus-Merzbacher disease. Patients with dementia, not otherwise speciﬁed, have been found to have genetic forms of Alzheimer’s disease, Creutzfeldt-Jakob disease, and frontotemporal dementia. MS has been an initial diagnosis for individuals later re-diagnosed as cerebral autosomal dominant arteriopathy with subcortal infarcts and leukoencephalopathy (CADASIL) and a variety of genetic leukodystrophies. Persons with movement disorders of uncertain cause have been found to have Huntington’s disease, hereditary ataxias, and genetic forms of dystonia. More detailed evaluation of patients with unexplained epilepsy has uncovered cases of myoclonus epilepsy associated with ragged-red ﬁbers (MERRF) and autosomal recessive myoclonic epilepsy. Many patients with Charcot-Marie-Tooth (CMT) disease have a diagnosis of peripheral neuropathy without recognition of its genetic nature. IMPORTANCE OF FAMILY HISTORY Because a positive family history is such an important element in the diagnosis of neurogenetic disorders, it is critical to pay careful attention to the family medical history and to record it appropriately for future reference (2). The physician should always inquire about the health of siblings, parents, and children. If clues begin to accumulate that a genetic disease may be present, then one inquires about more distant relatives such as grandparents, aunts, uncles, and cousins. Not only is it important to inquire about numerous family members, but the types of questions also need to be carefully tailored to the differential diagnosis. For example, if one is considering myotonic muscular dystrophy, you would ask about other family

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members with baldness, diabetes, developmental delay, or cataract. If considering (CMT) neuropathy, the questions would include difﬁculty in walking, high arched feet, braces, or foot surgery. Possible neuroﬁbromatosis would initiate questions about skin spots and skin lumps, and possible tuberous sclerosis would trigger questions about mental retardation, seizures, and kidney disease. A broad knowledge of the various possible syndromes is critical because nonobvious questions may become important; for example, the association of berry aneurysm with autosomal dominant polycystic kidney disease; diabetes and gall bladder disease with myotonic dystrophy; and schizophrenia with Huntington’s disease. Furthermore, general questions about other family members may add diagnostic support to the potential diagnosis. Figure 1 shows the pedigree of an index case with epilepsy. The occurrence of mental retardation, seizures, and renal tumors in other family members greatly increases the likelihood that the index case has tuberous sclerosis. There are standard protocols for

Figure 1 The index case (proband) in this family is a 16-year-old man with epilepsy. There has been no previous speciﬁc diagnosis of a genetic disease in his family. However, his family history reveals instances of seizures, mental retardation, renal tumor, developmental delay, and learning disability. This constellation of ﬁndings strongly suggests the possibility of tuberous sclerosis occurring in this family. This is an example of a careful family history contributing important diagnostic information regarding the physical complaints of the proband. The diagnosis of tuberous sclerosis is not trivial because it includes implications for the entire family regarding genetic risk, genetic testing, and the use of important medical procedures such as MRI, CT, and renal ultrasound.

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drawing and recording family pedigrees. The important elements are shown in the ﬁgure and more detailed information can be found in chapter 3 (2). ASSESSMENT OF SPORADIC CASES A sporadic or isolated case means a single occurrence of a disease without a positive family history. Table 3 lists most of the possible explanations for the sporadic occurrence of an apparent genetic disease. The most important possibility to consider is that the disease is not genetic but actually acquired (a so-called phenocopy). There are many nongenetic causes of chronic neuropathy, cerebellar ataxia, movement disorders, and spasticity that may mimic CMT disease, hereditary ataxias, Huntington’s disease, or familial spastic paraplegia, respectively. Our clinic has seen a dramatic case of lead poisoning masquerading as acute intermittent porphyria (3). On the other hand, sporadic or index cases may certainly have a genetic cause. Persons with autosomal recessive diseases are expected to have normal, heterozygous, carrier parents. In a small sibship, there may be only a single individual affected with an autosomal recessive disease. A sporadic case of an autosomal dominant disease may represent a new mutation (de novo). This is not uncommon in conditions such as neuroﬁbromatosis and tuberous sclerosis. In addition, many autosomal dominant and mitochondrial disorders have reduced penetrance, meaning a parent may carry the disease mutation but demonstrate no signs of the disease or may have very mild expression; carriers of the mutation may not be noticed by family or physicians. This is true of mutations in mitochondrial genes as well as diseases showing mendelian inheritance. A lack of biological family history information is a common situation for adopted persons, and some persons do not know the true identity of their biologic father (false paternity). Diseases caused by chromosomal aberrations are genetic in that they represent abnormalities of DNA, but they often occur only once in a family, such as typical trisomy 21 Down’s syndrome.

Table 3 Causes of a Sporadic Case Having an Apparent Genetic Phenotype Not genetic Autosomal recessive New mutation Decreased penetrance in family Mild expression in family Adoption False paternity Mitochondrial inheritance Chromosomal abnormality

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GENETIC COUNSELING Genetic counseling is an important aspect of the management of every patient with a neurogenetic disorder (4). It is discussed in detail in chapter 3 but will be brieﬂy reviewed here in the context of several speciﬁc neurogenetic disease categories. Genetic counseling may sometimes be relatively uncomplicated. For example, a patient with sporadic, well-documented multiple sclerosis (MS) may inquire about the risk for MS in his or her children. A brief discussion of multifactorial inheritance and reference to an empiric table of risks for MS (indicating that the risk is about 2–3%) is usually satisfactory. However, many families with neurogenetic diseases raise complex issues that require detailed and time-consuming counseling by an experienced professional with knowledge of both the disease process and the genetic factors. The various aspects of genetic counseling are outlined in Table 4. Accurate diagnosis is essential for genetic counseling. The wrong diagnosis will lead to inaccurate and inappropriate genetic counseling. Establishing the correct diagnosis is often a difﬁcult and sometimes incomplete process. It is better to state that the diagnosis is unclear and provide nonspeciﬁc counseling rather than assume an erroneous diagnosis and provide inaccurate counseling. It is important to recognize that genetic counseling is quite different for Duchenne muscular dystrophy versus limb girdle muscular dystrophy (X-linked vs. autosomal recessive or dominant), or CMT type 1A (autosomal

Table 4 Genetic Counseling Diagnosis Education Recurrence risks Expression Penetrance Natural history Prognosis Need for further testing DNA-based tests Other tests Other family members Genetic options Prenatal testing Adoption Artiﬁcial insemination Pre-implantation diagnosis Treatment options Referral to consultants Support groups Follow-up

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dominant) versus X-linked CMT, or MS (multifactorial) versus an autosomal recessive leukodystrophy. The diagnosis should be as speciﬁc as possible. For example, it is not enough to know that a patient has lower motor neuron disease. For genetic counseling, it is critical to know if the diagnosis is ALS or spinal muscular atrophy or Kennedy’s spinobulbar muscular atrophy. Given a correct diagnosis, an important part of genetic counseling becomes determination of recurrence risk, i.e., identifying who else in the family is at risk for inheriting the disease and the magnitude of that risk. Pattern of inheritance is the most important factor in determining risk and this is covered in detail in chapter 3. The most important part of this determination is the inheritance pattern associated with the accurate diagnosis. Single gene mendelian diseases have the recurrence risks associated with autosomal dominant, autosomal recessive and X-linked inheritance patterns. These risks are carefully explained and enumerated in several basic texts (5). These risks must also take into consideration the known penetrance of the relevant disease gene. Some disorders have a signiﬁcantly reduced penetrance, such that many persons inheriting the mutation never show signs of the disease. For example, the penetrance of the most common form of autosomal dominant dystonia (DYT1) is only about 40%. Also, penetrance depends on patient’s age and comprehensiveness of testing. Thus, the penetrance of the Huntington’s disease gene at age 20 is only about 10%, whereas it is greater than 90% by age 70. Similarly, many people with cerebral cavernous malformations seen on MRI will not have any clinical symptoms and appear not to have the disease. The family should also receive a discussion of prognosis and natural history of the speciﬁc neurogenetic disease. It is especially important to convey information of variable expression, that is, the range of both severity and types of clinical signs in individuals who have the disease gene. It is often helpful to describe a very mild case, an average or typical case, and a severe case. This requires that the counselor know the pertinent literature and have experience with families having the disease. The counselor also needs to determine the need for additional testing. It is frequently valuable to examine or test other family members to help arrive at the correct diagnosis or to determine who else is affected. Potential additional tests will be tailored to the disease entity, and might include EMG, EEG, MRI, or biochemical tests. DNA-based diagnostic tests are playing an increasingly important role in arriving at highly speciﬁc diagnoses in index family members and their relatives (see below). However, genetic or other diagnostic testing may not always be appropriate in asymptomatic at-risk relatives, especially when no treatment is available. Genetic counseling should also answer speciﬁc questions that families have. Many families ask for information regarding genetic options related to family planning. These options may include the availability of prenatal testing, adoption, artiﬁcal insemination, and pre-implantation diagnosis.

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Table 5 Genetic Testing Risks Depression Hypervigilance to symptoms Altered relationships Trouble with insurance Trouble with employment Social isolation

Beneﬁts Speciﬁc diagnosis Family planning Financial planning Decreased anxiety Eligible for treatment or prevention trials

The availability and relevance of such genetic options depends on the speciﬁc disease and an ever-changing technology. Family members will also want the latest information regarding treatment options. Traditionally, neurogenetic diseases have been considered ‘‘untreatable.’’ However, that perspective is changing and an expanding number of neurogenetic disorders will soon have available treatments. PKU and pyridoxine dependent seizures are models of neurogenetic diseases for which there have long been recognized treatments. New treatment options are appearing for an increasing number of neurogenetic diseases (one recent example being the Fabry disease) (6). Therefore, it is important to gather timely information on relevant clinical trails for patients. Finally, complete genetic counseling includes any appropriate referrals to diagnostic or therapeutic consultants, identiﬁcation of disease speciﬁc patient and caregiver support groups, and arrangement for long-term clinical follow-up. GENETIC TESTING Genetic testing is an ever-expanding component of the evaluation of families with neurogenetic diseases. The number and variety of DNA-based diagnostic tests is rapidly increasing. Table 5 lists many of the risks and beneﬁts of genetic testing. It is important to recall that there are several different kinds of genetic tests including chromosome karyotypes, biologic assays, DNAbased tests and linkage analysis. Genetic testing is reviewed in more detail in the chapter by Dr. Nance in this book (7). NEUROGENETICS INFORMATION RESOURCES No neurologist can possibly have a detailed knowledge of all neurogenetic diseases. It is impossible to remain up to date on all aspects of differential diagnosis, gene identiﬁcation, genetic testing, and available support groups. Table 6 lists additional resources providing speciﬁc and detailed information about neurogenetic diseases. The table also lists four websites.

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Table 6 Neurogenetic Information Resources www.geneclinics.org www.genetests.org www.ncbi.nlm.nih.gov/Omim/ www.neuro.wustl.edu/neuromuscular/ Pulst S. Neurogenetics. New York: Oxford Press, 2000. Rosenberg RN, Prusiner SB, DiMarco S, Bardin RL. The Molecular and Genetic Basis of Neurological Disease. 3rd ed. Boston: Butterworth–Heinemann, 2003. Lynch DR, Farmer JM, eds. Neurologic Clinics: Neurogenetics. Vol. 20:3. Philadelphia: W.B. Saunders Co Elsevier Science, 2002. This text

Geneclinics (www.geneclinics.org) has expert authored and peer reviewed proﬁles on more than 300 of the most important genetic diseases including many neurogenetic disorders. The proﬁles are organized in a user-friendly way that includes diagnostic criteria, differential diagnosis, genetic testing, genetic counseling, molecular background, support groups, and literature references. A valuable resource for genetic testing information is the website www.genetests.org. This site is an annotated index of hundreds of genetic diseases, the availability of genetic testing for each disease, and a listing of associated testing laboratories with contact information. The site helpfully divides laboratories into those doing commercial/clinical or research testing and the information is constantly updated. Online Mendelian Inheritance in Man (OMIM) is an extensive listing of all known human genetic diseases with a selected bibliography for each entry. The Washington University website (http://www.neuro.wustl.edu) is speciﬁcally designed for neuromuscular diseases. Rosenberg et al. and Pulst are text books that contain an overview of neurogenetics. Finally, this present text now provides another up-to-date resource regarding neurogenetic diseases.

AN INTEGRATED CLINICAL NEUROGENETICS STRATEGY The comprehensive evaluation and management of patients with neurogenetic diseases has four main components (Table 7). The ﬁrst is primarily neurological. This relates to taking a history, examining the patient, formulating a differential diagnosis, establishing a correct diagnosis, understanding the natural history and potential complications of the disease, and treatment and management. The second is primarily genetic. This relates to obtaining a detailed pedigree, establishing the correct pattern of inheritance, determining genetic risks, identifying the importance of the diagnosis to other family members, understanding issues of penetrance and variable expression, interpreting genetic test results, and giving advice about genetic

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Table 7 Integrated Strategy for Clinical Neurogenetics Neurological Genetic Psychosocial Whole family perspective

options. The third is psychosocial counseling. This refers to the frequent necessity for helping patients and their families deal with the often complex mental, emotional, and social implications of their diseases. The fourth aspect is having a family perspective. This means recognizing that genetic diseases have important implications for more than just the initial index case. When a genetic diagnosis is made, other family members are quickly drawn into the cascade of events that ﬂow from the initial diagnosis out into the larger family. Other family members often require evaluation and counseling. Practioners of medical genetics clearly practice ‘‘family medicine.’’ No individual can provide all of these services. A core neurogenetics clinical unit would include a neurologist, genetics counselor, social worker, and psychologist or psychiatrist. Consultations to nurses, rehabilitation medicine specialists, radiologists, and relevant subspecialties of internal medicine are common. This team approach will provide the best long term care for patients with neurogenetic diseases and form an integrated evaluation and management strategy.

REFERENCES 1. Bird TD. Approaches to the patient with neurogenetic disease. In: Lynch DR, Farmer JM, eds. Neurologic Clinics: Neurogenetics. Vol. 20. Philadelphia, PA: W.B. Saunders, 2002:619–626. 2. Bennett R. The Practical Guide to the Genetic Family History. Wiley-Liss, 1999. 3. Bird TD, Wallace DM, Labbe RF. The porphria, plumbism, pottery puzzle. JAMA 1982; 247(6):813–814. 4. Pagon RA. Genetic diagnosis and counseling. In: Dale DC, Federman DD, eds. Sci Am Med. Vol. 2. Sect 9: Subsect VIII. New York, NY: Scientiﬁc American, Inc., 2001. 5. Nussbaum R, McInnes R, Willard H. Genetics in Medicine. 6th ed. Philadelphia: W.B. Saunders, 2001. 6. Eng C, Guffon N, Wilcox W, Germain D. Safety and efﬁcacy of recombinant human a-galactosidase a replacement therapy in Fabry’s disease. N Engl J Med 2001; 345:9–16. 7. Bird T. Risks and beneﬁts of DNA testing for neurogenetic disorders. Seminars in Neurology 1999; 19:253–259.

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2 Genetic Testing for Neurological Disorders Martha A. Nance Department of Neurology, University of Minnesota, Minneapolis, Minnesota, U.S.A.

INTRODUCTION Historically, the most important legacy of the Human Genome Project in the ﬁeld of neuroscience will be the era of experimental human neurogenetics: the ability to create experimental animal models of otherwise exclusively human neurologic disorders, and to perform experimental research using those animal models. However, while neurologists and their patients and families await the improved treatments that experimental neuroscience promises, a direct and immediate consequence of the discovery of disease genes, genetic testing is already available in clinical practice. We review below the role of genetic testing in neurology, and the role of the neurologist in ensuring that genetic tests are used and interpreted properly. PRINCIPLES OF GENETIC INHERITANCE A full discussion on the principles of genetics is beyond the scope of this chapter, but a few points are important to consider. We will assume that the concepts of autosomal dominant, autosomal recessive, and X-linked inheritance are known to the reader, and will brieﬂy discuss other concepts that are important to the appropriate use and interpretation of gene tests.

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Reduced Penetrance, Variable Expressivity, Manifesting Carriers The concepts of reduced penetrance and variable expressivity are relevant to dominantly inherited genetic conditions, for which a single copy of a mutation-bearing gene is sufﬁcient to cause the disease. Individuals carrying mutations known to cause diseases such as Huntington’s disease occasionally remain symptom free throughout a long lifetime; in these individuals, the gene mutation is said to be nonpenetrant. Apparent nonpenetrance may be seen in a young patient carrying a gene mutation for tuberous sclerosis or neuroﬁbromatosis type 1 (NF1) (1,2); complete penetrance for NF1 by age ﬁve, however, has been documented (2). The terms ‘‘reduced’’ and ‘‘incomplete’’ penetrance refer to the behavior of a gene mutation in a population, while ‘‘nonpenetrance’’ applies to a single individual or group of individuals who have no disease symptoms despite carrying the gene mutation. A related concept is that of variable expressivity. A parent with ﬁve cafe´-au-lait spots who is otherwise normal in every way can have a child with severe manifestations of neuroﬁbromatosis; a clinically asymptomatic sibling of a child who has many manifestations of tuberous sclerosis may show periventricular heterotopias on brain magnetic resonance imaging (MRI) (3,4). Striking genotypic and phenotypic heterogeneity has been reported for the myotonic dystrophies (5,6), hereditary spastic paraplegias (7), hereditary neuropathies (8), and many other dominantly inherited conditions. Variable expression of disease symptoms in patients carrying the same gene mutation is related to other speciﬁc genes, environmental inﬂuences, and genetic (ethnic) background, among other factors. For the polyglutamine disorders, a group of neurodegenerative disorders caused by expansion of the trinucleotide cytosine–adenine–guanine (CAG) within the coding portion of the causative genes, variable expression takes the form of an inverse correlation between the size of the repeat expansion and the age of symptom onset—including the possibility of nonpenetrance for individuals with borderline expansions of the trinucleotide repeat sequence (9,10). As noted above, reduced penetrance and variable expressivity occur because of other genetic and environmental factors that modify the effects of the disease gene mutation. Identifying these modiﬁers is one of the goals of current research for many neurogenetic diseases, but clinically important successes have yet to be reported. As clinicians provide genetic testing for patients with single-gene disorders, they must help the patients understand to what extent the presence of a disease gene does or does not explain or predict the onset age, severity, or nature of disease symptoms, as well as the potential psychosocial implications of the results (11,12). Manifestation of disease symptoms is sometimes reported in female carriers of genes responsible for X-linked disorders. Carriers of mutations in the CMTX gene, for example, can have symptoms of neuropathy, and

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carriers of ABCD1 (adrenoleukodystrophy) or proteolipid protein gene (spastic paraplegia/Pelizeaus-Merzbacher disease) mutations have been reported to have white matter changes on brain MRI, and sometimes to have disease symptoms (13,14). Depending on the gene and disease, the classic teaching that ‘‘females are carriers for X-linked disorders but only males can develop the disease’’ may oversimplify the relationship between the gene and the disease, and is likely to be inaccurate. In most situations, female carriers will have milder disease symptoms than males, presumably because of the presence of a second X chromosome bearing a normal copy of the gene. The severity of disease manifestations in a female relates at least in part to the vagaries of X chromosome inactivation (a mechanism by which one of the X chromosomes is ‘‘turned off ’’ in each cell), a phenomenon that cannot be measured currently in clinical laboratories (15). A new example of the complex relationship between genes and diseases is the recently described Fragile X Tremor-Ataxia Syndrome (FXTAS). In this syndrome, premutations in the FMR1 gene—a gene in which full mutations are associated with male mental retardation—have been associated with the development of tremor and gait disturbance in later life, in both men and women (16,17). Splitting Identiﬁcation of disease genes often leads to ‘‘splitting’’ of clinically deﬁned neurologic entities into genetically deﬁned, often clinically indistinguishable subgroups. What was once Charcot-Marie-Tooth disease (CMT) is now a group of over 25 genetically deﬁned entities (8). At least 20 different genes responsible for autosomal recessive and dominant and X-linked forms of hereditary spastic paraplegia have been localized; gene tests are clinically available for at least seven of these genes (7). Identiﬁcation of disease genes and the development of clinically useful tests remains a work in progress. For example, although spinocerebellar ataxia types 1–3,6,7, and 8 account for about 50% to 70% of autosomal dominant hereditary ataxias, a signiﬁcant proportion of patients who undergo testing for all of these genes will remain without a speciﬁc diagnosis, as a number of ataxia genes remain to be localized, and others that have been localized remain to be identiﬁed (18,19). Although a speciﬁc genetic diagnosis may not lead to changes in therapy, there are three important clinical beneﬁts to a speciﬁc genetic diagnosis (e.g., spinocerebellar ataxia type 2) in comparison to a general clinical diagnosis (e.g., hereditary ataxia): (i) an end to the search for a ‘‘correct’’ diagnosis allows patients to focus on disease management or treatment, (ii) accurate prognosis often depends on the speciﬁc diagnosis, and (iii) accurate genetic risk assessment and counseling, as well as the availability of predictive, prenatal, or carrier testing for the patient or relatives, critically depends on a speciﬁc diagnosis.

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In addition to the possibility of multiple genocopies, for some neurogenetic conditions, apparently nongenetic phenocopies also exist. For example, the vast majority of Parkinson’s disease and about 90% of Alzheimer’s disease are sporadic, or (apparently) nongenetic, in nature. Although a higher proportion of ataxia is genetic, sporadic phenocopies are also very common. This heterogeneity complicates the diagnostic evaluation for many neurogenetic disorders. GENOTYPE CHANGES AND THEIR SIGNIFICANCE IN PHENOTYPIC APPEARANCE The relationship between gene mutations and the diseases they are associated with can be more or less straightforward. About 70–80% of Charcot-MarieTooth I is caused by duplications of the PMP22 gene (CMT 1A), but about 1% to 2% of cases—often with more severe symptoms—are caused by point mutations in the same gene. Furthermore, a clinically distinct condition, hereditary neuropathy with liability to pressure palsies, is caused by deletions in the same gene. CMT types 1B, C, and D may be genetically distinguished from CMT 1A by the presence of mutations in the MPZ, EGR2, and LITAF genes, respectively (20). Larger trinucleotide repeat expansions in the DM1 and FRDA genes have been associated, in general, with more severe myotonic dystrophy and Friedreich’s ataxia phenotypes, respectively, than are smaller expansions (21,22). Trinucleotide repeat expansions in the SCA6 gene cause a neurodegenerative ataxia, while point mutations cause episodic ataxia type 2 or familial hemiplegic migraine (23,24). Even more remarkably, trinucleotide repeat expansions in the gene encoding the androgen receptor cause Kennedy syndrome (spinobulbar muscular atrophy), while point mutations cause an androgen insensitivity syndrome (25,26). On the other hand, mutations in the gene coding for dystrophin are of many types, leading to a continuum of phenotypes ranging from the classic Duchenne to Becker muscular dystrophy to isolated dilated cardiomyopathy (27). Similarly, attempts to correlate disease symptoms or severity with the location or type of mutation in the neuroﬁbromatosis gene NFI have not been successful (3). For some genes, the relationship between gene mutation and the development of a disease is clear: 100% of Huntington’s disease (HD) is caused by CAG repeat expansions in the gene encoding huntingtin, for example, and all CAG repeat expansions in this gene beyond a certain size cause HD (there is reduced penetrance for small CAG expansions). The relationship between mutations in parkin and Parkinson’s disease, however, is less clear. Homozygous parkin mutations (mutations in both copies of the gene) cause autosomal recessive juvenile onset Parkinson’s disease. Heterozygous parkin mutations are also seen in about 20% of patients with young-adult onset Parkinson’s disease, and 2% of those with later onset disease, and may

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be associated with evidence of dopaminergic dysfunction in asymptomatic carriers, but are neither necessary nor sufﬁcient to cause Parkinson’s disease (28,29). The Apo E4 allele, likewise, is neither necessary nor sufﬁcient to cause a disease, but has clearly been associated with the presence of Alzheimer’s disease, and may serve as a determinant of onset age for both Alzheimer’s and Parkinson’s diseases (30,31).

GENE MUTATIONS AND ASSAYS TO DETECT THEM There is a tendency to refer to genetic testing as though it is a single entity, when, of course, each gene and test is unique. Different kinds of gene mutations require different detection techniques in the laboratory. Table 1 lists some types of gene mutations, with examples for each. Barriers to the transition from gene identiﬁcation in the research laboratory to clinical assay in a service laboratory have been discussed recently (32). Mutation detection methods used in molecular diagnostic laboratories have also been reviewed (33). The most straightforward situation is when a single mutation is always responsible for the disease, allowing the laboratory to create a speciﬁc assay for that mutation. This might be the case for a common point mutation, a gene duplication (as in the PMP22 gene in CMT), or for the CAG repeat expansion in a trinucleotide repeat disorder. Techniques used for mutation analysis depend on the speciﬁcs of the particular gene and mutation. Duplications of the PMP22 gene are large enough to be detected using the molecular cytogenetic technique of ﬂuorescence in situ hybridization (FISH). Most gene tests currently performed in molecular diagnostic laboratories, however, use the polymerase chain reaction (PCR) to selectively increase

Table 1 Types of Gene Mutations Mutation type Point mutations Single base pair or small deletions, insertions Large deletions Gene duplications Trinucleotide repeat CAG repeat Other repeats Abbreviation: CAG, cytosine–adenine–guanine.

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Example Many recessively inherited neurogenetic disorders Many neurogenetic disorders Duchenne muscular dystrophy Charcot-Marie-Tooth 1A Fragile X syndrome, myotonic dystrophy 1 Huntington’s disease, spinocerebellar ataxias 1–3 Myotonic dystrophy 2; spinocerebellar ataxia 10

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the abundance of the DNA segment of interest among all the other DNA sequences in the sample, followed by various gel electrophoresis techniques to separate DNA sequences by size, and the use of speciﬁc probes, stains, or ﬂuorescent markers to detect the mutation or size variation of interest. Different methods are required when there are many known or possible point mutations or small deletions within a gene. In order to detect ‘‘private’’ (family speciﬁc) mutations, laboratories may have to sequence the gene, a laborious and expensive task. More often, techniques such as single-strand conformation polymorphism (SSCP) or denaturing gradient gel electrophoresis (DGGE) are ﬁrst used to screen for differences between the patient DNA sequence and a normal gene sequence. In some laboratories or for some genes, the testing process may stop after the screening SSCP analysis, with a report stating that a variation was found between the patient DNA and a normal sequence, but without specifying what that difference is and whether it is pathogenic or simply a polymorphism (normal variation). At other times, or in some laboratories, additional efforts may be undertaken after SSCP to sequence the relevant part of the gene to identify the speciﬁc mutation. In some clinical and laboratory situations, once a novel gene mutation is identiﬁed in a patient, an allele-speciﬁc oligonucleotide (ASO) can be developed to test for the mutation in other family members. Communication between the clinician and the laboratory may be necessary to ensure that both parties understand what test has been done, the detection rate for the speciﬁc method, what its clinical implications are, and whether further testing is possible either in that laboratory or elsewhere if there is a negative or informative result. There are certain technique-imposed limitations to the resolution of some gene tests. An assay designed to be sensitive to CAG repeat expansions in the range of 20 to 80 repeats could miss a very large repeat expansion, and an assay designed to detect repeat expansions will not detect point mutations (34). Undetected variations in the DNA sequence at the primer site used in the PCR reaction to ‘‘cut out’’ the DNA region to be assayed could reduce or prevent ampliﬁcation of that region, and loss of detection of that gene (35). Alleles carrying large trinucleotide repeat expansions or gene insertions may also amplify poorly in the PCR reaction, ultimately resulting in the detection of only the normal allele. In the laboratory, detection of a single band on the gel may mean either that only a single allele was identiﬁed or that both alleles were of the same size. If genetic test results are discordant with the clinical situation or diagnosis, the laboratory may be able to utilize special techniques to resolve the discrepancy. Not all genes responsible for neurogenetic disorders have been identiﬁed. For genes that have been localized but not yet identiﬁed, or for genes in which the speciﬁc mutation is uncertain, analysis using markers genetically linked to the gene of interest may be possible in a family. Genetic linkage testing is not widely used for clinical purposes, as it is time consuming, expensive,

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and requires the cooperation of multiple family members. Identiﬁcation of families and genetic linkage analysis, however, remains an important research tool for the identiﬁcation of new disease genes. The gene encoding dystrophin provides an interesting example of the varying methodologies used in molecular diagnostic testing. About 65% of patients with Duchenne muscular dystrophy (DMD) have large deletions in the DMD gene, which can often be detected by FISH analysis or by electrophoretic techniques that can resolve large DNA fragments (Southern blotting) (36). About 30% of patients have small deletions, point mutations, or insertions that can be detected by mutation screening or scanning techniques. About 6% of patients have duplications of a portion of the gene. Novel or combined strategies can help increase the yield of diagnostic testing (37). If Duchenne muscular dystrophy is strongly suspected but a gene mutation has not been conﬁrmed by molecular genetic analysis, then a muscle biopsy specimen can be examined for dystrophin protein content. Identiﬁcation of a gene mutation is not the only way to diagnose a genetic condition. For any number of inborn errors of metabolism or other enzymopathies, measuring the blood or tissue level of the enzyme encoded by the gene, or the metabolite that builds up in the absence of the enzyme, may be a more efﬁcient and informative path to diagnosis than a gene assay. For example, the diagnosis of adrenoleukodystrophy in males is usually secured by measurement of plasma very long chain fatty acid levels, with mutation analysis of the ABCD1 gene providing a diagnostic backup procedure (13). CLINICAL USE OF GENE TESTS Genetic testing is used in four different clinical situations: (i) as part of a diagnostic evaluation, (ii) predictive testing for an adult-onset disease in an asymptomatic person, (iii) prenatal or preimplantation testing for an at-risk couple, and (iv) carrier testing for an X-linked or autosomal recessive condition. The neurologist is most often involved in the evaluation of a patient who has neurologic symptoms, and thus is most likely to order gene tests for diagnostic purposes. However, it is important for the physician who diagnoses or treats a genetic condition to be aware of the direct implications of the patient’s diagnosis for other relatives, and to offer or refer the patient and/or these relatives for further genetic counseling or testing as appropriate. Diagnostic Genetic Testing In the appropriate clinical situation, a gene test affords a rapid, efﬁcient, and precise diagnosis. Whether or not the precise diagnosis leads to any change

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in treatment, it allows the physician to better deﬁne the patient’s prognosis and provide speciﬁc information about the disease, as well as allowing the patient and family to focus on symptom management. From the physician’s perspective, it is difﬁcult to escape the view that more information is better, although some patients (and importantly, some health insurers), noting that gene test results may not alter the patient’s treatment, are reluctant to bear the cost of such tests. Patients should always be informed about and consent to genetic testing, and should ideally have genetic counseling prior to undergoing the test. Genetic counseling typically includes a review and documentation of the family history, a discussion of relevant aspects of basic genetics, including a description of how the gene test is done and how accurate it is, what genetic condition(s) is being tested for, as well as a discussion of the potential emotional impact of a genetic diagnosis on the patient and family (see chap. 3). Gene test results should be provided in the context of supportive counseling, as for the diagnosis of any chronic condition, with the added recognition that other family members may have concerns about their own genetic risks and testing possibilities. Some neurogenetic conditions are common enough that one or more molecular diagnostic laboratories offer diagnostic testing, while others are so uncommon or difﬁcult to test for that clinical service laboratories do not perform the test. Tables 2–4 list some of the trinucleotide repeat disorders, hereditary neuropathies, and spastic paraplegias for which genetic testing is clinically available. These tables provide a perspective on the relative availability and completeness of a clinical testing. For uncommon diseases, diagnostic testing can sometimes be arranged through a research laboratory. Many research laboratories in the United States are no longer making research test results available to patients because of patient privacy and ethical concerns, as well as legal liability concerns regarding the release of research data to subjects by a non-Clinical Laboratory Improvement Amendments (CLIA)-certiﬁed laboratory (38). Before sending a patient sample to a research laboratory, the physician and patient should be clear whether the sample is to be used for research purposes, whether any ‘‘results’’ are expected to return to the physician or patient, and what the expected time frame is for receipt of results. The physician who orders a gene test also bears the burden of interpreting the test results for the patient and family (39). While a laboratory report often includes an interpretation, standardized reports may not reﬂect the most recent ﬁndings in this rapidly evolving area, or may use terminology that is confusing or vague. For example, a laboratory report might describe a result of 37 CAG repeats within the HD gene as being within an ‘‘intermediate range’’ or ‘‘indeterminate,’’ implying that the clinical relevance of the result is entirely uncertain. A more appropriate term might be ‘‘incomplete penetrance,’’ meaning that a result in this range in a symptomatic person conﬁrms the diagnosis, while a result in this range in an asymptomatic person

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Table 2 Repeat Expansion Diseases Disease CAG repeat (polyglutamine) disorders Huntington’s disease Spinocerebellar ataxia type 1 (SCA1) Spinocerebellar ataxia type 2 (SCA2) Machado Joseph Disease (SCA3) Spinocerebellar ataxia type 6 (SCA6) Spinocerebellar ataxia type 7 (SCA7) Spinocerebellar ataxia type 12 (SCA12) Spinocerebellar ataxia type 17 (SCA17) Dentatorubral-pallidoluysian atrophy (DRPLA) Spinobulbar muscular atrophy (Kennedy disease) Others Fragile X (FRAXA) Myotonic dystrophy type 1 (DM1) Myotonic dystrophy type 2 (DM2) Friedreich’s ataxia Progressive myoclonic epilepsy type 1 (EPM1) Spinocerebellar ataxia type 8 (SCA8) Spinocerebellar ataxia type 10 (SCA10) Oculopharyngeal muscular dystrophy

Repeat CAG CAG CAG CAG CAG CAG CAG CAG CAG

Inheritance Autosomal Autosomal Autosomal Autosomal Autosomal Autosomal Autosomal Autosomal Autosomal

dominant dominant dominant dominant dominant dominant dominant dominant dominant

CAG

X-linked

CGG CTG CCTG GAA Dodecamer

X-linked Autosomal dominant Autosomal dominant Autosomal recessive Autosomal recessive

CTG ATTCT GCG

Autosomal dominant Autosomal dominant Dominant/recessive

neither rules out nor makes certain the later clinical development of HD. For clinical entities for which there are multiple causative genes, the challenge may be in helping the patient understand that a normal gene test result does not prove that the condition is nongenetic, simply that the condition was not caused by a mutation in that particular gene. For genetic disorders for which there are multiple causative mutations within the same gene, the physician must understand and explain whether normal test results ruled out all possible mutations within the gene, or just one or some of them. The ﬁrst genes identiﬁed were those for single-gene disorders, for which there was a clear relationship between the presence of the gene and the development of the disease, according to well-understood genetic inheritance patterns (autosomal dominant, recessive, and X-linked). The genetic waters grow ever murkier, however, as clinicians develop the tools to test for genetic mutations or polymorphisms that confer an increased risk of disease without directly causing the disease (e.g., the Apo E4 allele in Alzheimer’s disease), or whose relationship to the disease is uncertain (e.g., the gene encoding parkin in juvenile, earlyonset, and late-onset Parkinson’s disease). Detailed and careful counseling

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Table 3 Clinically Testable Hereditary Neuropathies Disease

Protein

Mutation type

CMT1A

Peripheral myelin protein 22

HNPP

Peripheral myelin protein 22

CMT1B CMT1D CMT2E CMT4A

Myelin protein zero Early growth response protein 2 Neuroﬁlament triple L protein Ganglioside-induced differentiation protein 1 Early growth response protein 2 Periaxin Connexin-32

CMT 4E CMT4F CMTX

Duplication (98%), pt mutation (2%) Deletion (80%), pt mutation/ small del (20%) pt mutation pt mutation pt mutation pt mutation pt mutation pt mutation pt mutation, small del

Abbreviations: CMT, Charcot-Marie-Tooth disease; pt, point; del, deletion; HNPP, hereditary neuropathy with liability to pressure palsies.

is required to explain to the patient the clinical relevance of these types of tests (40). Predictive Testing Predictive testing refers to the use of a gene test in a patient at risk for a genetic disease (typically an adult-onset disease) of which the patient does Table 4 Clinically Testable Spastic Paraplegias Locus

Protein

Inheritance

SPG1

L1CAM

X-linked

SPG2

PLP2

X-linked

SPG3A SPG4 SPG6 SPG7 SPG10 SPG13 SPG17 SPG20 SPG21

Atlastin Spastin NIPA1 Paraplegin KIF5A HSPD1 Seipin Spartin Maspardin

Autosomal Autosomal Autosomal Autosomal Autosomal Autosomal Autosomal Autosomal Autosomal

dominant dominant dominant recessive recessive dominant dominant dominant dominant

Condition SP with MR, aqueductal stenosis/ hydrocephalus SP, Pelizaeus-Merzbacher disease Pure SP Pure SP SP SP Pure SP Pure SP Silver syndrome Troyer syndrome Mast syndrome

Abbreviations: SPG, spastic paraplegia locus; L1CAM, L1 cell-adhesion molecule; SP, spastic paraplegia; MR, mental retardation; PLP2, proteolipid protein 2.

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not currently have symptoms. Until treatments are found to forestall or prevent such conditions, which are usually dominantly inherited neurodegenerative disorders, the only purpose of predictive testing is the psychosocial beneﬁt perceived by the patient. For this reason, the principles underlying predictive testing are somewhat different than those guiding the use of gene tests for diagnostic purposes. Detailed genetic counseling is necessary to ensure that the patient understands clearly what the gene test means and what it does not mean (the presence of the abnormal gene, for instance, does not determine that a person is symptomatic, nor does it determine at what age the person will become symptomatic), as well as the potential adverse psychological and social outcomes of the test. The decision to undergo predictive testing should be voluntary and informed, meaning that parents should not request predictive testing for their underage children, adoptive agencies should not require predictive testing duplicates prior to adoption, and other third parties—spouses, employers, insurance agencies, or courts—should not require or perform predictive testing without the consent of the person being tested. Protocols have been established for predictive testing in HD, the ﬁrst condition for which predictive testing was performed with any frequency; these protocols are associated with a low risk of adverse outcomes in those at risk for HD, and can easily be applied to other neurogenetic conditions (41,42). The main points within HD predictive testing protocols include (i) the provision of genetic counseling prior to the test, (ii) pretest psychological assessment and counseling, (iii) the availability of a neurological examination if a patient requesting predictive testing is concerned about possible symptoms of the disease, (iv) freely given consent for the predictive test, (v) the opportunity to opt out of having the test or being informed of the results, even after consent is given, and (vi) results to be given in person in a supportive counseling environment. Many patients choose to selfpay for most or all of the procedures and visits related to predictive testing, to provide an additional layer of protection against the possibility of insurance discrimination. Some patients request anonymous or pseudonymous testing to provide additional privacy protection; providers and their institutions vary in the degree of anonymity they are able or willing to provide (in some medical centers, patients cannot register to be seen without providing a social security number, for example, and some physicians and genetic counselors are uncomfortable providing predictive testing to a person who is unwilling to provide a name, family history information, or some amount of personal information) (43). A more detailed discussion of the ethical issues raised by predictive testing can be found in chapter 5. Predictive testing has been reported for an increasing number of diseases, including hereditary ataxias, neuropathies, muscular dystrophies, hereditary Alzheimer’s disease, CADASIL, spastic paraplegias but by far the greatest experience has accrued with its use in HD (44–47).

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Prenatal and Preimplantation Testing Prenatal testing refers to the use of a gene test by a parent on behalf of an at-risk fetus, while preimplantation testing involves the combination of in vitro fertilization techniques with single-cell DNA assays, with implantation into the uterus of an embryo proven to be free of the abnormal gene in question. Prenatal testing has focused in the past on life threatening or disabling conditions of infantile or childhood onset, but can also now be applied to adult-onset neurogenetic conditions (48–50). Prenatal or preimplantation testing could be requested by a patient who currently suffers from a neurogenetic condition, by a patient who is known to carry a gene mutation that causes a disease but who is not currently symptomatic, by a couple both known to be carriers of a recessive disease gene, or by a patient who is at risk for carrying an abnormal gene but unaware of his or her genetic status. Preimplantation testing could also be requested by a patient who is known to have an abnormal disease-causing gene, or by a patient who is at risk but who does not wish to know his or her own gene status (51). Prenatal and preimplantation testing both require detailed counseling about the risks of the procedures used to obtain fetal tissue, the potential outcomes of the testing process, and the medical choices to be made based on those outcomes. In general, the only ‘‘therapeutic’’ procedure to be offered if a prenatal test shows the presence of a disease-causing gene mutation is termination of the pregnancy. If this is not a permissible alternative for the patient or physician, then it may not be appropriate to perform the prenatal test. Preimplantation testing avoids the issue of abortion, but requires intensive testing, medications, and procedures which are usually not covered by insurance, and thus requires a ﬁrm commitment from the patient or couple. It should be noted that genetic counseling for at-risk couples includes a discussion not only of options that include gene tests, but other reproductive options such as childbearing without testing, artiﬁcial insemination, surrogate mother, adoption, or sterilization procedures to permanently prevent the possibility of pregnancy. Carrier Testing The term ‘‘carrier’’ refers to an individual who carries a single copy of an abnormal gene that alone does not cause disease symptoms in the presence of a second, normal copy of the gene. The term is relevant for genetic disorders inherited in an autosomal recessive fashion, and for X-linked disorders. Both biological parents of a child who is affected with an autosomal recessive disorder are obligate carriers of a single copy of the abnormal gene, barring rare and unusual genetic events, and the mother of a boy with an X-linked disease is also likely to be a carrier of the abnormal, disease-causing gene. Others at risk for being carriers in either of these

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scenarios include clinically normal siblings of the affected person, as well as aunts (and uncles for autosomal disorders) and grandparents. Genetic testing of individuals who do not have a disease diagnosis or any neurological symptoms must be thought out carefully, with discussion of the implication of the carrier state to their own health and that of their descendants, as well as the possibility that others will misunderstand or misinterpret the signiﬁcance of the carrier state. RESOURCES FOR THE CLINICIAN The clinician must identify appropriate candidates for genetic testing, select the correct test, ensure that the patient is properly counseled about the test and its potential outcomes, understand the signiﬁcance of the results for the patient, and recognize the potential importance of the results for other relatives. It is very difﬁcult for any physician to remain current in this rapidly moving ﬁeld, and the use of on-line resources is encouraged. Online Mendelian Inheritance in Man (OMIM) (www.ncbi.nlm.nih.gov), and GeneTests (www.genetests.org) both provide descriptions of neurologic disorders and the gene mutations associated with them. GeneTests also provides detailed information about laboratories that perform clinical or research tests for many conditions, including contact information for the laboratories, and summarizes both the clinical and the genetic aspects of diseases and groups of diseases in a clinically useful fashion. Disease-speciﬁc lay organizations often provide accurate and up-to-date information about the genetic aspects of a disease. Finally, genetic counselors and genetics physician specialists can be an invaluable resource to the neurologist, as they can provide both the genetic education and the supportive counseling that patients and families undergoing genetic testing require, as well as assisting with referrals for prenatal, preimplantation, or carrier testing. REFERENCES 1. Kwiatkowski J, Jozwiak S, et al. Comprehensive mutational analysis of the TSC1 gene: observations on frequency of mutation, associated features, and nonpenetrance. Ann Hum Genet 1998; 62:277–285. 2. Huson SM, Compston DA, et al. A genetic study of von Recklinghausen neuroﬁbromatosis in south east Wales: I. Prevalence, ﬁtness, mutation rate, and effect of parental transmission on severity. J Med Genet 1989; 26:704–711. 3. Carey JC, Viskochil DH. Neuroﬁbromatosis type 1: a model condition for the study of the molecular basis of variable expressivity in human disorders. Am J Med Genet 1999; 89:7–13. 4. Viskochil D. Genetics of neuroﬁbromatosis 1 and the NF1 gene. J Child Neurol 2002; 17:562–570. 5. Day JW, Ricker K, et al. Myotonic dystrophy type 2: molecular, diagnostic and clinical spectrum. Neurology 2003; 60:657–664.

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6. Mankodi A, Thornton CA. Myotonic syndromes. Curr Opin Neurol 2002; 15:545–552. 7. Fink JK. Hereditary spastic paraplegia. Neurol Clin 2002; 20:711–726. 8. Berciano J, Combarros O. Hereditary neuropathies. Curr Opin Neurol 2003; 16:613–622. 9. Langbehn DR, Brinkman RR, et al. A new model for prediction of the age of onset and penetrance for Huntington’s disease based on CAG length. Clin Genet 2004; 65:267–277. 10. Brinkman RR, Mezei MM, et al. The likelihood of being affected with Huntington’s disease by a particular age, for a speciﬁc CAG size. Am J Hum Genet 1997; 60:1202–1210. 11. Origone P, Bonioli E, et al. The Genoa experience of prenatal diagnosis in NF1. Prenat Diagn 2000; 20:719–724. 12. Bird TD. Risks and beneﬁts of DNA testing for neurogenetic disorders. Semin Neurol 1999; 19:253–259. 13. Moser H, Dubey P, Fatemi A. Progress in X–linked adrenoleukodystrophy. Curr Opin Neurol 2004; 17:263–269. 14. Battini R, Bianchi MC, et al. Unusual clinical and magnetic resonance imaging ﬁndings in a family with proteolipd gene mutation. Arch Neurol 2003; 60:268–272. 15. Heard E. Recent advances in X-chromosome inactivation. Curr Opin Cell Biol 2004; 16:247–255. 16. Hagerman RJ, Leavitt BR, et al. Fragile X-associated tremor/ataxia syndrome (FXTAS) in females with the FMR1 premutation. Am J Hum Genet 2004; 74:1051–1056. 17. Hagerman PJ, Hagerman RJ. Fragile X-associated tremor ataxia syndrome (FXTAS). Ment Retard Dev Disabil Rev 2004; 10:25–30. 18. Schols L, Bauer P, et al. Autosomal dominant cerebellar ataxias: clinical features, genetics, and pathogenesis. Lancet Neurol 2004; 3:291–304. 19. Taroni F, DiDonato S. Pathways to motor incoordination: the inherited ataxias. Nat Rev Neurosci 2004; 5:641–655. 20. Bird TD. Charcot-Marie-Tooth Type 1 in Gene Reviews. www.genetests.org, accessed February 6, 2005. 21. Ranum LP, Day JW. Myotonic dystrophy: RNA pathogenesis comes into focus. Am J Hum Genet 2004; 74:793–804. 22. Mateo I, Llorca J, et al. Expanded GAA repeats and clinical variation in Friedreich’s ataxia. Acta Neurol Scand 2004; 109:75–78. 23. Jen J, Kim GW, Baloh RW. Clinical spectrum of episodic ataxia type 2. Neurology 2004; 62:17–22. 24. Mantuano E, Veneziano L, et al. Spinocerebellar ataxia type 6 and episodic ataxia type 2: differences and similarities between the two allelic disorders. Cytogenet Genome Res 2003; 100:147–153. 25. Greenland KJ, Zajac JD. Kennedy’s disease: pathogenesis and clinical approaches. Intern Med J 2004; 34:279–286. 26. Zitzmann M, Nieschlag E. The CAG repeat polymorphism within the androgen receptor gene and maleness. Int J Androl 2003; 26:76–83. 27. Muntoni F, Torelli S, Ferlini A. Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet Neurol 2003; 2:731–740.

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28. Khan NL, Scherﬂer C, et al. Dopaminergic dysfunction in unrelated asymptomatic carriers of a single parkin mutation. Neurology 2005; 64:134–136. 29. Poorkaj P, Nutt JG, et al. Parkin mutation analysis in clinic patients with earlyonset Parkinson’s disease. Am J Med Genet A 2004; 15:44–50. 30. Zareparsi S, Camicioli R, et al. Age at onset of Parkinson disease and apolipoprotein E genotypes. Am J Med Genet 2002; 15:156–161. 31. Kamboh MI. Molecular genetics of late-onset Alzheimer’s disease. Ann Hum Genet 2004; 68:381–404. 32. Amos J, Grody W. Development and integration of molecular genetic tests into clinical practice: the US experience. Exp Rev Mol Diagn 2004; 4:465–477. 33. Taylor CF, Taylor GR. Current and emerging techniques for diagnostic mutation detection: an overview of methods for mutation detection. Methods Mol Med 2004; 92:9–44. 34. Mao R, Aylsworth AS, et al. Childhood-onset ataxia: testing for large CAG repeats in SCA2 and SCA7. Am J Med Genet 2002; 110:338–345. 35. Wong LJ, Chen TJ, et al. Novel SNP at the common primer site of exon IIIa of FGFR2 gene causes error in molecular diagnosis of craniosynostosis syndrome. Am J Med Genet 2001; 102:282–285. 36. Korf BR, Darras BT, Urion DK. Dystrophinopathies. In GeneTests, www. genetests.org, accessed February 6, 2005. 37. Schwartz M, Duno M. Improved molecular diagnosis of dystrophin gene mutations using the multiplex ligation-dependent probe ampliﬁcation method. Genet Test 2004; 8:361–367. 38. Schwartz MK. Genetic testing and the clinical laboratory improvement amendments of 1988: present and future. Clin Chem 1999; 45:739–745. 39. McInerney-Leo A, Hadley DW, et al. Genetic testing in Parkinson’s disease. Mov Disord 2005; 20:1–10. 40. Bird TD. Apolipoprotein E genotyping in the diagnosis of Alzheimer’s disease: a cautionary view. Ann Neurol 1995; 38:2–4. 41. Anonymous. Guidelines for the molecular genetics predictive test in Huntington’s disease. Neurology 1994; 44:1533–1536. 42. Nance MA, Myers R, et al. Genetic guidelines for Huntington’s Disease: Its Relevance and Implications (Revised). New York: Huntington’s Disease Society of America, 2003. 43. Visintainer CL, Matthias–Hagen V, et al. Anonymous predictive testing for Huntington’s disease in the United States. Genet Test, 2001; 5:213–218. 44. Prevost C, Veillette S, et al. Psychosocial impact of predictive testing for myotonic dystrophy type 1. Am J Med Genet 2004; 126:68–77. 45. Yoshida K, Tamai M, et al. Analysis of 14 individuals who requested predictive genetic testing for hereditary neuromuscular disorders. Rinsho Shinkeigaku 2002; 42:113–117. 46. Lesca G, Goizet C, Durr A. Predictive testing in the context of pregnancy: experience in Huntington’s disease and autosomal dominant cerebellar ataxia. J Med Genet 2002; 39:522–525. 47. Creighton S, Almqvist EW, et al. Predictive, prenatal, and diagnostic genetic testing for Huntington’s disease: the experience in Canada from 1987 to 2000. Clin Genet 2003; 63:462–475.

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48. Dean NL, Tan SL, Ao A. The development of preimplantation genetic diagnosis for myotonic dystrophy using multiplex ﬂuorescent polymerase chain reaction and its clinical application. Mol Hum Reprod 2001; 7:895–901. 49. de Die Smulders CE, Land JA, et al. Results from 10 years of preimplantationgenetic diagnostics in The Netherlands. Ned Tijdschr Geneeskd 2004; 148: 2491–2496. 50. Piyangmonkol W, Harper JC, et al. A successful strategy for preimplantation genetic diagnosis of myotonic dystrophy using multiplex ﬂuorescent PCR. Prenat Diagn 2001; 21:223–232. 51. Schulman JD, Black SH, et al. Preimplantation genetic testing for Huntington disease and certain other dominantly inherited disorders. Clin Genet 1996; 49:57–58.

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3 Genetic Counseling Robin L. Bennett University of Washington, Medical Genetics, Seattle, Washington, U.S.A.

INTRODUCTION No matter what a person’s age, the diagnosis of a neurodegenerative disorder can be devastating. When such a disorder is inherited, effects can ripple through the family and often through many generations. The child or adult, their spouse or partner, their friends and family will have a plethora of questions: What is this condition? How rare is it? Will it get worse? Is there a cure? Is there a decreased life expectancy? Will my children or other relatives be affected? Is there any prenatal testing? Is there a test to diagnose this condition before a person has symptoms? Will I or my family face discrimination in social situations, in seeking insurance coverage, or in employment? Genetic counseling can make a difference in how individuals and their families cope with the diagnosis, make decisions about genetic testing (particularly presymptomatic or predictive testing), and make reproductive choices and other important life decisions. Genetic counseling may be a one-time meeting or occur over several sessions, even over many years (1). ARE GENETIC DISORDERS UNIQUE? Health professionals are accustomed to giving ‘‘bad news;’’ so what, if anything, makes providing care and information to individuals with genetic disorders unique? All diseases probably have a genetic component, and it is essential that all health professionals develop genetic competency (2). Although an aura of ‘‘genetics exceptionalism’’ is counter-productive to incorporating

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genomic medicine into primary and specialty health care, there are several aspects of genetic disorders that carry important distinctions from disorders without a strong genetic component because of their personal, family, and social consequences (3–5). The most obvious characteristic of genetic disorders is that they are familial—a diagnosis in one person has implications for the rest of the family, offspring, parents, siblings, and extended relatives. When a person has a herniated disk or back pain, no other relatives are involved; this is a private conversation between that individual and the health care provider(s) regarding diagnostic evaluation and management. In contrast, if a person has a neurogenetic disorder, the whole family may become ‘‘the patient.’’ This brings challenges in communicating medical and risk information to relatives, which may have accompanying emotional consequences, as well as raising unique issues of conﬁdentiality and privacy of health and personal information. A complicating factor is that often genetic testing entails ﬁrst testing a person who is known to have the condition before testing unaffected relatives. For example, there are many different forms of autosomal dominant ataxia (chap. 15), and a mutation must ﬁrst be identiﬁed in an affected family member before presymptomatic testing can be offered to unaffected relatives. Families often reﬂect back on their ancestors with pride, as evidenced by the millions of people worldwide that are involved in genealogy research. Genealogist David Hey notes, ‘‘Knowing one’s ancestors is not a matter of mild curiosity; it is often part of an attempt to explain life and to understand how we have come to be what we are, not just physically through inherited genes, but how we have come to believe in certain principles or to have acquired the attitudes, prejudices, and characteristics that mould our personality. For many people, tracing a family tree and discovering the lives of their ancestors is not a task that is undertaken lightly’’ (6). The identiﬁcation of a genetic disorder in a family may be perceived as ‘‘a blemish’’ in the family lineage. Over 100 years ago, Francis Galton observed that ‘‘There are some disorders of which we barely wish to speak, as if timidity of utterance could hush thoughts’’ (7). Unfortunately, many genetic disorders continue to be stigmatizing, particularly if the condition (such as Huntington’s Disease) is associated with mental illness or dementia. Individuals and their relatives may worry about genetic discrimination. Fears of labeling, stigma, and genetic discrimination may hinder communication in the family in relation to a genetic diagnosis or make relatives wary about participating in research studies. These worries may be so profound that an individual may be dissuaded from disclosing important health information to health-care providers. Providing patients with information about current federal and state laws protecting against discrimination of individuals with disabilities and in some states, protection for individuals with genetic disorders, can be reassuring (8–10).

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The familial nature of genetic disorders can also be associated with feelings of guilt. This may take the forms of both parental and survivor guilt. A parent may feel guilty for having passed a condition on to their offspring, even though a parent has no control over which gene alterations are passed to one’s children. A couple often experiences conﬂicting emotions about knowingly ‘‘passing’’ a condition to future offspring instead of making the choice not to have biological children, or choosing prenatal diagnosis to identify an affected fetus. A person or couple may feel that the genetic disorder is punishment by God or a higher power for some past deed. Feelings of guilt can be exacerbated by casting blame. For example, a child may bluntly query a parent, ‘‘Will I be like you?’’ Survivor guilt may be experienced by a person who is unaffected by the genetic condition but feels guilty for having ‘‘escaped’’ the condition, whereas other relatives have been less fortunate. One client at risk for Huntington’s disease described her experience of survivor guilt as feeling like the client was standing outside of a burning house with the rest of her family inside. The ‘‘survivor’’ may feel on the outskirts of the ‘‘family team’’ despite knowing it is irrational to desire ill health (11). The diagnosis of a genetic condition can alter reproductive plans for not just the initial person diagnosed with the condition, but many other relatives. Often, the perceived parental role is threatened by ﬁnding out genetic carrier status (12). The religious and ethical belief systems of couples and their extended families may be challenged, particularly as couples wrestle with core values on how they regard life and health and their relationship to their own and their family’s views on biological parenting. Couples at risk to have an offspring with a neurogenetic disorder face difﬁcult decisions about adoption, assistive reproductive technologies (including use of donor egg or sperm, and preimplantation diagnosis), prenatal diagnosis and possible abortion of an affected fetus, or taking their spin at genetic roulette. Despite advances in management of genetic disorders, currently no cures exist for most genetic diseases. The permanent nature of genetic disease may bring a sense of fatalism or hopelessness accompanying the diagnosis or the results of genetic testing. As genetic disorders are chronic diseases, there may be a continual array of new health and physical challenges over a person’s lifetime. The individual and the family may experience ‘‘chronic sorrow’’ for the ‘‘healthy person who will never be.’’ Results of genetic testing may alter the person’s perception of health, self-concept and self-esteem, as well as their perceived genetic or social identity (12–14). Genetic counselors are speciﬁcally trained to deal with the spectrum of scientiﬁc and psychosocial issues that are involved with genetic diagnosis and testing for individuals and their families who wrestle with neurological disorders having a genetic etiology.

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WHO ARE GENETIC COUNSELORS? Genetic counseling is a distinct medical specialty with the role of providing clinical health care, education and emotional support to individuals and families challenged by congenital and inherited diseases. The focus is on how genetic conditions affect the psychological, medical, ﬁnancial and social aspects of the lives of individuals and their families diagnosed with, or at risk for, disorders with a genetic component. The term ‘‘genetic counselor’’ is generally reserved for masters-level health professionals with extensive training in human genetics and counseling skills. The ﬁeld began with the ﬁrst group of 10 genetic counselors who graduated from Sarah Lawrence College in 1971. Currently, there are 30 programs in the United States and Canada accredited by the American Board of Genetic Counseling (15), with similar programs in place in the United Kingdom, South Africa, and Australia (8). As of 2004, there are over 2400 members of the professional society of genetic counselors, the National Society of Genetic Counselors. Other health professionals who are trained speciﬁcally to provide genetic counseling services include physician geneticists (medical geneticists) and clinical nurse specialists in genetics. Medical geneticists attend a fellowship or residency program accredited by the American Board of Medical Genetics (16) or the Canadian College of Medical Genetics. Advance Practice Genetic Nurses and Genetics Clinical Nurses meet genetic competencies through a portfolio process established by the Genetic Nursing Credentialing Commission (17). Table 1 includes a list of resources for locating health professionals with expertise in genetic counseling. Given the complexity of neurogenetic disorders, a multi-disciplinary team approach is ideal. Many advocacy organizations for various genetic disorders are recognizing ‘‘Centers of Excellence,’’ where such teams have assembled to provide comprehensive care for individuals with genetic disorders. The Genetic Alliance is a network of genetic advocacy organizations with a listing of disease-speciﬁc support groups whose websites note the growing number of disease focused centers of excellence (18).

Table 1 Resources for Locating a Clinical Genetics Professional American Board of Genetic Counseling http://www.abgc.org American College of Medical Genetics http://www.acmg.net Canadian College of Medical Genetics http://ccmg.medical.org International Society of Nurses in Genetics http://www.isong.org National Society of Genetic Counselors http://www.nsgc.org GeneClinics http://www.geneclinics.org

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THE PROCESS OF GENETIC COUNSELING Genetic counseling is important across the lifespan—from preconception counseling and prenatal diagnosis, to the diagnosis of inherited disorders in newborns and children, and the diagnosis of adults with inherited predisposition to a variety of neurodegenerative disorders. The approach to genetic counseling involves assessing family and environmental history (e.g., occupational exposures and embryonic teratogens) to determine disease risk; assisting in genetic testing, diagnosis, and disease prevention and management; and offering psychosocial support and ethical guidance to help patients make informed, autonomous health decisions and reproductive choices. The process of genetic counseling is outlined in Table 2. Genetic counseling has a tradition of not directing patient decisionmaking (nondirective counseling) particularly with regard to reproductive choices, and demonstrating respect for patient autonomy. There has been a clear effort to distance genetic counseling from the eugenics movement of the 1930s and 40s. The current approach to genetic counseling favors a psychosocial approach that emphasizes shared deliberation and decisionmaking between the counselor and the client (1,19–21). GENETIC FAMILY HISTORY—THE PEDIGREE A primary component of genetic counseling is obtaining and interpreting a medical family history that is usually recorded in the form of a pedigree (3).

Table 2 The Process of Genetic Counseling Obtaining and assessing family history (3–4 generation pedigree) Assessing environmental exposures (e.g., occupational, embryonic teratogens) Psychosocial assessment Interpretation of medical and family history Conﬁrmation of diagnosis of disease in the family [may necessitate obtaining family medical records, death certiﬁcates, and/or genetic testing of a patient’s relative(s)] Assessment of chance of disease occurrence or recurrence Education about pattern(s) of inheritance Discussion of options of genetic testing (including costs, speciﬁcity, and sensitivity) Discussion of possible emotional consequences of genetic diagnosis and/or testing Review of management options and prevention Review and discussion of reproductive options Discussion of key ethical issues (e.g., testing minor children for adult onset conditions, testing individual at 25% risk for autosomal dominant condition) Education about resources (e.g., disease speciﬁc support groups) Referral to specialists as needed Referral to research as appropriate Counseling to promote informed choices and adaptation to the condition

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Figure 1 Pedigree symbols of relationships. Source: Adapted from Ref. 3.

Standard pedigree symbols (Figs. 1–3) are used to record relationships of relatives and to track diseases and demographic characteristics (22). The square or circle representing an affected relative can be shaded to document a disease, and more than one condition can be shown by partitioning the symbol into more than one sector and using different ﬁll patterns. The age of onset of the condition should be noted on the pedigree as well as the age and cause of death of relatives. Ideally, conﬁrmation of a clinical

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Figure 2 Common pedigree symbols. Source: Adapted from Ref. 3.

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Figure 3 Pedigree symbols of pregnancy and pregnancies not carried to term. Source: Adapted from Ref. 3.

diagnosis with medical records or death certiﬁcates provides the most accurate information from which to base pedigree analysis and genetic risk assessment (3). A pedigree key or legend is used to identify the medical conditions that are traced in the pedigree, and to explain unusual abbreviations or less commonly used symbols. Typically, a pedigree includes two generations of ascent from the consultand (the person requesting the medical or genetic information) or proband (the affected individual who brings the family to medical attention), and two generations of descent. Information is usually collected on ﬁrstdegree relatives (children, siblings, and parents), second-degree relatives (half-siblings, aunts, uncles, nieces, nephews, grandparents, and grandchildren), and sometimes third-degree relatives (e.g., ﬁrst cousins). For example, pedigree assessment of a 50-year-old male with a hereditary neuropathy may include information about the man’s children and grandchildren, parents and grandparents, siblings, nieces and nephews, aunts and uncles, and cousins; whereas the pedigree assessment of a 10-year-old girl with ataxia would likely only include information about her siblings, parents, aunts and uncles, cousins, and grandparents. Certain genetic disorders are more common in various ancestral groups; for example, Canavan disease or Tay-Sachs disease are found in individuals of Ashkenazi Jewish ancestry. The ancestral origins of both sets of grandparents should be noted on the pedigree (e.g., German, Vietnamese, Italian, Amish, Czekoviz, African American). The offspring of consanguineous

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unions (relationships between individuals with a closely related common ancestor, e.g., ﬁrst cousins) are more likely to share a deleterious recessive gene in common; therefore, noting if consanguinity is present in the family is important for genetic risk assessment (23). A pedigree provides complicated and comprehensive medical information about a family in a simple-to-obtain format. A pedigree can be the key to making a genetic diagnosis and can help identify other relatives at risk for the genetic disease who may beneﬁt from genetic counseling and genetic testing. However, a pedigree is more than a diagnostic tool (3,24). The process of obtaining a pedigree can help develop client rapport. It can be used as a teaching tool to demonstrate to a patient the evidence of reduced penetrance, variable expression of the disease phenotype (phenotype is the physical characteristics of a disease), and varied age of onset of the disease symptoms. A pedigree can also be used to clarify patient misconceptions or family myths. For example, a patient may believe that only men have the condition in the family or that only the ﬁrst person in every generation is affected, and the pedigree is often a graphic means of teaching the pattern of inheritance. PEDIGREE ANALYSIS AND RISK PERCEPTION One of the major purposes of drawing a pedigree is to recognize patterns of inheritance that may aid in clinical diagnosis (e.g., distinguishing recessive ataxias such as Friedreich ataxia, from dominant ataxias such as the many spinocerebellar ataxias). The common patterns of inheritance as well as the variables that can mask recognition of these patterns are reviewed in Table 3. Communicating the concept of risk to patients is a challenge in genetic counseling. Explaining the meaning of risk in multiple ways is the most effective approach; for example, distinguishing an absolute risk from a relative risk (e.g., a 5% absolute risk but a two-fold increased risk), and using percentages to frame the magnitude of risks from different perspectives. For example, a couple at risk to have a child with an autosomal recessive condition would be counseled that the risk is 25% chance or a one in four chance for an affected child, but a 75% chance, or a three in four chance of having a child who is unaffected. Comparing individual risk to population risks can help clients put their personal risk in perspective. When presenting risk ﬁgures, the clinician should be careful not to relay any personal bias as to whether a risk is high or low. Usually, it is best to give a result as a fact, rather than prefacing the result with ‘‘I have good news’’ or ‘‘I have bad news’’ (25). Understanding the patient’s perception of risk is more important than the actual number. Patients may have trouble accepting risks that are different from their preconceived notion of whether the ‘‘chances’’ were high or low. Each patient will have different notions on what is an acceptable chance or risk. The interpretation of risk will be inﬂuenced by patients’ prior experience with the disease, and with their social, religious, and ethnocultural views of

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Table 3 Examples of Clues for Recognizing Patterns of Inheritance and Variables That Can Mask Recognition of These Patterns Inheritance pattern

Mode of transmission

Pedigree clues

Confounding factors

Disease examples

50% risk of disease to each son/daughter

Condition in consecutive generations Males/females affected Observe male–maletransmission Often variability in disease severity Homozygotes may be affected more severely Homozygous state may be lethal

Reduced penetrance Can miss diagnosis in relatives if mild expression for disease New mutations may be mistaken for sporadic if small family size

Charcot-Marie-Tooth 1 Huntington’s disease Neuroﬁbromatosis 1 Myotonic muscular dystrophy Tuberous sclerosis complex Frontotemporal dementia (FTDP-17) CADASIL Spinocerebellar ataxias

AR

25% risk of disease to each son/daughter 50% risk of passing on carrier state to each son/daughter Parents are carriers (healthy—no symptoms)

Usually one generation Males/females affected Often seen in newborn, infancy, childhood Often inborn errors of metabolism May be more common in certain ethnic groups (e.g. Tay-Sachs disease and Ashekenazism) Sometimes parental consanguinity

May be mistaken as sporadic if small family size If carrier frequency high, can look autosomal dominant (e.g., hemochromatosis)

Friedreich ataxia Metachromatic leukodystrophy Niemann Pick disease Spinal muscular atrophy Wilson disease

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AD

Heterozygous women are affected and their daughters have a 50% chance of being affected/sons have a 50% chance of being affected (lethal)

XLR

Sons of female carriers have a 50% chance of being affected Daughters of female carriers have a 50% chance of being carriers

Chromosomal

Increased risk for trisomy seen with advanced maternal age

No-male-to-male transmission Often lethal in males so see paucity of males in pedigree May see multiple miscarriages (due to male fetal lethality) Females usually express condition but have milder symptoms than males No male-to-male transmission Males affected Females may be affected, but often milder and/ or with later onset than males Suspect if individuals has 2þ major birth defects or 3þ minor birth

Small family size

Rett syndrome

May be missed if paucity of females in pedigree. Symptoms in female carriers is thought to be caused by lyonization (imbalance of X-inactivation)

Duchenne/Becker muscular dystrophy Fabry disease Fragile X syndrome Spinal bulbar muscular atrophy Adrenoleukodystrophy Trisomy 21 (Down’s syndrome) Trisomy 18

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XLD

(Continued)

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Table 3 Examples of Clues for Recognizing Patterns of Inheritance and Variables That Can Mask Recognition of These Patterns (Continued ) Inheritance pattern

Pedigree clues

Risk for affected fetus depends on speciﬁc chromosomal rearrangement (ranges from 1% to 15% or higher)

defects Fetus with structural anomalies Unexplained MR (static) especially with dysmorphic features Unexplained psychomotor retardation Amgibuous genitalia Lymphedema or cystic hygroma in newborn Couples with 3 or more pregnancy losses Individuals with multiple congenital anomalies and family history of MR Unexplained infertility

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Confounding factors

Disease examples Turner syndrome (45,X) Robertsonian translocations Reciprocal translocations Subtelomeric defects

Bennett

Mode of transmission

0–100%

No male transmission to offspring, only maternal transmission Highly variable clinical expression Often central nervous disorders Males and females affected, often in multiple generations

Generally considered rare

Multifactorial

Based on empiric risk tables

Males and females affected No clear pattern Skips generations Few affected family members

May actually be single gene

Mitochondrial encephalopathy with ragged-red ﬁbers (MERRF) Mitochondrial encephalopathy, lactic acidosis, strokes (MELAS) Neropathy with ataxia and retinitis pigmentosa (NARP) Schizophrenia Bipolar disorder Epilepsy

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Mitochondrial

Abbreivations: AD, autosomal dominant; AR, autosomal recessive; MR, mental retardation; XLD, X-linked dominant; XL, X-linked recerssive Source: Adapted from Ref. 24.

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the disease. Providing information in several ways to accommodate various learning styles (e.g., audiovisual, verbal, and written) is helpful. Providing a summary letter after the clinic visit is a useful resource for the patient and family and can facilitate coordination of the patient’s care if it is sent to relevant health professionals (13,26).

SUMMARY Genetic counseling provides more than estimates of disease occurrence or recurrence. Genetic counseling is the process of helping people understand and adapt to the medical, psychological, and familial implications of genetic contributions to disease. It is multifaceted in that genetic counseling includes assessing personal, social, religious, and ethnocultural views on how a genetic diagnosis, genetic testing, and genetic test results potentially inﬂuence a person’s life. Pre- and post-test genetic counseling is important for individuals who undergo genetic testing. Interpretation of genetic testing must be placed in the context of the patient’s and the family’s medical history. Genetic counseling involves obtaining and interpreting a medical family history, and education about inheritance, testing, management, disease prevention, reproductive options, ethical issues, resources, and research. It is particularly important that individuals who are considering presymptomatic or prenatal genetic testing receive genetic counseling before genetic testing, and not only after a positive test result. The ultimate goals of genetic counseling are to facilitate patient decision-making to promote informed choices and adaptation to the condition. Patient decisions are supported in the context of individual values, beliefs, and goals. Genetic counselors and other clinical genetic specialists are speciﬁcally trained to serve as a resource to health professionals and their patients with regard to genetic diagnosis and management, and to provide psychological support for individuals and their families who are faced with the many issues surrounding a neurogenetic diagnosis. REFERENCES 1. Bennett RL, Hampel HL, Mandell JB, Marks JH. Genetic counselors: translating genomic science into clinical practice. J Clin Invest 2003; 112:1274–1279. 2. National Coalition of Health Care Professionals in Genetics, Genetic Core Competencies (http://www.nchpeg.org accessed January 2005). 3. Bennett RL. The Practical Guide to the Genetic Family History. New York: John Wiley and Sons, Inc., 1999. 4. Plumridge D, Bennett R, Dinno N, Branson C. The Student with a Genetic Disorder: Educational Implications for Special Education Teachers and For Physical Therapists, Occupational Therapists and Speech Pathologists. Springﬁeld: Charles C Thomas, 1973. 5. Schild S, Black RB. Social Work and Genetics. A Guide for Practice. New York: The Hawthorth Press, 1984.

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6. Hey D. The Oxford Guide to Family History. Oxford and New York: Oxford University Press, 1993. 7. Galton F. Natural Inheritance. London: MacMillan, 1889. 8. National Society of Genetic Counselors (http://www.nsgc.org, accessed January 2005). 9. National Human Genome Research Institute (http://www.nhgri.nih.gov/ 11510227 accessed January 2005). 10. National Conference of State Legislatures (http://www.ncsl.org/programs/ health/genetics/charts.etm.asp accessed January 2005). 11. Williams JK, Schutte DL, Evers C, Holkup PA. Redeﬁnition: coping with normal results from predictive gene testing for neurodegenerative disorders. Res Nurs Health 2000; 23:260–269. 12. McConkie-Rosell A, Devellis BM. Threat to parental role: a possible mechanism of altered self-consept related to carrier knowledge. J Genet Couns 2000; 9:285–302. 13. Baker DL, Schuette JL, Uhlmann WR, eds. A Guide to Genetic Counseling. New York: Wiley-Liss, 1998. 14. Weil J. Psychosocial Genetic Counseling. Oxford: Oxford University Press, 2000. 15. American Board of Genetic Counseling (http://www.abgc.net accessed January 2005). 16. American Board of Medical Genetics (http://www.abmg.org accessed January 2005). 17. Genetic Nursing Credentialing Commission, Inc (http://geneticnurse.org accessed January 2005). 18. Genetic Alliance (http://www.geneticalliance.org accessed January 2005). 19. Wiel J. Psychosocial genetic counseling in the post-nondirective era: a point of view. J Genet Couns 2003; 12:199–211. 20. Biesecker BB. Back to the future of genetic counseling: commentary on psychosocial genetic counseling in the post-nondirective era. J Genet Couns 2003; 12:213–217. 21. Biesecker B, Peter K. Genetic counseling: ready for a new deﬁnition? J Genet Couns 2003; 11:536–537. 22. Bennett RL, Steinhaus KA, Uhrich SB, O’Sullivan CK, Resta RG, Doyle DL, Markel DS, Vincent V, Haminishi J. Recommendations for standardized human pedigree nomenclature. Am J Hum Genet 1994; 56:745–752. 23. Bennett RL, Motulsky AG, Bittles A, Hudgins L, Uhrich S, Lochner Doyle D, Silvey K, Scott CR, Cheng E, McGillivray B, Steiner RD, Olson D. Genetic counseling and screening of consanguineous couples and their offspring: Recommendations of the National Society of Genetic Counselors. J Genet Couns 2002; 11:97–119. 24. Bennett RL. The family medical history. Prim Care Clin Ofﬁce Pract 2004; 31:479–495. 25. Bennett RL, Bird TD, Teri L. Offering predictive testing for Huntington’s disease in a medical genetics clinic: practical applications. J Genet Couns 1993; 2:123–137. 26. Hallowell N, Murton F. The value of written summaries of genetic consultations. Patient Educ Couns 1998; 35:27–34.

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4 Gene Therapy for Inherited Diseases of the Central Nervous System Tyler Mark Pierson Division of Neurology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

John H. Wolfe Division of Neurology, Children’s Hospital of Philadelphia and W. F. Goodman Center for Comparative Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

INTRODUCTION Gene therapy is a potential treatment regimen where exogenous genes or genetically engineered cells are introduced into individuals to relieve a disease phenotype. A number of methods for the delivery of DNA or RNA are under active investigation. The goal of therapy is to produce therapeutic proteins or alter gene expression in the host to stabilize or correct a potential disease state by providing appropriate intracellular homeostasis or by reducing the concentration of extracellular toxins. Difﬁculties with the cellspeciﬁc targeting of vectors, cell-speciﬁc transgene expression and regulation, the toxicity of delivery vehicles, and host immune responses have all hampered the achievement of these goals. To date, there has been limited success with human gene therapy for genetic disease and no successful therapy involving the central nervous system (CNS) (see http://www4.od.nih. gov/oba/rdna/htm for past and current gene therapy trials).

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The CNS is an especially difﬁcult target because of the need to circumvent the blood–brain barrier (BBB) to access the brain parenchyma, as well as the complex characteristics of its anatomy and cellular makeup. Most clinical trials for gene therapy in the CNS have been directed to neoplastic disease and adult neurodegenerative disorders, including Parkinson’s disease, amyotrophic lateral sclerosis; and Huntington’s disease (1–11). The only attempts to treat childhood-onset inherited disease have been in the Canavan and Batten diseases (12,13). This review will evaluate the potential application of gene therapy for the correction of genetic disease in the CNS. The major problem with genetic diseases affecting the brain is that the lesions are typically globally distributed, necessitating widespread delivery of either the gene or the protein. The discussion will focus on strategies for correcting different types of neurogenetic diseases based on their molecular and genetic pathology. Examples are provided for several types of disorders, including the effects of speciﬁc features associated with the diseases that affect gene therapy strategies and the various delivery vehicles. GENERAL PROPERTIES OF CNS GENE THERAPY Gene therapy involving the CNS must overcome a number of potential problems. Finding ways to circumvent the restricted entry of any compound into the CNS by the BBB is of primary importance. Once past the BBB, the delivery vehicle must reach the target cells via the complex microscopic and macroscopic structures of the brain. Another problem associated with CNS-based gene therapy strategies is the restriction on the volume of gene transfer vector preparation that can be injected. The BBB is especially problematic for the introduction of therapeutic genes or cells into the CNS. While many gene therapy protocols outside the CNS use intravenous delivery, the BBB effectively removes this option by restricting the movement of most substrates from the vasculature into the brain parenchyma (14). One approach to circumvent this restriction utilizes intravenously administered liposomal compounds to access the brain by active and passive transport across the intact BBB (15–17). Early data have shown some promise with this technique; however, cell-speciﬁc targeting of gene therapy may be difﬁcult to achieve with these methods. Liposomes have been used to deliver drugs in humans with some efﬁcacy (18). However, safety concerns regarding their use for gene delivery have not been determined (discussed in the section ‘‘Liposomal Delivery Systems’’). Another strategy is to temporarily open the BBB by using osmotic agents. These methods have not generally been very successful, and there is a signiﬁcant concern about the adverse side effects of the procedure. Viral vectors may enter the brain if they are administered intravenously to an individual prior to the complete maturation of the BBB (19).

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However, this method requires the diagnosis to be made in the prenatal or neonatal period, which can be difﬁcult even in individuals with a high suspicion of having a neurogenetic disease (20,21). Because of these obstacles, most protocols use a direct transcranial approach to deliver therapeutic agents to the parenchyma or ventricles. This may signiﬁcantly increase health risks in comparison to an intravenous injection, as neurosurgery can lead to serious complications, such as intracranial hemorrhage or fatal meningoencephalitis. Once the transgenic vehicle is injected into the parenchyma, diffusion may be inadequate for effective dispersion of the vehicle throughout the brain. Dispersion often is not uniform as viral vectors injected into speciﬁc parts of the brain may travel more easily along different neuroanatomical pathways than into some structures that are just millimeters away (22). Hyperosmolar solutions have been used to increase diffusion of delivery vehicles as they shift ﬂuid out of parenchymal cells, increasing the relative volume of the extracellular space (23). Another means of increasing the area exposed to the therapeutic vehicle includes the use of multiple injection sites; however, the additional areas of local injury may increase the risk of local damage or infection, making surgery even more risky and less attractive. Other difﬁculties include targeting speciﬁc cell types within the brain, the risk of direct injury to cells by the delivery vehicle, and the potential of organ-wide damage or inﬂammation leading to increased intracranial pressure and herniation. These problems have been addressed in several ways. Some vehicles (viruses in particular) have speciﬁc tropisms toward different cell types and so may allow for cell-speciﬁc targeting (24). In cases where delivery is unable to target speciﬁc cell types, cell-speciﬁc expression can be regulated by cell-speciﬁc promoters. The nestin promoter can be used to target neural stem cells (25,26), the glial ﬁbrillary acidic protein (GFAP) promoter can be used for astrocytes (27,28), and the neuron-speciﬁc enolase promoter can be used for neurons (29,30). Importantly, the vectors must not cause signiﬁcant toxicity as this may result in forming an epileptogenic focus or provoke a large inﬂammatory response that could increase intracranial pressure (31,32). Tumorigenesis of transplanted cells could also hinder progress. These examples emphasize that therapeutic complications in the brain may result in extremely deleterious effects. CANDIDATE DISEASES FOR GENE THERAPY IN THE CNS There are numerous genetic disorders of the CNS that are potential targets for correction by gene therapy. These disorders can be classiﬁed into two general categories: (i) diseases where only a subset of cells will need to express the therapeutic protein or RNA, with subsequent correction of nearby cells; and (ii) diseases in which most of the affected cells will need to express the therapeutic protein or RNA for amelioration of disease. The

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ﬁrst category includes disorders in which the therapeutic protein can be provided to other cells by secretion and endocytosis (33) or the toxic compound can diffuse into the cells expressing the therapeutic protein (34). In the ﬁrst type of correction, the therapeutic effect is supplied to affected cells by other cells and will be described as being ‘‘in trans’’ or via ‘‘trans-correction.’’ Cells capable of providing trans-correction could be targeted by a delivery vehicle in vivo or in vitro with subsequent transplantation into the CNS (also known as ex vivo therapy). In contrast, the second category consists of disorders with a need to express the therapeutic protein or RNA in a cellular compartment that is inaccessible from outside the cell. In these cases, the affected cells are required to possess the transgene and express the therapeutic protein or RNA themselves. In this type of correction, the therapeutic intervention will be described as being supplied ‘‘in cis’’ or via ‘‘cis-correction.’’ The diseases that can be corrected in trans have more ﬂexibility in their application than those requiring cis-correction because of less-speciﬁc targeting requirements and a lower number of transduction events. The following discussion of neurogenetic disease further categorizes disorders amenable to gene therapy by their genetic mechanisms and then hypothesizes whether they require cis- or trans-correction. Autosomal Recessive Mutations of Cytoplasmic or Nuclear Proteins Autosomal recessive (AR) mutations of cytoplasmic or nuclear proteins result in disease through the absence or diminished activity of a speciﬁc protein. Expression of the deﬁcient wild-type protein corrects the cellular pathology; however, the cytoplasmic or nuclear location of these proteins requires transduction of these cells with the therapeutic gene, as there are no other ways to trafﬁc proteins or RNA interference (RNAi) to these compartments. Because of the need to correct every affected cell, diseases like Pelizaeus-Merzbacher disease (35), vanishing white matter disease (36), or MNGIE (37) would be difﬁcult to correct as affected cells are located throughout the brain. In contrast, disorders with a more limited cohort of affected cells may be more amenable to correction. Pantothenate kinaseassociated neurodegeneration (one of numerous subtypes of a disorder formerly known as Hallavorden–Spatz disease) is an example of this type of disorder. An AR disease characterized by dystonia, pigmentary retinopathy, and psychiatric symptoms is caused by mutations in the gene encoding pantothenate kinase 2. Pantothenate kinase 2 is essential for coenzyme A biosynthesis and deﬁciency of this enzyme causes tissue-speciﬁc accumulation of cysteine-containing enzyme substrates that lead to iron accumulation in the basal ganglia and retina resulting in cell dysfunction (38). Correction

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of this disease would require transduction of the deﬁcient gene into the affected cells in the basal ganglia or retina in order to return coenzyme A concentrations to sufﬁcient levels. Because the affected cells are located in well-deﬁned areas of the brain, the delivery of genes to the affected areas may be less difﬁcult and yet still have a signiﬁcant impact on the disease pathology. In contrast, targeting AR disorders of cytoplasmic proteins with more widespread pathology may have a lesser impact on the disease state as widespread transduction may be more difﬁcult and correction less efﬁcient on the whole. Dominant Negative Mutations Dominant negative mutations cause pathology in the presence of wild-type proteins by disrupting their normal function. Correction of these disorders could involve the use of RNA interference (RNAi) technology by speciﬁcally blocking the translation of the dominant negative protein (39,40). This technology relies on the therapeutic transgene expressing small interfering RNAs (siRNAs) consisting of sequences speciﬁc to the mutations of the dominant negative alleles. The siRNAs then cause degradation of the mutant mRNA, while having little or no effect on the translation of the wild-type mRNA (41). This mechanism for degrading the mutant RNA must be present in the cytoplasm of every affected cell and so would require the siRNA-encoding transgene to be provided in cis for correction. Dominant negative disorders with focal pathology would have a greater chance of correction than those with more diffuse lesions. An example of a dominant negative disorder with focal pathology is idiopathic earlyonset torsional dystonia. The disorder is characterized by paroxysmal sustained muscle contractions in the legs or arms that can be unsettling and painful to the patient. The disease is due to mutations in the DYT1 gene, which encodes torsinA, an AAAþ ATPase that resides within the endoplasmic reticulum and nuclear membrane. The mutant gene product accumulates in the nuclear envelope leading to neuronal dysfunction (42,43). The pathology of this disorder is felt to be localized to the basal ganglia, and thus cis-correction of the affected cells may be able to ameliorate the phenotype. Other possible targets of this type of technology are the trinucleotide repeat (CAG) disorders that encode a polyglutamine tract. These are autosomal dominant diseases [spinocerebellar atrophies, Huntington’s disease, and Kennedy disease (44)] with pathology secondary to the expression of a toxic protein that accumulates in the nucleus of affected neurons. Targeting these proteins would be challenging for this technology secondary to the common presence of CAG repeats in many normal transcripts, but other methods or abnormalities could allow for successful interference (40,41).

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Autosomal Recessive Mutations Causing Accumulation of Diffusible Toxins There are a number of AR neurogenetic disorders affecting the function of cytoplasmic proteins that catabolize diffusible cellular toxins, including amino acidopathies, organic acidopathies, and peroxisomal diseases. Current treatment options include limiting the ingestion of foods that are rich in the toxin’s precursors (45), or removing circulating toxins from the bloodstream by liver or other organ transplantation (46). Unfortunately, in many cases these options are inadequate or too toxic to protect the brain from further injury. However, if the deﬁcient enzymes were present in the microenvironment of the brain, accumulation of the toxin might be limited, reducing the risk of further neurologic injury. Studies utilizing bone marrow transplantation (BMT) for many of these disorders indicate that removal of the circulating toxins by a cohort of cells expressing the deﬁcient protein may be a better method of correction (47). Unfortunately, BMT is associated with signiﬁcant morbidity and mortality and may require at least a year to show any beneﬁt while the disease continues to progress (47,48). Because of these disadvantages, the use of trans-corrective gene therapy may be a more attractive alternative. A cohort of cells would be engineered in vivo or ex vivo to express the deﬁcient protein and metabolize the accumulating toxins, allowing for immediate expression in the CNS and avoiding any need for immuno-ablation. Adrenoleukodystrophy is an example of a peroxisomal disorder where the accumulation of toxic fatty acids causes disease, and removal of toxins has relieved the disease phenotype (47,48). Due to mutations of lignoceroyl-CoA synthetase, the disease is the result of the defective transport of very long chain fatty acids (VLCFAs) into the peroxisome. It presents insidiously with diminished attentiveness and cognition and these symptoms are associated with signiﬁcant white matter changes. As the disease progresses, the patient becomes severely incapacitated and death occurs within 10–20 years of diagnosis (or even more rapidly in some patients) (49,50). Refsum disease is another peroxisomal disease caused by the accumulation of phytanic acid leading to ataxia and polyneuropathies (51). Relief of the disease phenotype is also associated with decreased circulating toxin. Maple syrup urine disease is an amino-acidopathy due to the accumulation of branched chain amino acids in the brain. Dietary restrictions are the primary means of therapy; however, removal of circulating toxins has also been shown to ameliorate disease and decrease progression of neurologic symptoms (46). Type I glutaric aciduria is an example of an organic acidemia, due to a deﬁciency in glutaryl-CoA dehydrogenase that leads to the accumulation of glutaric acid. Experience indicates that removal of these compounds by trans-correction may protect against any further CNS damage with metabolic exacerbations (52,53). These examples represent a few of the different types of amino acidopathies, organic

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acidemias, or peroxisomal disorders that may be amenable to a transcorrective gene therapy approach. All are attractive targets because in each case the corrected cells may be able to break down the toxins for the rest of the brain. Questions that need to be answered with this strategy include: (i) How many cells expressing the deﬁcient proteins are required to relieve CNS pathology? (ii) Do these cells need to be scattered throughout the CNS or will a concentrated group be enough to remove the toxins? (iii) Will the removal of the circulating toxins be enough to correct pathology or would signiﬁcant metabolic deﬁciencies still be present causing other types of pathology? Lysosomal Storage Disease Lysosomal diseases are the result of AR mutations in genes encoding lysosomal acid hydrolases (Hunter disease is an X-linked exception) and other proteins that affect lysosome function. The acid hydrolases are posttranslationally modiﬁed with the addition of a mannose-6-phosphate residue, which binds speciﬁc mannose-6-phosphate receptors, enabling the enzyme to be sorted to the lysosome during its passage through the vesicular system. Modest quantities of these enzymes are secreted into the extracellular space as well, where they can bind to mannose-6-phosphate receptors located on the extracellular surface of the plasma membrane of neighboring cells, allowing the enzymes to be endocytosed and translocated to their lysosomes. Intravenous enzyme replacement therapy (ERT) has used this feature to correct Fabry and the non-neuronopathic subtype of Gaucher disease in humans (although the enzyme used in correcting Gaucher disease is mannose-terminated) (54–58). However, the intravenous delivery of the deﬁcient enzyme does not allow transport into the brain due to the restriction of the BBB (19). Direct infusion of protein into the brain via indwelling intracranial catheters carries a high risk of infection and makes this strategy an unattractive option. BMT has been used as well, with some success (47,48), but the CNS disease is not improved in many engrafted patients. These data have led to numerous studies of CNS trans-correction in animal models of lysosomal disease, many of which have shown promising results (59). Lysosomal disease is subcategorized into mucopolysaccharidoses, lipidoses, mucolipidoses, and neuronal ceroid lipofucsinoses by the type of substrate that accumulates in lysosomes. Though each disease is rare, cumulatively these disorders are present in approximately 1 of 8000 live births (60–62). Examples of the mucopolysaccharidoses are Hurler disease, Hunter disease, San Filippo disease, and Sly disease. These disorders accumulate glycosaminoglycans (previously known as mucopolysaccharides) in their lysosomes with associated alterations in cell size and physiology. The resulting phenotypes include coarse facies, bony malformations, mental retardation, and hydrocephalus (63). Examples of the sphingolipidoses are

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metachromatic leukodystrophy, Krabbe disease, and Tay-Sachs disease. Patients with these disorders accumulate toxic lipids in their cellular membranes resulting in myelin dysfunction. Individuals are not dysmorphic, but experience relentless progression of spasticity and cognitive decline (49,64). Mucolipidoses are due to errors in the post-translational modiﬁcation of lysosomal enzymes. Lysosomal enzymes in these disorders lack a mannose-6-phosphate residue and are trafﬁcked through the secretory pathway and not to the lysosome. Examples of these disorders include I-cell and pseudo-Hurler diseases that have characteristics of both the mucopolysaccharidoses and sphingolipidoses (65). Neuronal ceroid lipofuscinoses are a diverse group of neurodegenerative diseases associated with intractable epilepsy, dementia, and brain atrophy (66,67). Disease Severity, Diagnosis, and Age of Treatment Initiation Genetic diseases affecting the CNS generally follow a progressive degenerative course, making it important to diagnose and treat patients prior to the onset of debilitating symptoms (47,48). Individuals with infantile-onset disorders often carry mutant alleles that encode little or no enzyme activity, while later-onset disorders usually have partial enzyme activity. Infantileonset disorders are more difﬁcult to treat because the lack of enzyme activity leads to rapid progression of the disease with signiﬁcant disabilities being present by the time a deﬁnitive diagnosis is made. These patients may be immunologically na€ve to the transgenic protein, thus immune responses may complicate therapy. Alternatively, juvenile- and adult-onset disease may be more capable of responding to therapy because of the expression of mutant enzymes with partial activity. These mutant enzymes allow for a slower accumulation of the toxic substrate and a less aggressive progression of symptomatology. They may also expose the immune system to a variant of the wild-type protein making it less likely to elicit an immune response to the therapeutic protein.

ANIMAL MODELS OF GENETIC CNS DISORDERS Animal models of neurogenetic disease that accurately reﬂect the human phenotype are very important in evaluating the efﬁcacy and safety of any gene therapy prior to its use in humans. Many animal models have been discovered as naturally occurring mutations in domestic stocks. Germ-line gene mutation technology (knockout mice) has made many more mouse models available. Animal models with naturally occurring mutations can be very accurate in their phenotypic representation of human disease, mostly because their phenotype is what brought them to attention. However, with molecular genetic analysis, some of these models had mutations that were inconsistent

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with their representative human disease. For example, one of the most common human mutations of Krabbe disease (I546T) is inconsistent with the galactocerebrosidase mutation (W339X) found in the twitcher mouse, a model of Krabbe disease (68–70). Dog and monkey models of the disease were also inconsistent with this common human mutation (69,70). In contrast, knockout animals have been very successful at reproducing human gene mutations, but may have different phenotypic characteristics than the human or other animal models (70). Both the naturally occurring mutants and knockout animals provide investigators with a large amount of information and means by which to study gene therapy. The majority of these illnesses use mouse models; However, large animal models, however are much closer in brain size to humans and will be important translational models. Mice are the most utilized species since they can be generated rapidly and have well-characterized genetic backgrounds. Examples of naturally occurring mouse models of human neurogenetic disease include the jimpy and rumpshaker mice, which have different mutations in the myelin proteolipid protein gene. Human genotypic and phenotypic variants are PelizeausMerzbacher disease and X-linked Paraplegia type 2, respectively (71,72). Other examples of naturally occurring mouse models of human disease include the sulfamidase-deﬁcient mouse (San Filippo disease) (73) and beta-glucuronidase-deﬁcient mouse (Sly disease) (74). The model of Sly disease [mucopolysaccharidosis type (MPS) VII] has become widely used in the ﬁeld of gene therapy in the CNS because of the number of experimental assays that are available to both localize and quantify the transferred gene and enzyme (discussed further) (59). Rats have many of the same advantages as mice in regard to breeding and genetic background, but may be more useful for certain behavioral studies. Rat models involving deﬁciencies in proteolipid protein (75), N-acetylgalactosamine-4-sulfatase (MPS VI) (76) and acid lipase (Wolman disease) (77) have been characterized; however, rat models are not as numerous as the naturally occurring mouse models. The disruption of genes by inserting intervening sequence into their coding regions (the knockout mouse) is a valuable tool for the generation of animal models of human neurogenetic disease. Examples of this technology include mice deﬁcient in arylsulfatase A (metachromatic leukodystrophy) (78), the hexosaminidase a-subunit (Tay-Sachs disease) (79), the hexosaminidase b-subunit (Sandhoff disease) (80,81), superoxide dismutase (familial amyotrophic lateral sclerosis) (82), a-mannosidase (83), and a-L-iduronidase (Hurler disease) (84). The technology has also enabled researchers to produce exact genotypic models by replacing wild-type genes with genes carrying speciﬁc human mutations. These mice are termed ‘‘knock-in’’ mice and examples include models for superoxide dismutase and the Gaucher disease (85,86). Many of the knockout or knock-in mice have been shown to accurately mimic the phenotype of their homologous human disease. For example,

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the arylsulfatase A deﬁcient mouse has increased urine sulfatides, gait abnormalities, and white matter signal changes that are similar to lateinfantile onset metachromatic leukodystrophy (78). Other examples include the Sandhoff and Hurler mice having very similar clinical, pathological, and biochemical characteristics when compared to the human disease (80,81,84). Knockout mice do not generate protein products from the disrupted locus and this may be signiﬁcant in how the immune systems of these animals react to the presence of a previously unseen therapeutic protein. In comparison, knock-in models may produce near full length proteins, which are non-functional but may decrease the immunogenicity of the therapeutic protein (59). In addition, the presence of mutant proteins identical to those found in human disease may be valuable in modeling the subtle aspects of the disease or in revealing interactions between the mutant and normal proteins when they are co-expressed. Knockout or knock-in mice do not always accurately depict the human phenotype. For example, disruption of the gene encoding the b-hexosaminidase a subunit, which is defective in Tay-Sachs disease, had no affect on the neurologic development or life span in mice. This occurred because mice accumulate toxic gangliosides at much lower levels than their human counterparts, as a result of degradation via an alternative biochemical pathway not present in humans (81,87). Similar results occur in hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deﬁcient mice, the gene involved in Lesch–Nyhan disease, due to higher ﬂux through a salvage pathway in mice (88). Knockout mice may also be inaccurate models for disease correction when the loss of the protein is not the pathophysiologic cause of the disease, as experienced with the Cu/Zn superoxide dismutase in SOD1 null mice (82). These animals were created to model familial amyotrophic lateral sclerosis (ALS), but did not develop motor neuron disease. Subsequent generation of knock-in mice with the human G37R or G85R or G93A mutations exhibited the appropriate phenotype. The greater accuracy of the knock-in was due to a ‘‘gain of toxicity’’ mechanism speciﬁc to the mutant allele (85,89–91); thus, reproducing the speciﬁc human mutation was crucial in generating an accurate model. Even this method may be inadequate. Speciﬁc point mutations of the b-glucocerebrosidase gene associated with the less severe forms of Gaucher disease were generated in knock-in mice, but were found to express a phenotype consistent with the more devastating forms of the disease (86). However, while many knockout or knock-in mice can be very useful in modeling some human diseases for gene therapy, each genetic change must be evaluated for its similarities to the human disease. Transgenic and knock-in technology has also been used to generate animal models of dominant negative disease. These models are valuable in evaluating strategies to disrupt the production of the dominant negative

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protein, such as interfering RNA (RNAi). Numerous animal models of trinucleotide repeat disorders have been generated expressing transgenes containing pathologic amounts of the CAG repeats. Functional models of human disease include the Machado-Joseph disease (spinocerebellar ataxia type 3) (MJD) and Huntington’s disease mice. MJD mice express transgenes possessing 79 CAG repeats in the open reading frame (ORF) of the MJD1 protein. Expression of this mutation in Purkinje cells resulted in the development of severe ataxia, wide-based hind-limb postures, and cerebellar atrophy (92). Huntington’s disease mice were engineered to contain the 50 upstream region and exon 1 of the huntingtin gene containing CAG expansions of 115–150 repeats. These mice have progressive neurodegeneration and limb dyskinesias consistent with the human disease (93). However, other trinucleotide repeat studies have been less successful. A model of spinobulbar muscular atrophy expressing the testosterone receptor gene containing 45 CAG repeats exhibited no pathology and was inadequate for study because of locus stability, as well as the low expression of the transgenic mRNA (94). Expression of the mutant genes from their natural promoter or locus may be a remedy to this problem, as the natural loci may favor more accurate tissue-speciﬁc levels of gene expression, which would provide a more physiologic target for testing RNAi-based gene therapies. A major drawback of using rodents to model human neurogenetic disease is the relatively small and simple structure of their brains. This simplicity may not allow gene therapy protocols to function more effectively in brains of animals with signiﬁcantly larger volume and physical complexity (59). Cats and dogs possess brains that are more similar in physical characteristics to human brains than rodents. They have longer life spans and their relatively large size allows them to be manipulated more easily with surgical and non-invasive imaging modalities. Animal models of neurogenetic disease in larger mammalian species are exclusively naturally occurring, as knockout technology is not yet available for these species. Naturally occurring models exist for a number of LSDs, for example, both cat and dog models have been discovered for MPS I [Hurler disease (95,96)], GM1 gangliosidosis (97,98), and MPS VII [Sly disease (99,100)]. In addition, cat models exist for a-mannosidosis (101), MPS VI (102), and Tay-Sachs disease (103), while dog models exist for MPS IIIa (104), Krabbe disease (105), and one of the subtypes of neuronal ceroid lipofuscinosis (Batten disease) (106,107). Other types of genetic disease affecting the CNS include dog models of narcolepsy (108) and juvenile-onset epilepsy (Lafora disease), which was recently shown to be due to a triplet repeat (109). These models are important for studies to scale up gene therapy for human neurogenetic disease. However, the slow generation times, increased cost of medical management, and other difﬁculties usually restrict their use to translational studies after proof-of-principle experiments in mice.

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LYSOSOMAL STORAGE DISEASE: ANIMAL MODELS AS A PARADIGM FOR CNS GENE THERAPY Animal models of lysosomal storage disease have been the most abundant and widely used genetic disease models for the CNS. Lysosomal diseases have been identiﬁed in numerous species, including the mouse, rat, cat, dog, guinea pig, quail, cattle, goat, pig, sheep, and even the emu (59). These naturally occurring mutations are rare, as in humans, and were discovered in domestic stocks because the phenotypic abnormalities are stereotypical and dramatic. Some of these mutants were identiﬁed many years ago, and are some of the most well-understood illnesses in animals. They have played a major role in evaluating the natural diversity of these diseases. Many of these mutations were among the ﬁrst disease genes to be characterized in neurogenetic disease. Lysosomal diseases possess many features that make them attractive candidates for gene therapy (discussed in the section ‘‘Lysosomal Storage Disease’’). Most of them are caused by deﬁciencies in enzymes with similar traits, including the requirement for the acidic environment of the lysosome to function, intravesicular trafﬁcking via mannose-6-phosphate residues, and secretion and uptake by neighboring cells. In principle, therefore, vectors and strategies developed to treat one lysosomal disease should be applicable to others by changing only the transferred gene, although there are important differences in the physical characteristics among the enzymes, such as stability. This will be important in human disease because of the rarity of each disorder and the limited therapeutic options for the CNS pathology in these disorders. A type of trans-correction strategy for the CNS is to use secretion of the therapeutic enzyme from endogenous cells transduced in vivo or exogenous cells transduced in vitro and subsequently transplanted into the CNS. Trans-correction has been utilized with some success outside the CNS utilizing enzyme replacement therapy (ERT) in individuals with nonneuronopathic Gaucher (57,58) or Fabry disease (54,56). This method uses the intravenous delivery of the deﬁcient enzymes to trans-correct affected cells. Data from these studies indicated the therapeutic enzyme activity does not need to be restored to wild-type levels, as 10% to 20% of this amount can be adequate for correction. Although ERT usually does not work for CNS disease due to the BBB, these results suggest that enzyme expression within the brain by gene transfer has a reasonable chance of success in humans. Animal studies support this. One disorder that has been used extensively as a model system for gene therapy is MPS VII (Sly disease), which is caused by a deﬁciency of b-glucuronidase (GUSB). MPS VII is a multisystemic disorder affecting the liver, spleen, bones and joints, heart, eye, and CNS. It is a rare case of neurologic diseases in humans (110) and animals, but breeding colonies

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have been established from carriers in the mouse, cat, and dog for study. The animal models have very similar clinical presentations to affected children. Early studies on the biochemical genetics of GUSB in the mouse led to the relatively early isolation and use of the GUSB cDNA in some transfer studies (99,111,112). The GUSB enzyme has a number of features that enhance its usefulness for gene therapy experiments. Most importantly, the enzymatic activity can be localized within the brain by using a simple histochemical staining reaction for direct visualization. In combination with in situ hybridization, the enzyme-positive cells expressing the GUSB mRNA can be distinguished from cells that have taken up the enzyme (113–116). Correction of pathology can be monitored histologically by evaluating lysosomal distension and cellular size. Species differences in heat stability of the enzyme have been used to measure the human GUSB expressed from a vector, as opposed to the endogenous mouse or cat forms (113,114,117). This permits many studies to be done in normal animals prior to evaluating their diseased counterparts, which are more difﬁcult to produce and maintain. Cells can express GUSB at super-physiologic levels without obvious toxic side effects, thus engineering cells to overexpress enzyme may result in better delivery to the surrounding tissues (118,119). Finally, the animal models of MPS VII have signiﬁcant pathology with involvement of numerous organ systems on the macro- and microscopic level. This pathology accurately models the human disease in the CNS and non-CNS tissues. The technical advantages of this model system have allowed a number of principles of gene therapy to be demonstrated ﬁrst in MPS VII, which are applicable in principle to this whole class of disorders: (i) It is only necessary to transfer the gene to a small number of cells because the normal enzyme can be secreted in three dimensions to correct the surrounding diseased cells, with less than 5% of normal activity needed to fully reverse the metabolic defect in some cases (120). (ii) Lysosomal storage can be reversed after the disease is advanced, including in large animals (120,121). (iii) The brain with advanced lesions can respond, but needs to have the enzyme present within the brain matter (122). (iv) Behavioral abnormalities can be corrected after the disease is advanced (123). (v) Despite the global distribution of storage lesions, selective neurodegeneration occurs in brain areas (hippocampus and cerebral cortex) that are important for mentation and can be reversed by gene transfer, thus it may not be necessary to correct all of the brain disease to have a positive effect in a clinical setting (124). (vi) More extensive distribution of enzyme can occur by neuronal transport in certain pathways to distal sites (22), which may be clinically important due to the limited number of injection sites that can be used. Many of these ﬁndings were subsequently extended to other experiments in models of MPS VII models and other LSDs (125,126). When applying these principles to other diseases, there will be variations in speciﬁc

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responses due to inherent differences in the speciﬁc enzymes. For example, the percentage of normal levels that will be needed for correction may vary, since there are signiﬁcant differences in the stability among the different enzymes. Another example is that dogs mount a vigorous immune response to alpha-L-iduronidase (defective in Hurler disease, MPS I) (127), whereas they respond only with humoral immunity to foreign GUSB and the response does not appear to interfere with graft survival, vector gene expression, or enzyme transfer to diseased cells (121,128). GENE DELIVERY VEHICLES Gene delivery vehicles can be divided into four general categories: naked DNA, liposomes, viral vectors, and transplanted engineered cells. Naked DNA transfer has been used mostly in muscle and has not been very effective in the brain. The other three methods have speciﬁc advantages and disadvantages as delivery methods in the CNS. The viral and liposomal methods of delivering genes involve introducing an exogenous nucleic acid to the subject’s own CNS cells, which express the gene to supply a therapeutic protein or inhibitory RNA in cis or trans. Transplanted cells deliver the therapeutic activity via inactivating diffusible toxins or supplying the therapeutic protein in trans. All three of these methods can be combined to increase the ﬂexibility and complexity of delivering transgenes to their targets. Liposomal Delivery Systems Liposomal delivery systems were ﬁrst developed by using cationic polyplexes that formed complexes with anionic DNA, but the compounds are much more stable in water than in saline, thus they accumulated in the lungs in vivo (129–131). When naked DNA was encapsulated in a very small liposome (<85 nm) and conjugated with polyethyleneglycol (PEG) strands, the new complex was stable in the blood for extended periods of time (17). These ‘‘PEGylated’’ liposomes can traverse the BBB if they are conjugated to a monoclonal antibody (mAb) targeting plasma membrane receptors and are termed PEGylated immuno-liposomes (PILs) (15,16). The mAbs can potentially be directed at various target molecules involved with the BBB. Neuron-speciﬁc enzyme activity was seen in Rhesus monkeys intravenously administered PILs with a mAb against the human insulin receptor and containing a plasmid expressing b-galactosidase from a neuron-speciﬁc promoter (132). Studies with the transferrin receptor have produced similar brain-speciﬁc expression patterns (133). This evidence indicates liposomal delivery using promoters driving expression within speciﬁc cells in the brain that can provide unique expression patterns to genes even when gene delivery is non-speciﬁc. This technology has been used to express

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the gene for tyrosine hydroxylase (134,135) and siRNAs in protocols for cancer gene therapy (136). Although these systems possess great potential for widespread gene delivery in the CNS, gene expression is very short-lived (only a few days). Viral Delivery Systems Viral vectors for gene delivery consist of a genetically engineered viral particle that contains a gene or cDNA in the viral genome encoding the therapeutic protein orsi RNA. The vectors are rendered incapable of replication by the removal of sequences required for viral replication from the vector. Most of these deleted sequences encode viral replication proteins that must be supplied in trans from ‘‘packaging’’ cells or plasmids transiently transfected into cells, which allows the therapeutic nucleic acid to be encapsulated into a viral particle. The resulting product is a replication-defective virus particle containing the desired transgene. When these vectors infect CNS cells, the modiﬁed viral genome either integrates into the endogenous DNA or is maintained as episomal DNA, depending on the type of virus. This characteristic is important because of the potential for the integrated viral genome to disrupt tumor suppressor genes or activate oncogenes that may lead to tumorgenesis (137–140). Episomal DNA vectors can also integrate, but do so at a much lower frequency. There are numerous characteristics that a viral vector must possess to be ideal for gene transfer. The most important is that it should not be toxic to the cells it infects. Toxicity can be in the form of viral-mediated effects or the induction of an immune response, which is especially dangerous in the brain (31,32). Both have caused numerous problems with gene therapy protocols and trials in the past. Another attractive feature is cell-speciﬁc tropism. This characteristic is governed by the surface proteins (capsid or envelope) of the virus, which bind to receptors on the cell surface. The ability of the virus to infect postmitotic cells is also a desirable quality, since many cells in the CNS are postmitotic. Finally, the ease of genetic manipulation due to a large transgene packaging capacity permits the inclusion of various gene regulatory elements and/or multiple target proteins. No single viral vector possesses all of these characteristics; hence, the investigator needs to determine which vector is most attractive to address the experimental requirements. Most viral vectors have had extensive modiﬁcations of their genomes for use in gene therapy (141). Most of the endogenous viral sequences are removed to make room for the target transgene and regulatory elements, including those responsible for replication or other pathogenic traits. Some of the other viral proteins are required for the production of viral particles or expression of the viral genome in the infected cells (e.g., reverse transcriptase in retroviruses). Because of the need to remove these from the target

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cell, most of these proteins are supplied in trans by a packaging cell line or helper virus that encodes the needed sequences in their DNA. There are also cis-acting viral sequences that are required in the viral vector for packaging of the nucleic acid or integration into the host genome. Much of the research involving the production of viral vectors for gene therapy has involved the delineation of which sequences are required in cis and which can be supplied in trans for each speciﬁc viral vector systems. Whether the viral genome integrates into the host genome or is maintained as an extra-chromosomal constituent of the nucleus is an important characteristic when deciding which viral vector to use. The two most important types of viral vectors that integrate into the nuclear genome are the retro- and the adeno-associated viruses, while adenovirus and herpesvirus genomes are mostly present in episomal forms. The following discussion highlights advantages and disadvantages of the various viral vector systems and their use in animal models of CNS gene therapy. Retrovirus Delivery Systems Retroviruses are single-stranded RNA viruses that require reverse transcriptase for the viral genome to be transcribed into DNA and incorporated into the host cell’s genome. This group includes oncoviruses and lentiviruses, consisting of the viral genome, reverse transcriptase, a protein core, and a phospholipid membrane envelope containing surface glycoproteins. Wild-type virus requires 50 and 30 long terminal repeats (LTRs) containing promoter/enhancer elements and sequences needed for reverse transcription and integration. Self-inactivating (SIN) retroviral vectors have functioning 50 promoter elements but their 30 LTRs have had the transcriptional regulatory sequences deleted. As a result of the reverse transcription mechanism, these alterations are duplicated in the 50 LTR in the double-stranded DNA provirus that integrates into the cellular genome, eliminating all promoters except those engineered with the transgene (142,143). The capacity for foreign genes in retroviral vectors is approximately 6–7 kb, which is an intermediate size when compared to other viral delivery vehicles. This capacity provides adequate space for several ORFs as well as additional regulatory elements. An attractive feature of the retroviral family is that certain envelope glycoproteins (env) from other viruses can be incorporated into the viral membrane. Different envelope proteins bind different receptors, giving each virus its particular tropism, thus exchanging the glycoproteins can alter the host cell range of the vector. Envelope proteins from Ebola, Marburg, LCMV, Mokola lyssavirus, vesicular stomatitis virus, and others been used in retroviral delivery systems (144–147). The vesicular stomatitis virus-G surface glycoprotein (VSV-G) is a particularly important envelope protein as it binds plasma membrane lipids instead of protein receptors, giving it a wide tropism in the brain (148). VSV-G also increases the stability of viral

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particles enough to allow ultracentrifugation, providing higher viral concentrations (149). One disadvantage of using retroviruses for gene therapy is their potential for integration site dependent mutagenesis, where insertion into oncogenes could transform the cell. This has occurred in a clinical trial for the X-linked form of severe combined immune deﬁciency (X-SCID) (138,150). Hematopoietic stem cells were infected with oncoretroviral vectors in cell culture in order to express the deﬁcient protein, the interleukin Z receptor gamma chain (IL-2Rg). Nine of ten patients had reconstitution of their immune systems by the genetically engineered cells, but regrettably, three of the patients developed T-cell leukemia after transplantation. The leukemia cells of two of the patients had a copy of the intact vector inserted in the vicinity of the LMO2 gene locus, a known oncogene (137). Subsequent study demonstrated the neoplasias were secondary to the transgenic promoter activating expression of LMO2, which kept some cells in a more undifferentiated state and increased their leukemic potential (138–140). However, these trials used retroviral vectors containing intact LTRs, which are powerful promoters and can be eliminated by using SIN conﬁgurations. Further transcriptional regulation of the integrated transgene’s promoter by utilizing insulators to block promoter effects of or by adjacent loci, may limit this possibility in the future (151–153). Retroviral vectors were some of the ﬁrst viral vectors used in protocols for gene therapy (154), and can be divided into two main subtypes based on their ability to infect nondividing cells. Oncoretroviruses cannot infect nondividing cells, while the lentiviruses can infect both dividing and nondividing cells. Lentiviral systems are the more widely used vector in gene therapy of neurogenetic disease. The Maloney murine leukemia virus (MLV) is the prototypic subtype of the oncoretroviral vectors, and was one of the ﬁrst vectors used in human trials of gene therapy for severe combined immunodeﬁciency due to adenosine deaminase deﬁciency (ADA–SCID) (154–157). All other oncoretroviral vectors are derived from this basic prototype and have similar means of function, but with enhanced capabilities. One of the biggest drawbacks of using oncoretroviral vectors is their inability to infect nondividing cells (158). Because of the relative paucity of mitotic cells in the CNS, most oncoretroviruses are not very useful for CNS gene therapy. However, they are used for ex vivo manipulation of cells (ﬁbroblasts, hematopoietic, and neural stem cells) (122). Lentiviruses can infect postmitotic cells but have more viral elements that must be supplied in trans than the oncoretroviruses (114). HIV-1 is the prototypical lentivirus, but FIV, HIV-2, EIAV, and SIV viruses are also being used in gene therapy studies (123,159–162). Lentiviruses have been pseudotyped with a number of different envelope glycoproteins to expand their tropism in the brain (146,163). Vectors containing the envelope proteins of the rabies-related Mokola virus, lymphocytic choriomeningitis

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virus (LCMV), and VSV transduce similar patterns of cells in the striatum, thalamus, and white matter of the rodent brain. In contrast, the MuLVpseudotyped vectors had a less extensive transduction pattern, but had selective expression in the striatum and hippocampal dentate gyrus. Ebola-pseudotyped vectors had no recognizable pattern of transduction in the CNS (163). In the rat, lentivirus vectors pseudotyped with the VSV Indiana strain, wild-type ERA, and CVS strains had strong transduction in the striatum, while Mokola- and LCMV-pseudotyped vectors were less effective (164). The use of cell-type speciﬁc promoters in the CNS showed that even if lentiviruses infected a wide variety of cell-types, the promoters could limit expression of the transgene to the expected target cells (165). This study compared neuronal and glial speciﬁc promoters [neuron-speciﬁc enolase (rNSE) and the glial ﬁbrillary acidic protein (hGFAP)] with the more ubiquitously expressed human cytomegalovirus promoter (hCMV), a hybrid CMV/beta-actin promoter (CAG) and human elongation factor 1a (EF1a) promoters from VSV-G pseudotyped lentiviruses. The GFAP promoter was expressed only in astrocytes and the NSE promoter was expressed only in neurons, while the hCMV, CAG, and EF1a were ubiquitously expressed. These results indicated that pairing pseudotypes with low cell-type speciﬁcity to various promoters can confer cell-speciﬁc expression of the transgene in the brain. The most extensive use of lentivirus vectors in the CNS has been in Parkinson disease models. For neurogenetic diseases, the widest application has been in mouse models of lysosomal storage diseases. Multiple injections result in spread of the secreted enzyme and have been demonstrated to correct storage lesions in MPS VII and an metachromatic leukodystrophy model (arylsulfatase-A deﬁciency), and can mediate improvements in some behavioral tasks (125,166). Cognitive improvement in the MPS VII model was associated with alteration in expression of genes associated with neuronal plasticity (123). Other studies have used lentivirus vectors to deliver trophic factors to rescue genetically diseased neurons, for example, in the mouse model of ALS (167). These data suggest that it may be feasible to use lentivirus vectors in human gene therapy trials for neurogenetic diseases. Adeno-associated Virus Delivery Systems Adeno-associated virus (AAV) is a single-stranded DNA virus of the parvovirus family. It can be maintained as an episome or be integrated into the nuclear DNA. AAV is classiﬁed as a dependovirus because it requires a helper virus (adenovirus or herpesvirus) for active replication in infected cells. The required helper functions can be supplied in trans by packaging plasmids to make vector virus. AAV is particularly attractive for gene transfer because the wild-type virus has no known pathology in humans. The major limitation of AAV vectors is that they have a cloning capacity for foreign genetic

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sequences of only 4.5 kb. This relatively small size can accommodate many cDNAs, but limits the choice of promoters, introns, and other regulatory elements in the vector genome (168). This limitation can be overcome by dual infection of the same cell with multiple vectors containing different but overlapping sequences because the vector genome forms head-to-tail concatemers in the cell, which is maintained in an episomal state (169,170). However, the efﬁciency of this type of vector is generally low. The outer coat of AAV viruses is formed from the capsid protein. The capsids are grouped by serotype, and 11 different serotypes have been identiﬁed. A vector genome only requires the inverted terminal repeat (ITR) elements (approximately 150 nucleotides at each end of the vector) from the viral genome. The AAV2-derived ITRs are the most commonly used for vector construction in gene therapy protocols (24). The vector genome can be packaged with any of the capsid serotypes, and AAV serotypes have a wide range of tropisms in the CNS. In mice, AAV-2 and AAV-3 predominately infect neuronal cells, AAV-4 infects ependymal cells, and AAV5 effectively transduces neuronal and glial cells (171,172). Intraventricular injections of neonatal mice with AAV1 or AAV2 vectors result in very widespread gene delivery that is maintained in the adult for at least one year (173,174). AAV5 transduces very efﬁciently when injected into the adult mouse brain (175), but is very limited when injected into the ventricles of neonates (176). These characteristics make AAV vectors very promising tools in any potential gene therapy in the CNS and they have been used successfully in a number of animal studies of CNS gene therapy (168,177). AAV-2 vectors have been used in many experiments in the MPS VII mouse brain, showing long-term expression and reversal of storage (178–181). AAV vectors containing the alpha-N-acetylglucosaminidase gene driven by a CMV or neuron-speciﬁc enolase promoters were used to treat the brains of adult MPSIIIB mice using similar procedures (182). These mice have their cellular pathology corrected in regions up to 1.5 mm from the injection site. Knockout mice that model ALS have been improved by intramuscular injection with AAV vectors expressing IGF-1. They had reduced disease progression after retrograde delivery of the transgenes to the motor neurons in the spinal cord (183). N-acetylaspartylase (ASPA) knockout mice (Canavan disease) had an AAV vector expressing genes for both ASPA and green ﬂuorescent protein (GFP) injected into their striatum and thalamus (184). Three to ﬁve months after the injection, ASPA activity and GFP expression were elevated. MRI of injected animals showed a remarkable lack of spongiform degeneration in the thalamus, while magnetic resonance spectroscopy showed decreased N-acetoaspartate levels near the injection site. In parallel to these studies, a protocol for human gene therapy of the Canavan disease utilized the neurosurgical administration of AAV vectors containing the ASPA gene into 21 patients. These patients are currently

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being evaluated with a variety of biochemical, radiological, and neurological tests and the ﬁnal results are still pending. These gene transfer studies represent the ﬁrst clinical use of AAV in the human brain (12). Studies of children with Batten disease have been initiated (13). Both of these studies use AAV2 vectors, but other serotypes may be better for clinical use if they are safe. In studies on the cat brain, AAV-1 transduced a greater number of gray matter cells than AAV-2, and AAV-5 was ineffective even though it works well in the rodent brain (117,185). AAV-1 was the only serotype that also transduced white matter cells (117,185). Subsequently, cats with the lysosomal storage disease a-mannosidosis were injected with AAV-1 vectors expressing feline a-mannosidase (185). Signiﬁcant improvements were noted in the animal’s neurologic signs. MRI revealed improvement in myelination and postmortem evaluation revealed reduction of storage lesions throughout the brain, even in areas relatively distant from the injection sites (185). The experiments in mammals with brains more similar in size to humans may suggest more effective strategies for clinical trials. Adenovirus Delivery Systems Adenovirus is a double-stranded DNA virus with a protein capsid that confers a speciﬁc serotype to the individual viruses. Most of the adenoviral vectors used in gene delivery are based on serotype 5 (Ad5), but Ad2, Ad4, and Ad7 are also used. Ad5 affords a greater capacity for retrograde transport in the brain than other serotypes. Adenoviral vectors are generally episomal and thus most result in fewer integration dependent alterations in cellular homeostasis or oncogenesis than vectors requiring integration for function (186,187). Adenovirus also has a large number of serotypes (>50, primarily on the basis of neutralization assays) that can infect a very diverse set of cell types (188). It has no requirement for helper virus or complex post-infectious processing (i.e., reverse transcription, etc.) (189). The ﬁrst adenoviral systems had transgene capacities of 6 kb to 8 kb, but newer adenoviral vector systems (discussed further) have an increased transgene capacity of up to 25 kb to 40 kb, which is one of the largest of the viral vectors. Although these traits make adenovirus useful for experiments, adenovirus has been linked to a fatality in a human gene therapy trial. This death resulted from the patient’s aggressive immunologic response to the adenoviral vector (190–192). Because adenovirus is a common human pathogen, it is not surprising that over 55% of subjects tested express antibodies to Ad5 serotype (193,194). Ways of reducing the immunogenicity of adenovirus include the removal of most of the endogenous viral sequences from the vector. The viral proteins must then be expressed in trans by packaging cells. Capsid proteins may also be engineered to decrease their cross-reactivity with wild-type adenoviruses. A variation of adenoviral vectors called high capacity (HC) or ‘‘gutless vectors’’ can be derived in which a substantial portion of the endogenous

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adenovirus genome has been deleted, with the vector only containing some small cis-acting sequences and the transgene (195). The optimal size of these vectors for stable packaging into the capsid is within 27 kb to 42 kb in length. Smaller genomes experience signiﬁcant gene rearrangements. This range produces a large transgene capacity for genetic manipulation, which can accommodate additional regulatory elements within the vector genome, such as insulators or regulable promoters or multiple transgenes. If the vector is less than 27 kb, ‘‘stuffer’’ sequences of non-coding DNA sequence may be added to ensure it is large enough for packaging. Adenoviral vectors have been studied in a number of animal models (196,197). In utero intravenous injection of rats with adenovirus expressing LacZ led to widespread expression of the reporter gene (198). Transduction occurred primarily in the telencephalon and hypophysis, with expression persisting for at least one week after birth. In another study, neonatal mice with GM1 gangliosidosis were given a single intravenous injection of adenoviral vector encoding murine beta-galactosidase. After 30 days, the treatment attenuated GM1 ganglioside accumulation, and corrected mice reported 10% to 20% of normal enzyme activity in the brain (199). Intracerebral injections of adenovirus encoding the b-subunit of hexosaminidase into Sandhoff mice resulted in expression of near normal levels of hexosaminidase A and B (23). Studies with adenoviral vectors expressing GUSB in MPS VII mice have also been performed. Intravenous and intraventricular delivery of adenovirus encoding GUSB in fetal, neonatal, adult mice has led to correction of storage lesions throughout the brain (200–202). HC-adenoviral vectors have also been successfully used in the CNS to express transgenes (196,203–207). Adenoviral vectors will continue to play a signiﬁcant role in future CNS gene therapy experiments, especially when large cloning capacity is required (196,205–207). Herpesvirus Delivery Systems Type I herpes simplex virus (HSV-1) is a common human pathogen with a strong tropism for neurons (208). It is a double-stranded enveloped DNA virus that exists in latent and replicating states, which have unique subsets of gene expression from latency- or replication dependent promoters. Many of these genes must be expressed for viral replication and maintenance, but are toxic to cells. These genes must be expressed from regulable promoters for vector production in packaging cell lines. The only gene actively transcribed during latency is the latency associated transcript (LAT) gene from the LAT promoter (209,210). This promoter has shown expression of transgene in vivo for extended periods of time (211). One variant of HSV-1 that is used in gene therapy experiments has a deletion in a gene associated with pathogenicity, allowing it to be injected directly into the brain at titers that would be lethal with a wild-type vector (212). Another herpesvirus vector

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system is the amplicon vector, in which many of the viral-encoded genes are removed except for cis-acting sequences required for packaging the capsid. Once these sequences are removed, vectors have a transgene capacity of 15 kb to 20 kb and form concatemers up to the 150 kb genome length of wild-type HSV-1 (213,214). This large transgene capacity is desirable when complex regulatory systems and/or multiple ORFs are needed. Another favorable quality of herpesviruses is their extensive ability to infect neurons via the axon and be transported back to the nucleus along the axonal microtubules. This makes it an attractive method for delivery of transgenes to spinal cord neurons in models of neuropathy and motor neuron disease. Examples of its use in animals include studies where HSV vectors were injected into the gastrocnemi of rats with a latencyassociated promoter upstream of b-galactosidase (215). Subsequently, b-galactosidase was expressed in anterior horn cells of the spinal cord bilaterally, with 90% of the anterior horn motor neurons having enzyme activity for up to 182 days after the injection. Other related studies used herpesviral vectors indirect intracranial injections of mice with a transgene expressing GUSB from a latency-associated promoter (216). The herpesvirus has a great deal of potential for gene delivery, but much more research is required to more fully understand the regulation of its gene expression and its infectivity. Poliovirus Delivery Systems Poliovirus is an example of a vector system that can infect neural cells, but which has limited applications due to its basic biology. Poliovirus is a nonenveloped single-stranded RNA virus and does not have a DNA form in the cell. The hallmark feature of poliovirus is its tropism for anterior horn cells (217). However, poliovirus may be a very difﬁcult vector system to use in studies of gene therapy for several reasons. A major obstacle in using the system in humans is the almost worldwide immunity to the virus. Indeed, poliovirus may be the one virus in which the successful efforts of world health organizations for global vaccinations makes it a safety risk for use in human gene therapy. Another difﬁculty with poliovirus is the limited ability to use the virus in modeling studies as many animals lack the poliovirus receptor and cannot be infected. Transgenic mice expressing the poliovirus receptor are available and can be infected with the live virus (218). One of the biggest disadvantages of using poliovirus is its lack of a DNA form. This means the genome is relatively short-lived in infected cells and possible longterm correction of disease may be difﬁcult to achieve. This may be advantageous for use in gene therapy where short-term expression may be desirable, such as tumor therapy. Replication-competent poliovirus containing RNA encoding brainderived neurotrophic factor (BDNF) were injected in intramuscularly transgenic mice expressing the human poliovirus receptor (219). At three days

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post-infection, antigens of poliovirus and BDNF were detected in the motor neurons of the spinal cord; however, these antigens were no longer detectable four days later. Other studies focused on the use of poliovirus-capsid encapsulated RNA replicons in anterior horn cells or intarsia inoculation. GFP, luciferase, and murine tumor necrosis factor alpha have been expressed in the spinal cord with some success (217,220). These studies suggest that poliovirus can be used as a vector for the delivery of neurotrophic factors or deﬁcient enzymes to motor neurons. However, the signiﬁcant difﬁculties involved with the immunogenicity, the short-lived expression of transgenes, and a need for transgenic mouse models for infection will need to be overcome. Ex Vivo Methods in Gene Therapy Another means of delivering therapeutic enzyme to the brain is by transplanting cells genetically engineered in vitro to express or overexpress proteins capable of trans-correcting pathologic cells. This ex vivo method would provide the therapeutic proteins via secretion, as with lysosomal enzymes (122), or as cytoplasmic proteins capable of detoxifying diffusible substrates or trophic factors to maintain cells. It could also allow for endogenous neuronal cells deﬁcient in cytoplasmic proteins to be genetically engineered to express the protein with subsequent repopulation of pathologic sites. Hematopoietic cell transplantation (HCT) uses a similar trans-corrective approach to treat patients with lysosomal or peroxisomal disorders affecting the CNS (48,221). Utilizing the migration of hematopoietic cells into the brain and subsequent differentiation of these cells into microglia as the enzyme donors has had some success alleviating these disorders (48,221). However, HCT is often not effective because too few of the cells cross the BBB and so inadequate amounts of protein reach the brain. HCT is also associated with the potentially fatal risks of graft versus host disease or infection during periods of immunocompromise. In addition, the use of relatives as donors often means that the grafts are heterozygous for the gene of interest and do not produce optimal amounts of the therapeutic protein. The goal of ex vivo gene therapy is to use stem cells derived from the affected individual and genetically engineer them to express the deﬁcient enzyme in order to avoid many of these transplantation barriers. The use of NSCs in ex vivo protocols has the potential of being used in the treatment of neurodegenerative disease. However, this will require removing cells from the patient’s brain, engineering them in culture, and expanding them to large numbers enough for transplantation back into the brain. The most likely regions to obtain NSCs postnatally with the lowest level of surgical risk will be the cerebellum (up to two years of age) and the olfactory bulbs.

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In animal models, ex vivo manipulated neural stem cells (NSCs), neuronal progenitors, astrocytes, ﬁbroblasts, and bone marrow stromal cells have been transplanted into the CNS and successful provided therapeutic enzymes to endogenous cells (122,222–224). A detailed discussion of neural stem cells is beyond the scope of this review, but several excellent reviews have been written discussing their properties in detail (225–230). Neural stem cells are undifferentiated cells that exist in developing and adult nervous systems that are self-renewable, while also having the potential to differentiate into many of the cell types of the CNS (227–230). When NSCs are transplanted into the CNS, they are capable of becoming integrated into the local milieu, where they can differentiate into glia or neurons depending on the site of engraftment and the local microenvironment (229). NSCs elaborate neurotrophic factors, which can aid in alleviating or repairing disease pathology (231). They can be expanded in vitro to provide signiﬁcant numbers of cells for ex vivo genetic manipulation (232) and possibly large scale transplantation into large animal brains. NSCs preferentially migrate to regions of pathology and damage, which may be valuable in correcting neurogenetic diseases with focal pathology (e.g., type I glutaric aciduria) (229). They can also replace CNS cells lost in the pathogenesis of the disease with cells expressing the deﬁcient protein, making them more resistant to the disease process (233). An important factor regulating the ability of NSCs to migrate from the graft site and disperse throughout the brain is the developmental stage of the brain at the time of transplantation. In mouse models, fetal and neonatal brains have been shown to be more amenable to widespread dispersion of NSCs than adult brains. For example, NSCs overexpressing GUSB were injected into the ventricles of newborn MPS VII mice and were subsequently found throughout the brain (225). These cells corrected disease pathology and were a source of therapeutic enzyme throughout the life of these mice. NSCs capable of expressing the a-subunit of b-hexosaminidase were also injected into the ventricles of fetal mice with similar results (234). NSCs were also used to replace lost oligodendroglia in the shiverer mouse, which has mutations in myelin basic protein that cause widespread oligodendroglia loss (235). Neonatal injections of wild-type NSCs into the ventricles of these mice resulted in widespread engraftment and a predisposition toward forming oligodendroglia, which led to a signiﬁcant decrease in clinical symptomatology (233). In the adult mouse brain, the migrational properties of NSCs are much more limited. NSCs injected into the ventricles of adult mice follow a less widespread migratory pattern than in neonates with a large number of NSCs accumulating in the olfactory bulbs (236,237). NSCs expressing b-galactosidase were injected into the subventricular zone, striatum, cortex, and olfactory bulb of adult mice. Several weeks after transplantation, the cells were found near the transplant sites, but extensive migration of grafted cells was observed in the olfactory bulb only (229,238–241). The dispersion

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of NSCs in the adult brain is less widespread than in the fetal and the neonatal brain and is likely to be inadequate for correction of the diffuse pathogenesis of most neurogenetic diseases in the adult brain. Mechanisms to increase the migratory potential of these cells may allow them to be more capable of widespread correction of pathogenesis in human neurogenetic disease (242,243). The studies suggest that ex vivo methods of gene therapy may be more readily used in the CNS of neonates affected by neurogenetic disease than at later ages. The predisposition of NSCs to migrate to areas of injury or tumors may make them more useful in adult brains with focal lesions. The neurogenetic disorders typically have diffuse CNS pathology, thus transplants of NSCs into the developing neonatal brain may result in better dispersion of cells for trans-correction than in adults. SUMMARY Inherited diseases of the CNS produce a wide variety of metabolic and structural lesions. Each type of disease has unique characteristics that can be exploited, or are barriers to genetic correction. There have been signiﬁcant improvements in vector design over the past several years, which have been applied successfully to a number of animal models. However, human gene therapy trials have shown that there are a number of signiﬁcant obstacles still to be overcome. Safety is a major concern: insertional mutagenesis by a retrovirus vector, with subsequent malignant transformation of the targeted cells, has occurred in three children receiving therapy for X-linked severe combined immunodeﬁciency, with one death; and a young adult patient with ornathine transcarbamylase deﬁciency died from an aggressive immune response to the adenovirus delivery vector. Thus, much of the current research is aimed at developing more effective means to safeguard against these and other complications of gene delivery. The safety of vector systems has been signiﬁcantly improved, such as the self-inactivating retroviruses that remove viral promoter elements from the integrated provirus. However, the insertion of foreign DNA into cells still carries a risk of altering genes to deregulate normal cellular controls on growth. The use of targeted insertion of foreign DNA is commonplace in transgenic technology, but is currently too inefﬁcient for somatic gene therapy applications. Further reﬁnement of transgene expression driven by cell-speciﬁc promoters or inducible systems associated with transcriptional insulators may more precisely regulate production of the transferred gene and avoid inappropriate expression from mistargeted tissues. Temporary immunosuppression to block the host’s immune response until the gene transfer process is completed has been demonstrated, which may improve safety (224). Gene delivery in the brain presents additional difﬁculties. Although techniques to deliver genes across the BBB would obviate the need for

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surgery, the methods developed so far are either inefﬁcient or have only resulted in transient gene delivery. For the inherited diseases, the goal is permanent correction. Many genes associated with inherited CNS disease have been discovered, isolated, and characterized, and there is increasing understanding of the mechanisms of pathogenesis at the cellular and biochemical levels. The development of accurate genotypic and phenotypic animal models has led to the development of trans-corrective strategies for certain categories of disease, which may signiﬁcantly decrease the total number of cells to which the gene must be transferred to correct the disease process. The size of the human brain compared to rodents makes translation much more difﬁcult than in other organ systems and is particularly important in genetic diseases due to the global distribution of the lesions. The ﬁrst study demonstrating that gene transfer into the brain could be effective in ameliorating clinical neurological signs in a large animal model of a human genetic CNS disease was published only recently (185). The potential for transferring normal copies of genes into defective cells to effect permanent correction was recognized when the ﬁrst gene was cloned. Much like the ideas of organ or tissue transplantation, gene therapy will develop over time to deliver on its promise. This development must occur with caution to better predict potential obstacles, but gene therapy is likely to be at least partially effective in the CNS for some diseases. This will require special approaches and methods because of the inherent complexity of the brain, as well as its relative isolation from the rest of the body, but advances in animal models have demonstrated in principle that this goal in all likelihood will be accomplished.

ACKNOWLEDGMENTS We thank Drs. M. Romanko and R. Walton for excellent review of the manuscript. The work in J.H.W.’s laboratory is supported by NIH grants DK42707, DK46637, DK47757, DK63973, NS29390, and NS38690; T.M.P. is supported by a training grant from the National Institute of Child Health and Development (HD43021). REFERENCES 1. Trask TW, Trask RP, Aguilar-Cordova E, Shine HD, Wyde PR, Goodman JC, Hamilton WJ, Rojas-Martinez A, Chen SH, Woo SL, Grossman RG. Phase I study of adenoviral delivery of the HSV-tk gene and ganciclovir administration in patients with current malignant brain tumors. Mol Ther 2000; 1:195–203. 2. Eck SL, Alavi JB, Alavi A, Davis A, Hackney D, Judy K, Mollman J, Phillips PC, Wheeldon EB, Wilson JM. Treatment of advanced CNS malignancies

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203. Thomas CE, Schiedner G, Kochanek S, Castro MG, Lowenstein PR. Preexisting antiadenoviral immunity is not a barrier to efﬁcient and stable transduction of the brain, mediated by novel high-capacity adenovirus vectors. Hum Gene Ther 2001; 12:839–846. 204. Thomas CE, Schiedner G, Kochanek S, Castro MG, Lowenstein PR. Peripheral infection with adenovirus causes unexpected long-term brain inﬂammation in animals injected intracranially with ﬁrst-generation, but not with highcapacity, adenovirus vectors: toward realistic long-term neurological gene therapy for chronic diseases. Proc Natl Acad Sci USA 2000; 97:7482–7487. 205. Akli S, Caillaud C, Vigne E, Stratford-Perricaudet LD, Poenaru L, Perricaudet M, Kahn A, Peschanski MR. Transfer of a foreign gene into the brain using adenovirus vectors. Nat Genet 1993; 3:224–228. 206. Davidson BL, Allen ED, Kozarsky KF, Wilson JM, Roessler BJ. A model system for in vivo gene transfer into the central nervous system using an adenoviral vector. Nat Genet 1993; 3:219–223. 207. Bajocchi G, Feldman SH, Crystal RG, Mastrangeli A. Direct in vivo gene transfer to ependymal cells in the central nervous system using recombinant adenovirus vectors. Nat Genet 1993; 3:229–234. 208. Jacobs A, Breakeﬁeld XO, Fraefel C. HSV-1-based vectors for gene therapy of neurological diseases and brain tumors: part II. Vector systems and applications. Neoplasia 1999; 1:402–416. 209. Mitchell WJ, Steiner I, Brown SM, MacLean AR, Subak-Sharpe JH, Fraser NW. A herpes simplex virus type 1 variant, deleted in the promoter region of the latency-associated transcripts, does not produce any detectable minor RNA species during latency in the mouse trigeminal ganglion. J Gen Virol 1990; 71(Pt 4):953–957. 210. Naito J, Mukerjee R, Mott KR, Kang W, Osorio N, Fraser NW, Perng GC. Identiﬁcation of a protein encoded in the herpes simplex virus type 1 latency associated transcript promoter region. Virus Res 2005; 108:101–110. 211. Wolfe JH, Deshmane SL, Fraser NW. Herpesvirus vector gene transfer and expression of beta-glucuronidase in the central nervous system of MPS VII mice. Nat Genet 1992; 1:379–384. 212. Wolfe JH, Martin CE, Deshmane SL, Reilly JJ, Kesari S, Fraser NW. Increased susceptibility to the pathogenic effects of wild-type and recombinant herpesviruses in MPS VII mice compared to normal siblings. J Neurovirol 1996; 2:417–422. 213. Wade-Martins R, Smith ER, Tyminski E, Chiocca EA, Saeki Y. An infectious transfer and expression system for genomic DNA loci in human and mouse cells. Nat Biotechnol 2001; 19:1067–1070. 214. Saeki Y, Fraefel C, Ichikawa T, Breakeﬁeld XO, Chiocca EA. Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artiﬁcial chromosome. Mol Ther 2001; 3:591–601. 215. Yamamura J, Kageyama S, Uwano T, Kurokawa M, Imakita M, Shiraki K. Long-term gene expression in the anterior horn motor neurons after intramuscular inoculation of a live herpes simplex virus vector. Gene Ther 2000; 7:934–941.

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216. Zhu J, Kang W, Wolfe JH, Fraser NW. Signiﬁcantly increased expression of beta-glucuronidase in the central nervous system of mucopolysaccharidosis type VII mice from the latency-associated transcript promoter in a nonpathogenic herpes simplex virus type 1 vector. Mol Ther 2000; 2:82–94. 217. Bledsoe AW, Jackson CA, McPherson S, Morrow CD. Cytokine production in motor neurons by poliovirus replicon vector gene delivery. Nat Biotechnol 2000; 18:964–969. 218. Koike S, Taya C, Kurata T, Abe S, Ise I, Yonekawa H, Nomoto A. Transgenic mice susceptible to poliovirus. Proc Natl Acad Sci USA 1991; 88:951–955. 219. Jia Q, Liang F, Ohka S, Nomoto A, Hashikawa T. Expression of brainderived neurotrophic factor in the central nervous system of mice using a poliovirus-based vector. J Neurovirol 2002; 8:14–23. 220. Jackson CA, Messinger J, Palmer MT, Peduzzi JD, Morrow CD. Gene expression in the muscle and central nervous system following intramuscular inoculation of encapsidated or naked poliovirus replicons. Virology 2003; 314: 45–61. 221. Krivit W. Stem cell bone marrow transplantation in patients with metabolic storage diseases. Adv Pediatr 2002; 49:359–378. 222. Snyder EY, Taylor RM, Wolfe JH. Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain. Nature 1995; 374:367–370. 223. Ridet JL, Sarkis C, Serguera C, Zennou V, Charneau P, Mallet J. Transplantation of human adult astrocytes: efﬁciency and safety requirements for an autologous gene therapy. J Neurosci Res 2003; 72:704–708. 224. Jin HK, Schuchman EH. Ex vivo gene therapy using bone marrow-derived cells: combined effects of intracerebral and intravenous transplantation in a mouse model of Niemann-Pick disease. Mol Ther 2003; 8:876–885. 225. Snyder EY, Macklis JD. Multipotent neural progenitor or stem-like cells may be uniquely suited for therapy for some neurodegenerative conditions. Clin Neurosci 1995; 3:310–316. 226. Snyder EY, Wolfe JH. Central nervous system cell transplantation: a novel therapy for storage diseases? Curr Opin Neurol 1996; 9:126–136. 227. Fischer A. Primary immunodeﬁciency diseases: natural mutant models for the study of the immune system. Scand J Immunol 2002; 55:238–241. 228. Vescovi AL, Snyder EY. Establishment and properties of neural stem cell clones: plasticity in vitro and in vivo. Brain Pathol 1999; 9:569–598. 229. Gage FH. Mammalian neural stem cells. Science 2000; 287:1433–1438. 230. Park KI, Ourednik J, Ourednik V, Taylor RM, Aboody KS, Auguste KI, Lachyankar MB, Redmond DE, Snyder EY. Global gene and cell replacement strategies via stem cells. Gene Ther 2002; 9:613–624. 231. Niles LP, Armstrong KJ, Rincon Castro LM, Dao CV, Sharma R, McMillan CR, Doering LC, Kirkham DL. Neural stem cells express melatonin receptors and neurotrophic factors: colocalization of the MT1 receptor with neuronal and glial markers. BMC Neurosci 2004; 5:41. 232. Heuer GG, Skorupa AF, Prasad Alur RK, Jiang K, Wolfe JH. Accumulation of abnormal amounts of glycosaminoglycans in murine mucopolysaccharido-

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5 Ethical Dilemmas in Neurogenetics Wendy R. Uhlmann Department of Internal Medicine, Division of Molecular Medicine and Genetics, and Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, U.S.A.

This chapter will discuss ethical dilemmas that arise from neurogenetic conditions and genetic testing for these conditions, with a focus on Huntington disease, as it has been a paradigm for such issues. The guidelines for predictive genetic testing for Huntington disease, initially published in 1989 (1–3) and revised in 1994 (4–6) after the cloning of the HD gene, set the global standard for this type of testing. These guidelines set a high bar for patient autonomy in testing decisions and in the comprehensiveness of information provided prior to testing. In recent years, these guidelines have served as a template for predictive testing for other neurogenetic conditions and hereditary cancers (7,8). While ordering a genetic test to make a diagnosis in a symptomatic patient is within a neurologist’s scope of practice, neurologists should only order predictive genetic testing on an asymptomatic patient if they are working on a team that includes a genetics specialist (e.g., genetic counselor, geneticist, genetics nurse). Otherwise, these patients should be referred to a genetics clinic. The counseling, decision making, and potential ethical issues involved in predictive genetic testing are complex and these are time-consuming cases. Generally, the initial counseling session is longer than one hour, not including the time spent prior to the appointment obtaining family history information, constructing a three-generation pedigree, reviewing medical records, and ascertaining pertinent information about genetic testing and support groups (9). Central to predictive genetic testing is the pretest and post-test counseling

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that is needed, which is described in the testing guidelines for Huntington disease (4–6), in a position paper by the National Society of Genetic Counselors (10) and in Chapters 1–3 of this book. While genetic testing can determine whether a gene mutation has been inherited, it generally cannot predict age of onset, type of symptoms, severity, or disease course. Predictive genetic testing for neurogenetic conditions usually does not change medical therapy because there currently is no treatment to delay the onset of symptoms. The decision to undergo predictive genetic testing must be made with great care as one is looking into a crystal ball to predict the future. Therefore, patient autonomy in deciding about predictive testing is paramount. It must be left up to the patient to determine whether this information will be helpful to know in living their life and making decisions about ﬁnances, marriage, children, and employment. It is important that patients understand the beneﬁts, risks, and limitations of genetic testing and have thought about the potential personal and familial impact that results will have in order to arrive at an informed decision. When a parent has been diagnosed or had a positive predictive test result for an autosomal dominant neurogenetic condition, each child is at 50% risk and the risk to other family members is signiﬁcantly increased as well. Given the potential familial implications of test results, family dynamics, and communication issues with relatives, there have been several ethical issues raised by predictive genetic testing for neurogenetic conditions including: 1. 2. 3. 4. 5. 6. 7. 8.

Patients’ rights versus relatives’ rights Right to conﬁdentiality versus right to information Right to know versus right not to know Communication of genetic information to at-risk relatives Duty of conﬁdentiality versus duty to warn Coercion of family members to be tested Childhood and prenatal testing for adult-onset conditions Potential use of results by insurers and employers.

Some of these ethical issues are either addressed or can be extrapolated from the predictive testing guidelines for Huntington disease while others have been handled on a case-by-case basis. Resolving these ethical issues requires consideration of core ethical principles and practice guidelines, evaluation of the locus of decision making, a careful determination of risks and beneﬁts, and establishing whose rights take precedence when there is a conﬂict. The case scenarios and approaches used to resolve the ethical issues that are presented in this chapter are based on actual Huntington disease cases. However, similar ethical dilemmas could arise with testing for other neurogenetic conditions including early-onset Alzheimer’s disease, frontotemporal dementia, spinocerebellar ataxias, dentatorubral-pallidoluysian atrophy (DRPLA), cerebral autosomal dominant arteriopathy with subcortical

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infarcts and leukoencephalopathy (CADASIL), amyotrophic lateral sclerosis (ALS) and others. The approaches used to resolve the Huntington disease cases described below could similarly be applied in such cases. TESTING MINORS Denise is 42 years old. She recently underwent predictive genetic testing for Huntington disease and her results were positive. Now she would like her teen-age children tested. Denise feels that knowing the results will be helpful in planning her children’s future and would be particularly helpful in their ﬁnancial planning and allocation of their limited ﬁnancial resources. Above all, Denise wants relief from the anxiety of not knowing whether or not her children are at risk for Huntington disease. Should children be tested for neurogenetic conditions that will not arise until adulthood? A parent may feel that it is their parental right to know this information and that it is needed both to prepare the child and plan for the child’s future. However, there is also the risk that positive test results could result in changes in family dynamics, differential treatment, stigmatization and adversely affect psychological well-being and perceptions of self (11). The potential beneﬁts and harms of genetic testing in children are summarized in the American Society of Human Genetics/American College of Medical Genetics 1995 report (11). Given that there currently is no medical beneﬁt to determining carrier status for adult-onset neurogenetic conditions in childhood, the consensus is that such testing should be deferred until the child reaches the age of majority and can decide whether to learn this information (Table 1). This applies whether the adult-onset neurogenetic condition follows an autosomal dominant, autosomal recessive, or other pattern of inheritance irrespective of whether the child is at risk to be affected or simply a carrier. What if a child has behavioral issues, learning disabilities, nonspeciﬁc neurological ﬁndings, or problems with the law? These ﬁndings are not characteristic symptoms of adult-onset neurogenetic conditions and are commonly seen in children in the general population. The development of symptoms in childhood for adult-onset neurogenetic conditions is rare. Children should only have genetic testing if a neurologist/physician familiar with the juvenile form of the condition thinks a child is truly symptomatic. While children should not be tested unless there is a medical indication, the guidelines clearly state that ‘‘the child be informed of his or her at-risk status upon reaching the age of reason’’ (comment 2.1, page 1533) (5). TESTING A CHILD FOR ADOPTION The woman on the phone was anxious and insistent. She shared that she wanted to adopt a 2-year-old girl but had just learned that the girl’s biological

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Table 1 Pertinent Guidelines Regarding Genetic Testing of Minors American Society of Human Genetics, American College of Medical Genetics (1995). Points to consider: ethical, legal and psychosocial implications of genetic testing in children and adolescents, page 1233. (11) ‘‘Timely medical beneﬁt to the child should be the primary justiﬁcation for genetic testing in children and adolescents.’’ ‘‘If the medical or psychosocial beneﬁts of a genetic test will not accrue until adulthood, as in the case of carrier status or adult-onset diseases, genetic testing generally should be deferred.’’ Huntington Disease Society of America (1994): Guidelines for Genetic Testing for Huntington’s Disease, page 10. (4) ‘‘Minors should not be tested unless there is a medically compelling reason for doing so, i.e., an at-risk child is believed to be showing symptoms. However, under no circumstances is testing a substitute for a thorough neurological and neuropsychological workup . . . Parental anxiety concerning a child’s risk does not constitute a medically compelling reason.’’ International Huntington’s Association and the World Federation of Neurology Research Group on Huntington’s Chorea (1994): Guidelines for the Molecular Genetics Predictive Test in Huntington’s Disease, page 1533. (5) ‘‘The decision to take the test is solely the choice of the individual concerned. No requests from third parties—family members or otherwise—shall be considered.’’ (Recommendation 2) ‘‘The test is available only to individuals who have reached the age of majority (according to the laws of the respective country).’’ (Recommendation 2.1)

mother had Huntington disease. Could we arrange for genetic testing on the child as soon as possible? The guidelines for testing children for adult-onset genetic conditions described above equally apply for children who are being considered for adoption. The American College of Medical Genetics and the American Society of Human Genetics jointly issued a position statement in 2000 (12), adopted by the National Society of Genetic Counselors in 2002 (13), which states: ‘‘Because the primary justiﬁcation for genetic testing of any child is a timely medical beneﬁt to the child, genetic testing of newborns and children in the adoption process should be limited to testing for conditions which manifest themselves during childhood or for which preventive measures or therapies may be undertaken during childhood’’ (page 761) (12). Only genetic testing that would be ordered on all children of a similar age should be performed on children in the adoption process (12). Performing genetic tests on children to be adopted outside of those that are considered standard could potentially lessen their opportunities for adoption. ‘‘Although a result indicating that the child does not carry the HD gene may facilitate an adoption (albeit one in which the adoptive parents have

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demonstrated less than wholehearted acceptance), a positive result may consign a child to permanent foster care’’ (page 11) (4).

TESTING AN ADULT CHILD WHEN THE AT-RISK PARENT REFUSES TESTING Adam, age 24, requested predictive genetic testing for Huntington disease. His maternal grandmother, two maternal aunts, a maternal uncle, and two cousins have all been diagnosed with Huntington disease. Adam’s 45-year-old mother has no symptoms and reportedly does not want to be tested. Newly married, Adam wants to know whether he is at risk for Huntington disease for family planning and career decisions. Can Adam proceed with genetic testing? If Adam tests positive for the Huntington disease gene mutation, then his mother’s positive status would also be known, even though she does not want her status ascertained. In essence, proceeding with the adult-child’s request to be tested could be viewed as coercing the at-risk parent to be tested. Given the laws of patient conﬁdentiality, the genetic counselor or physician cannot just contact Adam’s mother about his request to be tested. Even if Adam contacted his mother and she granted permission to be contacted, she may remain steadfast in not wanting her carrier status for Huntington disease determined. Whose rights then take precedence—the adult-child’s right to know or the at-risk parent’s right not to know? The predictive testing guidelines (Table 2) support proceeding with the adult

Table 2 Pertinent Guidelines Regarding Testing an Adult Child at 25% Risk for Huntington Disease Recommendation

Comment

2.4 ‘‘This issue will arise when a child at 25% risk 2.4 ‘‘Extreme care should requests testing with full knowledge that his or her be exercised when testing parent does not want to know his or her own status. would provide Every effort should be made by the counselors and information about the individuals concerned to arrive at a satisfactory another person who has resolution of this conﬂict. A considerable majority not requested the test.’’ of representatives from lay organizations feel that if no consensus can be reached, the right of the adult child to know should have priority over the right of the parent not to know.’’ International Huntington’s Association and the World Federation of Neurology Research Group on Huntington’s Chorea (1994): Guidelines for the Molecular Genetics Predictive Test in Huntington’s Disease, pages 1533–1534. Source: From Ref. 5.

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child’s request. What is unclear is how much effort should or can be extended by the health care professional ‘‘to arrive at a satisfactory resolution of this conﬂict’’ (pages 1533–1534) (5). The approach our clinic has taken has been to discuss all of these testing implications with the patient, to encourage the patient to discuss the desire for testing with the at-risk parent, to provide the patient with contact information for genetics clinics and supportive resources for the at-risk parent. We also document our counseling in a detailed letter, which is sent to the patient and can be shared with family members. Some patients are unaware of the implications and will subsequently decide to approach their at-risk parent and communicate their desire for testing. Other patients will either refuse to discuss testing with their at-risk parent or will be unsuccessful in their attempts (14,15). In some of our cases and other studies (14,15), when the child has subsequently discussed these issues with the at-risk parent, the at-risk parent has decided to proceed with genetic testing. The rights and obligations of the parent and at-risk child, conﬂicts of interests, and approaches to resolving these ethical issues are discussed in an article by Lindblad (16). Maat-Kievit et al. have proposed guidelines for predictive testing of 25% at-risk applicants, which address different testing strategies and communication issues and emphasize ﬁnding a balance between involvement of the parent with the child’s autonomy in the decision-making process (14).

IS THERE A ‘‘DUTY TO WARN’’ AND SHARE TEST RESULTS WITH OTHER AT-RISK FAMILY MEMBERS? In the case above, Adam proceeds with predictive genetic testing for Huntington disease and his results are positive. Adam indicates that he has no plans to share his test results with his mother or his siblings even though he knows that his mother will have Huntington disease and his siblings are at 50% risk. Are you obligated to share this information with Adam’s mother and his siblings? It is not unusual for a physician to care for multiple family members. When a physician is caring for a family member and knows health status information that has signiﬁcant implications for other family members who are also under their care or seen in their practice, how should this be handled? Regardless of whether or not other family members are under the physician’s care, is there an obligation to contact them when the patient has been diagnosed or had a positive predictive genetic test result for Huntington disease? According to the American Society of Human Genetics (ASHG) Statement on Professional Disclosure of Familial Genetic Information (17): ‘‘Genetic information, like all medical information, should be protected by the legal and ethical principle of conﬁdentiality’’ (page 474) (17).

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The ASHG statement provides stipulations for the professional disclosure of genetic information: ‘‘Disclosure should be permissible where: 1. Attempts to encourage disclosure on the part of the patient have failed. 2. The harm is highly likely to occur and is serious, imminent, and foreseeable. 3. The at-risk relative(s) is identiﬁable. 4. The disease is preventable, treatable or medically accepted standards indicate that early monitoring will reduce the genetic risk.’’ (page 474) (17). While in cases like the one above, the at-risk relatives are identiﬁable and attempts to encourage disclosure have failed, Huntington disease does not meet the other criteria for professional disclosure of genetic information. Huntington disease is highly likely to occur in individuals who inherit the gene mutation; however, the onset of symptoms is gradual, and currently there are no preventative measures or treatment that will reduce the risk. ‘‘At a minimum, the health care professional should be obliged to inform the patient of the implications of his/her genetic test results and potential risks to family members’’ (page 474) (17). The health care professional should document in the medical record and send a letter to the patient about the familial implications of these genetic test results and the fact that the patient was encouraged to share this information with relatives. Hakimian further examines the ethical and legal issues faced by physicians in disclosing or withholding information about Huntington disease from known but unknowing family members (18). The consideration of whether there is a duty to warn will become more of an issue when there are preventative measures or treatment for neurogenetic conditions. Even today, this might pose an issue for a physician caring for a patient with myotonic dystrophy. Individuals with myotonic dystrophy can have cardiac conduction defects and cardiomyopathy which can result in early mortality. In addition, there is also an increased risk for congenital myotonic dystrophy, with signiﬁcant morbidity and mortality, when this condition is inherited from a female parent. The duty to warn would need to be carefully considered if there were identiﬁable relatives who would signiﬁcantly beneﬁt from cardiac surveillance or knowing their reproductive risks. However, it should be emphasized that the duty to warn remains a challenging, controversial issue, and one that needs to be carefully assessed and decided. The issue of nondisclosure of test results to an identiﬁable at-risk relative also applies in the identical twin case discussed below. TESTING IDENTICAL TWINS Eric, age 25 and newly married, requested predictive genetic testing for Huntington disease. His father and several paternal relatives had Huntington

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disease. Eric has an identical twin, Daniel, who lives in a different state. Eric reported that he talked with Daniel and Daniel had no interest in ﬁnding out whether he was at risk for Huntington disease. In fact, he was angry at Eric for wanting to be tested. Should you proceed with Eric’s testing request? In such cases, it is important to inquire how it was determined that they are identical twins since twins can look alike and not be identical. There are no speciﬁc guidelines regarding predictive testing of identical twins. Heimler and Zanko published a report of a case where a twin requested predictive genetic testing for Huntington disease (19). The approach taken by Heimler and Zanko was to ﬁrst determine zygosity of the twins. Once monozygosity was established, arrangements for genetic counseling were made for the second twin with the understanding that both twins would have to make a voluntary decision and be in agreement about proceeding with genetic testing. The twins underwent predictive genetic testing simultaneously and great effort was extended by the genetic counselors to time appointments and results disclosure, a logistical challenge given that one twin resided on the East Coast and the other on the West Coast. Other genetic counselors challenged the approach of Heimler and Zanko and would have allowed the twin to be tested, even if the co-twin was not in agreement (20,21). The guidelines make it clear that an individual should independently make the decision to be tested and not be coerced. In essence, proceeding with an identical twin’s request to be tested could be viewed as coercing the other twin to be tested. Similar to cases where an adult child at 25% risk wants to be tested, there is the issue of a patient requesting testing where information could be learned about a family member who does not want this information known. By extension of the guidelines for individuals at 25% risk, this means that the identical twin should be made aware of the implications of the results for the co-twin. Efforts should be extended to try to resolve the conﬂict, but ultimately the right to know would take precedence over the co-twin’s desire not to learn the carrier status. This has been the approach taken by our clinic. We also provide the patient with contact information for genetics clinics and supportive resources for the twin and document our counseling in a detailed letter to the patient. NON-DISCLOSURE OF TEST RESULTS Paul, age 39, underwent predictive genetic testing for Huntington disease. Hours before his clinic appointment to receive his test results, he called to cancel, indicating that he was not sure when or if he would reschedule. In preparation for his clinic visit, the genetic counselor had opened his test results and knew that he had tested negative. Should she share this information with Paul?

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The guidelines make it clear that results should only be disclosed in person and that the tested patient has the right to decide not to receive these results: 1. ‘‘The participant has the right to decide, prior to the date ﬁxed for the delivery of the results, that these results shall not be revealed to him or her’’ (Recommendation 8.4, page 1536) (5). 2. ‘‘The results of the test should be revealed in person by the counselor to the individual and his or her companion. No result should ever be revealed by telephone or by mail. The counselor must have time to discuss any questions with the individual’’ (Recommendation 8.5, page 1536) (5). Even patients who receive the ‘‘good news’’ that they are negative for Huntington disease can experience adverse reactions to the results, potentially having misgivings about life decisions made based on thinking they would be affected or experiencing ‘‘survivor’’ guilt from having escaped the fate of other affected family members (22,23). What if the test results are positive for Huntington disease—is there an obligation to make sure the patient knows the results? Communicating test results when the patient has indicated the desire not to know the results would be contrary to the guidelines. Result disclosure would likewise not be supported by the ASHG Statement on Professional Disclosure of Familial Genetic Information (17) since there is no imminent harm, preventative measures, or treatment. TESTING SIBLINGS SIMULTANEOUSLY Two sisters, Lisa and Anne, called and asked to be seen together for predictive genetic testing for Huntington disease. Both in their mid 30s and married, they had watched their affected father’s symptoms progress to the point where he now resided in a nursing home and no longer recognized them. The sisters wanted to be tested together, without their husbands’ involvement. They felt strongly that this was an experience that they shared with each other, one which only they could fully understand. Should their request for simultaneous testing be honored? It is recommended in the guidelines that a support person accompany the patient throughout the testing process but it is stipulated that ‘‘It may not be appropriate for the companion to be another at-risk individual’’ (Comment 3, page 1534) (5). The concern is whether an at-risk individual would be able to fully provide support given the shared risk for the same condition. If a sibling or at-risk family member is serving as a support person, learning their relative’s results could have a signiﬁcant emotional impact. For siblings who request testing together, there is the added concern that siblings could receive discordant results. When we have to deliver discordant results, our approach is to disclose the negative result ﬁrst and then the positive result. Delivering test results simultaneously to siblings and

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providing appropriate counseling and support is challenging. At our clinic, we feel that siblings should be apprised of these issues but ultimately, the patient’s decision to be tested, either alone or with a speciﬁc support person present, related or unrelated, should be respected. TESTING INDIVIDUALS WHO WORK IN PROFESSIONS WHERE NEUROLOGICAL DEFICITS CAN JEOPARDIZE THE SAFETY OF OTHERS David, age 45, had a father and several paternal relatives with Huntington disease. His 43-year-old sister was recently diagnosed with Huntington disease. David is an airplane pilot and is asymptomatic. He proceeds with predictive genetic testing and his results are positive for Huntington disease. For patients who are airplane pilots, truck drivers, construction workers, heavy equipment operators, and work in other similar professions, receiving a positive test result will have signiﬁcant implications for the safe performance of their jobs. Close neurological follow-up is paramount and the frequency of evaluations should be determined by the neurologist. Patients should be informed that as long as they are asymptomatic, they can continue in their line of work but should be thinking of alternatives for their job responsibilities in anticipation that they will eventually become symptomatic.

TESTING SYMPTOMATIC INDIVIDUALS WHO THINK THEY ARE ASYMPTOMATIC Nancy, age 46, was interested in genetic testing because of her family history of Huntington disease. During the clinic visit, the genetic counselor noted that Nancy had involuntary movements. When she asked Nancy directly whether she had any symptoms of Huntington disease, she denied having symptoms. In a 2002 study, Goizet et al. found that of patients who requested predictive genetic testing, approximately 10% had subtle neurological or behavioral symptoms of Huntington disease (24). Of note, this study also found that a number of patients who had subtle symptoms actually tested negative for Huntington disease. This emphasizes the importance of being conservative in assessing patients and cautious in interpreting and communicating ﬁndings. In the guidelines it states that ‘‘Particular care should be taken with participants who are believed to be showing early symptoms of HD; however, individuals with established, unacknowledged symptoms should not automatically be excluded from the test and should receive additional counseling’’ (comment 5.2.5, page 1535) (5). A neurology evaluation is recommended in the guidelines for predictive testing for Huntington

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disease, although a patient can be tested without this evaluation. If a patient is symptomatic but not aware of symptoms, it is important to inform the patient that a diagnosis could potentially be made with the neurology exam so that there is advance preparation for this possibility. In addition, it is important for the patient to understand that a diagnosis will have signiﬁcant implications for other family members whose risk could be as high as 50%. A patient who undergoes diagnostic testing for Huntington disease is not required to meet with a therapist. Therefore, if symptomatic patients request predictive testing, thinking that they are asymptomatic, the requirement to meet with a therapist as part of the predictive testing protocol could be beneﬁcial and a helpful resource as they later adjust to this diagnosis. TESTING ‘‘ASYMPTOMATIC’’ INDIVIDUALS WHO HAVE RECENTLY ATTEMPTED SUICIDE The call came from the inpatient psychiatric unit. The 38-year-old female patient had a strong family history of Huntington disease and was just hospitalized following a suicide attempt with an overdose of medications. Could we see the patient and order genetic testing? Individuals with Huntington disease have an increased risk for suicide. Psychiatric symptoms can precede or occur simultaneously with neurological symptoms of this disease. The suicide attempt could in fact be indicative that the patient has symptoms of Huntington disease. Alternatively, the patient could have underlying depression or signiﬁcant anxiety from being at risk that resulted in the suicide attempt. In cases like the one above, the line becomes blurred between genetic testing for Huntington disease being diagnostic or predictive. A neurology consult can be helpful in determining whether the patient additionally has any neurological symptoms consistent with Huntington disease, which would increase suspicions that the suicide attempt is a manifestation of the disease. Testing should be performed at a time of low stress (4) and therefore, testing at the time of a suicide attempt is not optimal. A history of depression or suicide attempts, however, is not a contraindication for testing. It is stipulated in the guidelines that ‘‘For applicants with evidence of a serious psychiatric condition, it may be advisable that testing be delayed and support services put into place’’ (Recommendation 2.5, page 1534) (5). It is important to ensure that the patient is not being coerced into testing, is in a position to give informed consent, and understands the signiﬁcance and implications of the test results. The bottom line is that the patient’s suicide attempt needs to be medically addressed and that knowing whether or not the patient has Huntington disease will not signiﬁcantly impact this treatment. The results of genetic testing often are not available for two to three weeks. The patient may be released from the psychiatric unit well before genetic test results are known, so it is important to make sure

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that the patient has appropriate ongoing psychiatric support in place and that there are plans to disclose the Huntington disease test results in person. TESTING A BLOOD SAMPLE FROM SOMEONE WHO HAS JUST COMMITTED SUICIDE The caller was using her cell phone from a funeral home. Her 43-year-old husband had just committed suicide. His father had died of Huntington disease. A couple months prior to his death, her husband had become abusive and erratic in his behavior. The woman wanted answers: Did he have Huntington disease and was her daughter now at risk? The legal next-of-kin has the right to make such testing decisions. This can become challenging when family members differ strongly about proceeding with testing and no consensus can be reached. Sometimes, it is not technically possible to test a sample from a recently deceased individual if the sample was obtained in the wrong type of tube, has not been stored at the proper temperature, or kept too long before processing. The Huntington disease genetic test is highly sensitive so even if an affected individual is not tested, testing still remains an option for at-risk relatives. When positive test results for Huntington disease are obtained for an individual who has committed suicide, it can be of beneﬁt to family members’ grief process and help bring some closure by providing an explanation for the suicide. In addition, test results can provide useful information for determining whether relatives are at increased risk for Huntington disease. One can imagine similar test requests from family members of individuals with other neurogenetic conditions. If testing is not available, DNA banking can be offered; labs offering this service can be accessed at GeneTests (www.genetests.org). TESTING A PREGNANCY Sara, age 38, recently underwent predictive genetic testing for Huntington disease and received positive test results. Now 10 weeks pregnant, she wished to have her pregnancy tested. Sara knew that the pregnancy was at 50% risk for Huntington disease and wanted the pregnancy tested but was unsure what she would do if the results were positive. It is controversial whether prenatal testing should be offered for adult-onset genetic conditions. It raises the issue of the value of a life given that an individual can often live 30þ years before the onset of symptoms of Huntington disease and approximately 25% of individuals ﬁrst become symptomatic after age 50 (25). Even if a child is prenatally diagnosed today as having the HD gene mutation, it is anticipated that there may be more effective treatment options and possibly a cure before that child becomes symptomatic. Given the neurological deterioration that occurs in affected individuals and the familial impact of this disease, individuals may feel strongly about not wanting to

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Table 3 Pertinent Guidelines Regarding Prenatal Testing for Huntington Disease Recommendation 7.1 ‘‘It is essential that antenatal testing for the HD mutation be performed only if the parent has already been tested.’’

Comment 7.1 ‘‘It is highly desirable that both parents agree to an antenatal test. If there is a conﬂict, every effort should be made by the counselors and the couple to reach an agreement. Exceptional circumstances (e.g. rape or incest) may justify deviating from this recommendation.’’

7.2 ‘‘The couple requesting antenatal testing must be clearly informed that if they intend to complete the pregnancy if the fetus is a carrier of the gene defect, there is no valid reason for performing the test. Furthermore, this situation is contrary to recommendation 2.1, since a child thus born with the gene defect cannot elect not to take the test upon reaching majority’’ International Huntington’s Association and the World Federation of Neurology Research Group on Huntington’s Chorea (1994): Guidelines for the Molecular Genetics Predictive Test in Huntington’s Disease, page 1535. Source: From Ref. 5.

have children at risk for Huntington disease. Prenatal testing for Huntington disease is an option though the guidelines make it clear (Table 3) that such testing should only be performed if positive test results will impact the decision to continue the pregnancy; pregnancy continuation with known positive results would be tantamount to testing a minor, which is contrary to the guidelines. For some neurogenetic conditions (e.g., some of the spinocerebellar ataxias, myotonic dystrophy) where there are genotype–phenotype correlations, these prenatal decisions will be even more complex. A parent may wish to terminate a pregnancy when testing indicates a high number of trinucleotide repeats, consistent with the neonatal form, but continue a pregnancy when the repeat number is in the range for adult onset of symptoms. For the continued pregnancies, this means that there would be parental knowledge of the future child’s affected status in adulthood (depending on disease penetrance) which would deprive the child of autonomy in deciding whether to ascertain this information. What about testing a pregnancy when the at-risk parent has not already been tested? Some patients who we have seen have requested to rule out Huntington disease in the pregnancy but do not want to be tested themselves. They understand that they could learn of their positive carrier status

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through testing the pregnancy. Some of these patients have also said that a beneﬁt of learning their positive status this way is that the results would be in the pregnancy records, not in their personal records, which would limit insurers’ access to this information. In these cases and at other centers (26–28), if both parents have consented to testing and understand that positive results could be revealed simultaneously for the pregnancy and the atrisk parent, we have proceeded with their requests. Maat-Kievit et al. also advocate that the guidelines include the option of direct testing of the pregnancy given that it is simpler, faster and has just a 25% chance of revealing the status of the at-risk parent (26,29). What if the father of the pregnancy is at risk and does not want his carrier status determined but his pregnant partner wants the pregnancy, which is at 25% risk, tested? Does the woman’s right to independently make decisions about a pregnancy take priority over the father’s wishes not to have his status determined? Both our group (30,31) and Tassicker et al. (32) feel that efforts should be extended to try to resolve the conﬂict but generally, the at-risk father’s decision not to have his carrier status determined should be respected. GIVING INFORMED CONSENT FOR INDIVIDUALS WITH COGNITIVE/PSYCHIATRIC IMPAIRMENT Brandon, age 45, came to clinic with his wife and 24-year-old daughter to discuss genetic testing for Huntington disease. The genetic counselor noted that Brandon had some involuntary movements, was slow to respond to questions and seemed depressed. She was unsure as to whether his symptoms were due to anxiety and depression or in fact were early symptoms of Huntington disease. Brandon would not allow the neurologist to examine him. Brandon’s wife and daughter provided most of the family and medical history information. Newly engaged, Brandon’s daughter was particularly interested in ﬁnding out whether her father had Huntington disease. Brandon’s wife was concerned about some of the changes she was seeing in her husband and was looking for answers. Brandon was noncommittal when asked about whether he wanted to be tested for Huntington disease. A central issue is whether patients who have cognitive impairment yet still act as their own guardians can actually provide informed consent. The healthcare provider needs to assess whether the patient has the decision-making capacity that includes the abilities to understand, appreciate, communicate a choice, and reason. Recognition that a patient has cognitive impairment will likely be apparent from the health care provider’s interactions with the patient. Cognitive functioning can also be more formally assessed through the Mini-Mental State Examination (MMSE) (33), a standard component of most neurological evaluations, the Aid to Capacity Evaluation (ACE) (34), the MacArthur Competence Assessment Tool—Treatment (MacCAT-T) (35), or use of other similar instruments.

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There are guidelines from the American Medical Association (36,37) and American Psychiatric Association (38) regarding obtaining informed consent when there is impairment of the patient’s decision-making capacity. In these cases, it is important to work with the family, keeping in mind state laws of working with a proxy and who can serve in this role. Even in cases where there is impaired decision-making capacity, one should strive to obtain the assent of the patient and act in accordance with core ethical principles. While one should strive to maintain the autonomy of patients, this needs to be balanced with the fact that patients may no longer have the capacity to make decisions that are in their best interest. If the patient is truly suspected to have symptoms of Huntington disease or other neurogenetic condition, this information may be important for health care and safety issues (e.g., driving), and has implications for other family members. INSURANCE ISSUES Patients requesting predictive testing often have concerns about whether their insurance will cover the costs and what the implications will be for their insurance coverage if results are positive. Patients need to be informed about the costs of testing, evaluations (therapist, neurologist), and clinic fees, which often total several hundred dollars, and that some insurance companies may not cover testing unless it is ordered to make a diagnosis of a symptomatic individual. Prior to proceeding with predictive genetic testing, it is advisable that patients have in place their desired coverage for health, life, long-term disability, and long-term care insurance. It should be emphasized with patients that they need to acknowledge their family history of a neurological condition if this information is requested and that denying this history could be treated as insurance fraud. Some states have laws prohibiting insurance and employment discrimination based on genetic information, and there are bills being considered at a federal level (see Resources section). ANONYMOUS TESTING The man on the phone was hesitant and would not identify himself. His request: ‘‘I want to be tested anonymously for Huntington disease.’’ Should you facilitate this patient having anonymous testing for Huntington disease or other neurogenetic conditions? There are no speciﬁc guidelines regarding anonymous testing for Huntington disease. The limited experience with anonymous testing for Huntington disease has been addressed in articles by Burgess et al. (39) and Visintainer et al. (40). Our policy has been to not offer anonymous testing (41) though others feel this should be an option (42). Given the fact that neurogenetic conditions can have similar symptoms, it is important to conﬁrm the diagnosis in the family prior to proceeding with genetic testing for a speciﬁc condition. Anonymity can be a

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barrier in establishing rapport, providing accurate risk assessments, and appropriate genetic counseling. While most HD testing centers do not offer anonymous genetic testing (40), at our center and others, there have been patients who have had anonymous genetic testing by virtue of the fact that they only provided limited nonidentifying family history information with no medical record documentation of their family history of Huntington disease, used a post-ofﬁce box for all communication, and paid cash for their clinic appointments. LEGAL CASES There have been anecdotal reports of at-risk individuals being forced to have predictive testing for Huntington disease in divorce, child custody, criminal, and other legal cases. Genetic testing of individuals under these circumstances could be viewed as coercion. The guidelines clearly stipulate that ‘‘The individual must choose freely whether to be tested and must not be coerced by family, friends, partners or potential partners, physicians, insurance companies, employers, governments or others’’ (comment 2.1, page 1533) (5). In such cases, it is important to consult with your hospital’s attorney prior to agreeing to see patients involved in legal cases. Given that the results may or may not be in the best interest of the patient, ‘‘testing should only be performed with the consent of the patient and his/her understanding of the likely social and legal results of a positive test’’ (page 258) (43). RESOURCES It is recommended in the guidelines that predictive genetic testing be performed by a team that includes genetics specialists. To locate genetics specialists in your area, the National Society of Genetic Counselors (www.nsgc.org), GeneTests (www.genetests.org), and the American College of Medical Genetics (www.acmg.net) have searchable on-line directories of genetics providers and clinics. To locate support groups for your patients, the Genetic Alliance (www.geneticalliance.org) and National Organization for Rare Disorders (NORD) (www.rarediseases.org) have on-line directories. The National Human Genome Research Institute (www.genome.gov) and the National Conference of State Legislatures (www.ncsl.org/programs/ health/Genetics/charts.htm) have on-line access to information on state and federal laws regarding use of genetic information by insurers and employers. SUMMARY This chapter focused on the ethical issues encountered in predictive genetic testing for Huntington disease, which are also applicable to other adult-onset autosomal dominant neurogenetic conditions for which there currently is no

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cure. Genetics expertise is essential for providing the pretest and post-test counseling, addressing the complex counseling issues, and resolving the challenging ethical dilemmas. These cases demonstrate that the patient may not just be the person seated in front of you and that results may reveal carrier status and have signiﬁcant implications for other family members. Predictive genetic testing for neurogenetic conditions raises several core issues including autonomy, coercion, locus of decision making, weighing the right to know versus the right not to know, and a careful determination of whose rights take precedence when there is a conﬂict. To resolve ethical dilemmas, it is important to use testing guidelines, position statements from professional societies, and take into account basic ethical principles. Sometimes giving patients copies of the testing guidelines and position statements can be helpful in resolving ethical dilemmas by showing them that there are global standards and that their request is not being questioned or denied without basis. Consulting with the hospital’s ethics committee and attorney may also be helpful, particularly for legal cases. Advances in genetics will result in an increase in the number of genetic tests. As effective treatment options become available, some of the ethical issues associated with predictive testing will become less signiﬁcant and easier to resolve.

RECOMMENDED READING Smith DH, Quaid KA, Dworkin RB, Gramelspacher GP, Granbois JA, Vance GH. Early Warning: Cases and Ethical Guidance for Presymptomatic Testing in Genetic Diseases. Bloomington: Indiana University Press, 1998. REFERENCES 1. Huntington’s Disease Society of America. Guidelines for predictive testing for Huntington’s disease. New York, 1989. 2. World Federation of Neurology: Research Committee. Research Group on Huntington’s chorea. Ethical issues policy statement on Huntington’s disease molecular genetics predictive test. J Neurol Sci 1989; 94:327–332. 3. Went L. Ethical issues policy statement on Huntington’s disease molecular genetics predictive test. International Huntington Association. World Federation of Neurology. J Med Genet 1990; 27:34–38. 4. Huntington’s Disease Society of America Inc. Guidelines for genetic testing for Huntington’s disease. New York: Huntington’s Disease Society of America, Inc., 1994. 5. International Huntington Association and World Federation of Neurology Research Group on Huntington’s Chorea. Guidelines for the molecular genetics predictive test in Huntington’s disease. Neurology 1994; 44:1533–1536. 6. International Huntington Association and the World Federation of Neurology Research Group on Huntington’s Chorea. Guidelines for the molecular genetics predictive test in Huntington’s disease. J Med Genet 1994; 31:555–559.

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7. Burson CM, Markey KR. Genetic counseling issues in predictive genetic testing for familial adult-onset neurologic diseases. Semin Pediatr Neurol 2001; 8: 177–186. 8. Hayden MR. Predictive testing for Huntington’s disease: a universal model? Lancet 2003; 2:141–142. 9. Uhlmann WR. A guide to case management. In: Baker DL, Schuette JL, Uhlmann WR, eds. A Guide to Genetic Counseling. New York: Wiley, 1998:199–229. 10. McKinnon WC, Baty BJ, Bennett RL, Magee M, Neufeld-Kaiser WA, Peters KF, Sawyer JC, Schneider KA. Predisposition genetic testing for late-onset disorders in adults: a position paper of the National Society of Genetic Counselors. JAMA 1997; 278:1217–1220. 11. American Society of Human Genetics, American College of Medical Genetics. Points to consider: Ethical, legal, and psychosocial implications of genetic testing in children and adolescents. Am J Hum Genet 1995; 57:1233–1241. http:// www.acmg.net/resources/policies/pol-018.asp; http://www.ashg.org/genetics/ ashg/policy/pol-13.htm. 12. American Society of Human Genetics Social Issues Committee, American College of Medical Genetics Social, Ethical and Legal Issues Committee. ASHG/ACMG statement: Genetic testing in adoption. Am J Hum Genet 2000; 66:761–767. http://www.acmg.net/resources/policies/pol-017.asp;http://www.ashg.org/genetics/ashg/policy/pol-36.htm. 13. National Society of Genetic Counselors, Inc. Genetic testing and adoption position statement. 2002. http://www.nsgc.org/about/position.asp#testandadoption. 14. Maat-Kievit A, Vegter-van der Vlis M, Zoeteweij M, Losekoot M, van Haeringen A, Roos RAC. Predictive testing of 25 percent at-risk individuals for Huntington disease (1987–1997). Am J Med Genet (Neuropsych Genet) 1999; 88:662–668. 15. Benjamin CM, Lashwood A. United Kingdom experience with presymptomatic testing of individuals at 25% risk for Huntington’s disease. Clin Genet 2000; 58: 41–49. 16. Lindblad AN. To test or not to test: an ethical conﬂict with presymptomatic testing of individuals at 25% risk for Huntington’s disorder. Clin Genet 2001; 60:442–446. 17. American Society of Human Genetics Social Issues Committee on Familial Disclosure. Professional disclosure of familial genetic information. Am J Hum Genet 1998; 62:474–483. http://www.ashg.org/genetics/ashg/policy/pol-29.htm. 18. Hakimian R. Disclosure of Huntington’s disease to family members: the dilemma of known but unknowing parties. Genet Test 2000; 4:359–364. 19. Heimler A, Zanko A. Huntington disease: a case study describing the complexities and nuances of predictive testing of monozygotic twins. J Genet Counsel 1995; 4:125–137. 20. Hodge SE. Paternalistic and protective? Genet Counsel 1995; 4:351–352. 21. Reich E. Testing for HD in twins. J Genet Counsel 1996; 5:47–49. 22. Huggins M, Block M, Wiggins S, Adams S, Sucherowsky O, Trew M, Klimek ML, Greenberg CR, Eleff M, Thompson LP, Knight J, MacLeod P, Girard K, Theilmann J, Hayden MR. Predictive testing for Huntington disease in Canada: adverse effects and unexpected results in those receiving decreased risk. Am J Hum Genet 1992; 42:509–515.

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23. Meiser B, Dunn S. Psychological impact of genetic testing for Huntington disease: an update of the literature. J Neurol Neurosurg Psychiatry 2000; 69: 574–578. 24. Goizet C, Lesca G, Durr A, French Group for Presymptomatic Testing in Neurogenetic Disorders. Presymptomatic testing in Huntington’s disease and autosomal dominant cerebellar ataxias. Neurology 2002; 59:1330–1336. 25. Myers RH, Sax DS, Schoenfeld M, Bird ED, Wolf PA, Vonsattel JP, White RF, Martin JB. Late onset of Huntington’s disease. J Neurol Neurosurg Psychiatry 1985; 48:530–534. 26. Maat-Kievit A, Vegter-van der Vlis M, Zoeteweij M, Losekoot M, van Haeringen A, Roos R. Paradox of a better test for Huntington’s disease. J Neurol Neurosurg Psychiatry 2000; 69:579–583. 27. Simpson SA, Harper PS, United Kingdom Huntington’s Disease Prediction Consortium. Prenatal testing for Huntington’s disease: experience within the UK 1994–1998. J Med Genet 2001; 38:333–335. 28. Simpson SA, Zoeteweij MW, Nys K, Harper P, Durr A, Jacopini G, Yapijakis C, Evers-Kiebooms G. Prenatal testing for Huntington’s disease: a European collaborative study. Eur J Hum Genet 2002; 10:689–693. 29. Maat-Kievit A, Vegter-van der Vlis M, Zoeteweij M, Losekoot M, van Haeringen A, Kanhai H, Roos R. Experience in prenatal testing for Huntington’s disease in the Netherlands: procedures, results and guidelines (1987–1997). Prenat Diagn 1999; 19:450–457. 30. Uhlmann W, Markel D, Petty E. Prenatal testing for adult-onset conditions: A Huntington disease testing dilemma [abstr]. J Genet Counsel 1996; 5:229–230. 31. Uhlmann WR, Markel DS, Goldman EB, Petty EM. Prenatal testing for Huntington disease: Whose decision is it? Report of a testing dilemma and analysis of ethical and legal issues. Submitted for publication. 32. Tassicker R, Savulescu J, Skene L, Marshall P, Fitzgerald L, Delatycki MB. Prenatal diagnosis requests for Huntington’s disease when the father is at risk and does not want to know his genetic status: clinical, legal and ethical viewpoints. BMJ 2003; 326:331–333. 33. Mini-Mental State Examination (MMSE) http://www.minimental.com. 34. Aid to Capacity Evaluation. http://www.utoronto.ca/jcb/disclaimers/ace_main. htm. 35. MacArthur Competence Assessment Tool—Treatment (MacCAT-T) http:// www.prpress.com/books/mactfr.html. 36. American Medical Association. H-140.989 Informed consent and decisionmaking in health care. http://www.amaassn.org/apps/pf_new/pf_online?f_n¼ resultLink&doc¼policyﬁles/HnE/H140.989.HTM&s_t¼%22H140.989%22& catg¼AMA/HnE&catg¼AMA/BnGnC&catg¼AMA/DIR&&nth¼1&&st_p¼ 0&nth¼1&. 37. American Medical Association. E-8.081 Surrogate decision making. 2001. http://www.ama-assn.org/ama/pub/category/8489.html. 38. American Psychiatric Association. Principles of informed consent in psychiatry resource document. APA Document Reference No. 960001: 1996. http://www. psych.org/edu/other_res/lib_archives/archives/199601.pdf.

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39. Burgess MM, Adam S, Bloch M, Hayden MR. Dilemmas of anonymous predictive testing for Huntington disease: privacy vs. optimal care. Am J Med Genet 1997; 71:197–201. 40. Visintainer CL, Matthias-Hagen V, Nance MA, US Huntington Disease Genetic Testing Group. Anonymous predictive testing for Huntington’s disease in the United States. Genet Test 2001; 5:213–218. 41. Uhlmann WR, Ginsburg D, Gelehrter TD, Nicholson J, Petty EM. Questioning the need for anonymous genetic counseling and testing. Am J Hum Genet 1996; 59:968–970. 42. Mehlman MJ, Kodish ED, Whitehouse P, Zinn AB, Sollitto S, Berger J, Chiao EJ, Dosick MS, Cassidy SB. The need for anonymous genetic counseling and testing. Am J Hum Genet 1996; 58:393–397. 43. Bird TD. Risks and beneﬁts of DNA testing for neurogenetic disorders. Semin Neurol 1999; 19:253–259.

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6 Autosomal Dominant Charcot-Marie-Tooth Disease and Related Disorders Craig L. Bennett Division of Genetics and Developmental Medicine, Department of Pediatrics, University of Washington School of Medicine, Seattle, Washington, U.S.A.

SCOPE OF THIS CHAPTER At present, over 20 genes have been identiﬁed that through mutation cause various forms of Charcot-Marie-tooth (CMT) disease. The major division of CMT into type 1 (CMT1) and type 2 (CMT2) has been accepted for many years now, and this division still seems to be helpful in delineating the various types of CMT despite the ever increasing identiﬁcation of CMT genes and genetic loci. In this chapter, I will focus predominantly on the autosomal dominant (AD) forms of CMT1 (demyelinating) and CMT2 (axonal). The autosomal recessive (AR) forms of CMT are less common and will not be reviewed in this chapter for space constraints (discussed in Ref. 1). I will describe the basic clinical and genetic features of the major forms of AD CMT1 and CMT2, along with a discussion of the possible underlying pathogenetic mechanism for each CMT form, if it is known. CMT: BACKGROUND AND GENERAL CLINICAL FEATURES CMT neuropathy or hereditary motor and sensory neuropathy (HMSN) is a group of heterogeneous inherited diseases of peripheral nerves (2–4). CMT

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is a common disorder affecting both children and adults, causing signiﬁcant neuromuscular impairment. With an estimated 1 in 2500 persons affected, CMT is a major contributing category within all neurogenetic diseases (5). CMT contributes to signiﬁcant morbidity, and in one or two rare forms, CMT also contributes to mortality. A major diagnostic criterion of CMT is that both motor and sensory nerve functions are affected, which leads to clinical features that include distal muscle weakness and atrophy, impaired sensation, and absent or hypoactive deep tendon reﬂexes. The onset of CMT is typically within the ﬁrst or second decade of life, although it may be detected by neurophysiological methods in infancy. The spectrum of CMT clinical presentation is wide, ranging from individuals with clinical ﬁndings of pes cavus with minimal or no distal muscle weakness to patients with severe distal atrophy and marked hand and foot deformity. CMT patients most commonly present with signs and symptoms of muscle loss and weakness of the lower legs and feet. Later the disorder progresses to affect the hands and forearms. For many patients, a history of abnormal gait with frequent tripping is reported. Patients may also present with complaints related to foot deformities resulting from loss of intrinsic muscles (pes cavus). Less often a patient’s initial complaint may be a sensory-related problem. Patients will frequently beneﬁt from physical therapy where indicated and ankle-foot orthotics (AFOs) are often used to alleviate foot drop in some patients. Surgery should only be considered when pain or difﬁcult walking results from severe foot deformity, or cannot be managed by more conservative (less invasive) means. Recent data suggest that foot deformities need to be aggressively managed, particularly when the disease manifests early in adolescence. Prevention of deformities before they become ﬁxed can make it possible for a plantigrade foot that is capable of bearing normal body loads to ﬁt better into shoes, allowing for more comfortable ambulation which prevents future problems (6).

ELECTROPHYSIOLOGICAL CLASSIFICATION OF CMT The current CMT classiﬁcation system divides the disorder into two broad categories. The ﬁrst is demyelinating CMT (CMT type 1 or ‘‘CMT1’’) that likely results from Schwann cell (SC) dysfunction, leading to loss of peripheral nervous system (PNS) myelin. The second category are those forms of CMT resulting from putative axonal degeneration (axonal CMT, CMT type 2 or ‘‘CMT2’’) (7). Individuals with CMT1 show hypertrophic demyelinating neuropathy (‘‘onion bulbs’’) on pathological examination. Patients have reduced motor and sensory nerve conduction velocities (NCVs) (<38–40 m/sec) and prolonged distal latencies. The reduction of NCVs seen

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in CMT1 are uniform and symmetric, a feature that contrasts with the acquired demyelinating neuropathies. In contrast, CMT2 patients possess a relatively preserved myelin sheath and generate normal or near normal NCVs. More diagnostic of axonal CMT2 is that patients show very low motor compound muscle action potentials (CMAPs) and sensory nerve action potentials (SNAPs) that are either reduced in amplitude or absent. Other subdivisions of CMT classiﬁcation include the more severe forms of CMT such as Dejerine-Sottas disease and congenital hypomyelination. CMT4 is used to designate AR forms of demyelinating CMT. There is also the relatively new category of dominant intermediate CMT (DI-CMT) with features of both demyelinating and axonal CMT. Mutation of the Dynamin 2 gene (DNM2) has recently been identiﬁed as the molecular basis of DI-CMTB (8). The mechanism by which mutations in the DMN2 gene, which encodes a protein of the large GTPase family, lead to an intermediate form of CMT is not known. The various chromosomal locations and genetic mechanisms underlying the various forms of CMT and related disorders are given in Table 1.

MAJOR FORMS OF DEMYELINATING CMT1 Genetics of CMT1 The mode of inheritance in CMT1 is AD. The majority of CMT1 pedigrees are linked to chromosome 17p11.2-12 and are designated as CMT1A (9). Genetic linkage studies have indicated that some CMT1 pedigrees demonstrate linkage to the long arm of chromosome 1 and have been designated CMT1B (10). Rare AD CMT1 pedigrees that map to chromosome 16p13 have been designated CMT1C (11). Additional pedigrees have been identiﬁed that link to chromosome 10q21-q22 and have been assigned the designation CMT1D (12). The X-linked form of demyelinating CMT1 maps to chromosome Xq13.1 and is now termed CMT1X (13). The causal genes for each form of CMT1 will be described in detail further. CMT1: Etiology and Pathogenesis Much of what is known of the etiology and pathogenesis of CMT1 comes from early studies on CMT1A or CMT1B. More recently, the genes that cause other forms of CMT1 have been discovered. This chapter will focus on the genes that through mutation cause the major forms of AD CMT1 and CMT2. Particular attention will be given to those genes that have yielded new understanding of pathogenetic mechanisms leading to disease, and to the genes that account for a high percentage of total CMT disease.

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Table 1 Charcot-Marie-Tooth Genes, Genetic Loci and Mode of Inheritance Inheritance

Locus

Gene

Mechanism

17p11-p12

PMP22

AD AD AD AD

1q22-q23 16p12-p13 10q21-q22 1q22-q23

MPZ SIMPLE EGR2 MPZ

Duplication/ Point mutation Point mutation Point mutation Point mutation Point mutation

AD X-linked

8p21 Xq13

NF-L Cx32

Point mutation Point mutation

17p11-p12 1q22-q23 10q21-q22 8q23-q24 19q13

PMP22 MPZ EGR2 ? PRX

Point Point Point ? Point

17p11-p12 1q22-q23 10q21-q22

PMP22 MPZ EGR2

Point mutation Point mutation Point mutation

Hereditary neuropathy with pressure palsies HNPP A AD 17p11-p12

PMP22

Deletion/ point mutation

1p35-p36 1p35-p36 3q13-q22

KIF1Bb MFN2 Rab7

Point mutation Point mutation Point mutation

12q23-q24

?

?

7p14 8p21 1q22-q23 Xq13 3q13

GARS NF-L MPZ Cx32 ?

Point Point Point Point ?

Xq24-q26 Xq26

? ?

? ?

Charcot-Marie-Tooth Type 1 CMT1A AD CMT1B CMT1C CMT1D CMT1E (deafness) CMT1F CMT1E Dejerine-Sottas (HMSNIII) DSDA DSD B DSD C DSD D DSD E

disease AD(AR) AD(AR) AD AD AR

Congenital hypomyelination (CMT4E) CHN A AD CHN B AD CHN C AD (AR)

Charcot-Marie-Tooth Type 2 (HMSNII) CMT2A AD CMT2A AD CMT2B AD (ulcers) CMT2C (vocal AD cord) CMT2D AD CMT2E AD (CMT1B) AD (CMTX) X-linked CMT2G AD (HMSNP) CMT2XA X-linked CMT2XB X-linked

mutation mutation mutation mutation

mutation mutation mutation mutation

(Continued)

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Table 1 Charcot-Marie-Tooth Genes, Genetic Loci and Mode of Inheritance (Continued ) Inheritance

Gene

Mechanism

1q21

LMNA ?

Point mutation ?

8q13-q21 11q22 11p15 ? 5q23-q33 8q24

GDAP1 MTMR2 SBF2 ? KIAA1985 NDRG1

Point Point Point ? Point Point

19q13

PRX

Point mutation

Charcot-Marie-Tooth ‘‘dominant intermediate’’ DI-CMTA AD 10q24 DI-CMTB AD 19p12

? DNM2

DI-CMTC

?

? Deletion/ point mutation ?

CMT2 AR A CMT2 AR B (CMT4C)

AR AR

Charcot-Marie-Tooth Type 4 CMT4A AR CMT4B1 AR CMT4B2 AR CMT4B3 AR CMT1 AR C AR CMT1 AR D AR (HMSNLom) CMT1 AR F AR (CMT4F)

AD

Locus

1p34

mutation mutation mutation mutation mutation

Abbreviations: AD, autosomal dominant; AR, autosomal recessive.

CMT1A: The Peripheral Myelin Protein-22 Gene CMT1A is associated with a 1.5 megabase (Mb) tandem DNA duplication on chromosome 17p11.2-p12 (14,15), and has been detected in many ethnic groups (16). The duplication in most pedigrees is inherited as a stable Mendelian trait; however, in some instances, it may arise as a de novo event. Features consistent with CMT1A have also been detected in patients with either partial or complete trisomy 17p, providing further support for the hypothesis that the duplication in CMT1A results from a gene dosage mechanism (17,18). Further understanding of the disease mechanism in CMT1A was provided by studies of murine disease models. The trembler (Tr) mouse suffers a dominantly inherited hypomyelinating neuropathy and has subsequently been found to represent a murine model for CMT1A (19). The gene for the Tr locus was identiﬁed when a point mutation was found in the peripheral myelin protein-22 (PMP22) gene (20). These ﬁndings in the Tr mouse suggested that the human PMP22 gene might map within the CMT1A gene duplication region, which was subsequently proven (21). While rarer in comparison to the duplication mutation, point mutations of the PMP22 gene in CMT1A patients were subsequently identiﬁed (22,23).

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The PMP22 gene encodes a membrane-associated protein of 18 kD that is increased to 22 kD by glycosylation. PMP22 localizes to the compact portions of peripheral nerve myelin (Fig. 1, reproduced from Ref. 24), and contains four putative transmembrane domains (25–27). Chance et al. suggested that the neuropathy in patients with the 17p11.2–12 duplication may result from the additional copy of PMP22 gene leading to a 50% increase in expression at this locus (17). This was subsequently conﬁrmed by examining patients’ sural nerve biopsy for PMP22 message and protein levels (28–30). It should be noted that the meiotic events that lead to the duplication in CMT1A can also cause the reciprocal chromosome deletion. When this occurs, a 50% reduction in the copy number of the PMP22 gene results. Patients receiving the deletion chromosome suffer a disorder known as hereditary neuropathy with liability to pressure palsies (HNPP), a disorder characterized by painless episodic, recurrent, and focal demyelinating nerve palsies (31). The fact that duplication or deletion of PMP22 gene can lead to either CMT1A or HNPP highlights the dosage sensitivity of PMP22. A greater level of effort has gone into understanding the disease pathology of CMT1A and the biology of the PMP22 protein than for any other CMT gene to date. PMP22 proteins carrying missense mutations associated with CMT1A are retained intracellularly (32–35) and appear to belong to a growing class of mutations termed ‘‘endoplasmic reticulum (ER) retention mutations’’ (36). These are mutations that are recognized by ER-resident-folding proteins, molecular chaperones, and/or other enzymes that serve to monitor the ﬁdelity of protein synthesis and macromolecular assembly (37,38). In wild-type sciatic nerves, PMP22 associates in a speciﬁc, transient (t1/2 11 min), and oligosaccharide processing-dependent manner with the lectin chaperone calnexin (CNX). In sciatic nerves of Trembler-J mice (point mutation), prolonged association of mutant PMP22 with CNX is found (t1/2 > 60 min) (36). Regulation of myelination not only requires timely synthesis of sufﬁcient quantities of myelin constituents, but also their coordinated trafﬁcking and assembly. In SC, 80% of newly synthesized PMP22 is known to be rapidly degraded (39). This likely serves as a major burden on the cellular machinery responsible for clearing misfolded proteins and, if this balance is perturbed due to overexpression of PMP22, CMT1A results (40). This phenomenon of misfolded protein is also seen with the cystic ﬁbrosis membrane protein CTFR, where up to 75% of newly synthesized protein is rapidly degraded (41). Trembler mice, in which one PMP22 allele is mutated (G150D), are much more severely affected than PMP22þ/ heterozygous mice carrying a single PMP22 allele (42). They have less myelin than PMP22þ/ animals, and the steady state levels of their myelin-speciﬁc mRNAs are dramatically reduced (43). It has been suggested that PMP22 protein interactions with chaperone proteins within the ER may account for the observed decrease

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Figure 1 The organization of a myelinated axon. Panel (A) depicts an ‘‘unrolled’’ myelinating Schwann cell, revealing the regions that form compact and non-compact myelin. Tight junctions are depicted as two continuous (faint grey) lines; these form a circumferential belt and are also found in incisures. Gap junctions are depicted as grey ovals; these are found between the rows of tight junctions, and are more numerous in the inner aspects of incisures and paranodes. Adherens junctions are depicted as dark ovals; these are more numerous in the outer aspects of incisures and paranodes. The nodal, paranodal, and juxtaparanodal regions of the axonal membrane are all indicated by labeled arrows, respectively. Panel (B) depicts the proteins of compact and non-compact myelin. Compact myelin contains P0, PMP22, and MBP; non-compact myelin contains E-cadherin, MAG, DM20, Cx32, and an unknown claudin. Source: Adapted from Ref. 24.

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in myelin protein insertion of PMP22 protein. Misfolded proteins in the ER subsequently activate transcription of the gene encoding GRP78 (BiP), and other ER-resident proteins through the transcription factor, HAC1 (44,45). Mutant PMP22 is sequestered in the ER after transfection into Cos-7 cells, and the protein mislocalized to the ER in Trembler sciatic nerve (33). Therefore, PMP22 is a peripheral nerve protein that is highly dependent on the cellular machinery for detecting and degrading misfolded proteins. One hypothesis is that if the SC burden to remove misfolded proteins is increased through point mutations or through overexpression, a disease state can result. It is known that PMP22 has three major transcript lengths that all encode the same protein. One PMP22 transcript is expressed from a nerve-speciﬁc promoter by using an alternate untranslated exon, 1A (46). It has been suggested that this nerve-speciﬁc transcript could be the target for speciﬁc knock-down as a therapeutic strategy for CMT1A in future studies (47). CMT1B: The Myelin Protein Zero Gene The clinical presentation in CMT1B is virtually identical to that seen in CMT1A (48). Mutations in the human myelin protein zero (MPZ ) gene, which was mapped to chromosome 1q22-q23, are the molecular basis of CMT1B (49). The protein encoded by MPZ is the major structural component of PNS myelin (approximately 50% of total protein mass) and represents approximately 7% of total SC mRNA (50). MPZ is a member of the immunoglobulin super-family of cell adhesive molecules and localizes to the compact portion of peripheral nerve myelin (Fig. 1). MPZ is a 28-kD protein with a single transmembrane spanning domain. Analysis of MPZ as a candidate gene for CMT1B revealed point mutations in affected individuals from pedigrees with this disorder (49). Numerous additional mutations in MPZ associated with CMT1B pedigrees have been described [96 different MPZ mutations (HGMD)], ﬁrmly establishing mutations in this crucial component of peripheral nerve myelin as the molecular basis of CMT1B. The MPZ gene, like PMP22, may also be dosage-sensitive. With either murine models of MPZ overexpression or MPZ-null mice, myelination defects of varying degrees occur. For example, Wrabetz et al. (51) demonstrated that transgenic mice containing extra copies of MPZ manifested a dose-dependent, dysmyelinating neuropathy, ranging from transient perinatal hypomyelination to arrested myelination. Normal myelination could be restored by breeding the transgene into an MPZ-null background. Interestingly, while MPZ mRNA overexpression ranged between 30–700%, increased MPZ protein was only detected in nerves of low-copy-number animals and dysmyelination only occurred when MPZ overexpression was increased between 30% and 80%. It is well established that mice either homo- or heterozygous null for the MPZ gene, share pathological features

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with human patients suffering from MPZ-related Dejerine-Sottas syndrome (DSS) or CMT1B, respectively (52). CMT1C: The Small Integral Membrane Protein of the Lysosome/Late Endosome The CMT1C designation was originally assigned when two families with AD CMT1 did not link with either of the major CMT1 loci on chromosome 1 or chromosome 17 (7). A subsequent genome-wide linkage study mapped the locus for CMT1C to chromosome 16p13.1-p12.3 (11). Affected individuals in these families manifest characteristic CMT1 symptoms including high-arched feet, distal muscle weakness and atrophy, depressed deep tendon reﬂexes, sensory impairment, slow NCVs, and nerve demyelination. Point mutations in the small integral membrane protein of the lysosome/late endosome (SIMPLE) gene were found to be the cause of CMT1C (53). In the three families originally described, and in additional pedigrees subsequently identiﬁed, the CMT1C phenotype could not be distinguished from either CMT1A or CMT1B (53,54). SIMPLE protein localization has been examined in several studies, and interestingly, SIMPLE does not locate to the myelin membrane like PMP22 and MPZ. Using recombinant protein, Moriwaki et al. found that SIMPLE localized to the lysosome/early-endosome in several cells lines (55). Bennett et al. examined human sciatic nerve samples by immunohistochemistry (IHC) and found that SIMPLE was present in most types of cells of the nerve and that SIMPLE did not localize to the myelin sheath of SC, but rather showed a cytoplasmic localization (54). Another ﬁnding for SIMPLE that can be contrasted with other genes that cause CMT1 is that the SIMPLE message does not undergo characteristic changes in expression following Wallerian degeneration (53). Further, northern blot analysis has shown that SIMPLE mRNA is present in all tissues examined which contrasts with limited expression of other CMT1 genes (53). It has been proposed that altered lysosomal function and protein degradation may have an impact on myelin development and maintenance based on the up regulation of this pathway in the Tr-J mouse (56). Lysosomal involvement may prove to be a common feature of both CMT1A and CMT1C. Mutated SIMPLE may exert its effects through a loss-of-function or dominant-negative mechanism leading to defects in lysosomal degradation, a pathway in which SIMPLE protein is proposed to operate (53). Currently, DNA testing is available for SIMPLE mutations on a research basis only; however, testing will soon be available commercially. CMT1D: The Early Growth Response 2 Protein Gene CMT1D is a rare but severe form of CMT1. Mutations of the early growth response 2 (ERG2) protein gene have been found in patients with severe

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forms of demyelinating CMT (CMT1D) and Dejerine-Sottas neuropathy (DSN; OMIM:145900) (12,57). To date, only six different point mutations of the EGR2 gene have been identiﬁed (HGMD). The EGR2 protein is a transcription factor with three tandem C2H2type zinc ﬁngers, and plays a crucial role in the regulation of PNS myelination (58). Mice deﬁcient in the murine orthologue of EGR2 (Krox20) show a total lack of myelin (amyelination) (59). Overexpression of EGR2 in SC leads to increased expression of MPZ, PMP22, GJB1/Cx32, periaxin (PRX) and other myelin-related genes (58). The fact that ERG2 is involved in controlling expression of so many critical myelin proteins may explain why mutation of this protein often leads to severe forms of CMT1. DNA testing for EGR2 mutations is commercially available. CMT1X: The Connexin 32 (Cx32/GJB1) It may be that CMT1X would more appropriately be placed within a unique category of its own, as it possibly represents both a demyelinating and an axonal form of CMT. When CMT1X is included within the CMT1 category, it represents a major proportion, accounting for approximately 10% of all demyelinating CMT patients. CMT1X has features of a demyelinating CMT1 neuropathy, as well as an absence of male-to-male transmission, an earlier age of onset, and a faster rate of progression in males than in females. The electrodiagnostic (EDX) ﬁndings in CMT1X are complicated, with signiﬁcant gender differences. Motor NCVs in male CMT1X patients often fall in the intermediate range 30–40 m/sec in the upper extremities. Females may have normal NCVs or mild slowing only (60). CMAP amplitudes are often low in median, ulnar, and peroneal nerves with intermediate slowing. SNAPs are abnormal in most patients, especially in the lower extremities. EMG examination shows rare active denervation (ﬁbrillation potentials and, positive sharp waves) with chronic reinnervation (reduced recruitment of large motor unit action potentials) in distal muscles. It remains to be determined whether CMT1X is primarily an axonal loss or a demyelinating disorder. Some have reported pathologic ﬁndings consistent with axonal loss (60,61), whereas others have reported ﬁndings consistent with a primary demyelinating neuropathy (62,63). Associated central nervous system (CNS) ﬁndings have been reported in some patients, including transient and reversible neurologic deﬁcits (weakness, numbness, abnormalities of cranial nerves, dysarthria, and aphasia) associated with reversible, conﬂuent white matter lesions seen on MRI of the brain (64–66). CMT1X maps to chromosome Xq13-q21 and results from point mutations in the connexin-32 (Cx32) gene also known as the gap junction beta-1 gene (GJB1) (67). Cx32 encodes a protein of 284 amino acids and is a major component of gap junctions. Cx32 is structurally similar to the PMP22

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protein, both proteins containing four putative transmembrane domains in similar orientation. To date, 244 different mutations of the Cx32 protein have been described in CMT1X patients. Cx32 has a pattern of expression in peripheral nerve similar to that of other structural myelin genes which are immediately down regulated following crush or transection nerve injury experiments. IHC studies show that the Cx32 protein localizes to the uncompacted folds of SC cytoplasm around the nodes of Ranvier and at Schmidt–Lanterman incisures, unlike PMP22 and MPZ which are present in compact myelin (67) (Fig. 1). This localization suggests a role for gap junctions composed of Cx32 in providing a pathway for the transfer of ions and nutrients across the folds of the myelin sheath. DNA testing is clinically available for GJB1/Cx32 mutations causing CMT1X.

MAJOR FORMS OF DEMYELINATING CMT2 Genetics of CMT2 CMT2 is less common than CMT1, accounting for approximately 30% of all forms of CMT (68). Many loci have now been identiﬁed that cause rare forms of CMT2 in different families, with both AD (CMT2) and AR inheritance (AR-CMT2 or CMT4C). The fact that several of the genes that cause CMT have now been shown to cause either CMT1 or CMT2 through rare mutations, complicates these clinical designations. It was originally unclear if mutations in any single gene would prove to be a common cause of CMT2. It has recently become apparent that mutations in the Mitofusin 2 (MFN2) gene is the major cause of CMT2A and may ultimately prove to be the most common cause of CMT2 (69). It is also possible that mutations of the ganglioside-induced differentiationassociated protein 1 (GDAP1) gene may emerge as a relatively frequent cause of CMT4C or AR-CMT2 (not described in detail in this chapter). CMT2: Etiology and Pathogenesis In general, CMT2 has a later age-of-onset than CMT1, produces less involvement of the intrinsic muscles of the hands, and lacks palpably enlarged nerves. Extensive demyelination with ‘‘onion bulb’’ formation is not present in CMT2. Axonal pathological changes consist of a loss of large myelinated axons and signs of regeneration with abundant small thinlymyelinated axons (70). Clinical DNA testing is not widely available for most forms of CMT2, but a few reference laboratories will sequence the NEFL gene that causes CMT2E. Also, sequencing of the GDAP1 gene is available for recessive forms of CMT that span the range of phenotypes: severe

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demyelinating CMT4A, severe axonal CMT4C4 with vocal cord paresis, and severe axonal or intermediate CMT4C5 without accessory features. CMT2A: The Mitofusin 2 Gene CMT2A was ﬁrst mapped to chromosome 1p35-p36 in three CMT2 families (71). A mutation in the KIF1B gene, which encodes an axonal motor protein, was found in one Japanese pedigree (72,73). Subsequent analysis of additional pedigrees mapping within the original 9.6-cM interval have shown that CMT2A more commonly results from mutations of the MFN2 (69). All pedigrees with CMT2A are deﬁned by distal muscle weakness and atrophy, depressed deep tendon reﬂexes, pes cavus, and abnormal EDX exams compatible with CMT2. Like most CMT2 pedigrees, CMT2A is characterized with variable disease expression, variable age-of-onset, and NCVs that are normal or only slightly diminished. A total of 13 mutations have now been reported in the MFN2 gene (69). In fact, it may be that MFN2 mutations will prove to be the most common cause of CMT2. A small study of 36 families with axonal CMT, each too small for linkage analysis, uncovered seven additional individuals (19%) with MFN2 mutations as part of the initial study reporting the MFN2 gene as the cause of CMT2A. MFN2 is expressed predominantly in skeletal muscle and heart, with lower expression in brain, kidney, and liver (74). Message has also been detected in spinal cord and peripheral nerve by northern blot (69). MFN2 protein localizes to the outer mitochondrial membrane and regulates the mitochondrial network architecture by fusion of mitochondria. Six of seven MFN2 mutations were identiﬁed within or immediately upstream of the GTPase domain of MFN2. It has been shown that an intact GTPase domain is essential for the function of mitofusins (75–77). Mitochondria undergo a dynamically regulated balance between fusion and ﬁssion reactions and have a tubular and branched membrane network (78). An efﬁcient mitochondrial network is required for fundamental cell functions, such as equilibrating mitochondrial gene products to overcome acquired somatic mutations of mitochondrial DNA (79), and establishing a uniform membrane potential for energy supply (80). These essential functions of mitochondria may be perturbed by mutations of MFN2. Zuchner et al. (69) suggest that MFN2 may also be involved in the process of apoptosis as MFN2 protein colocalizes with the proapoptotic protein, Bax (81). Homozygous Mfn2 knockout mice die in mid-gestation owing to placental defects (75). Although heterozygous Mfn2þ/ mice were reported to have a normal phenotype, murine embryonic ﬁbroblast cultures from Mfn2þ/ mice had markedly lower mitochondrial mobility (75). To date, it is not known if Mfn2þ/ mice will develop a CMT2A-like phenotype with

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age. Zuchner et al. hypothesize that mobility and transport of mitochondria, which are key elements to the functional health of the extended neuronal axons, may be affected by MFN2 mutations and lead to CMT2A (69). Looking toward a future therapy for CMT2A, a virally transported MFN2 construct in a Mfn2þ/ murine cell line was shown to rescue mitochondrial fusion–ﬁssion imbalances (75), raising the intriguing possibility that CMT2A may be amenable to future intervention. Currently, DNA testing for MFN2 is available on a research basis only. CMT2B: Ras-Associated Protein CMT2B is one form of CMT where limb ulceration is a prominent feature (OMIM: 600882) and onset is in the second or third decade of life. The degree of neuropathy ranges from mild to severe, with distal muscle weakness and wasting often preceding the onset of sensory loss, foot ulceration, infections, and amputations of the toes. It has been argued that CMT2B could be classiﬁed as a hereditary sensory and autonomic neuropathy, but motor features are also common to the families with Ras-associated protein (RAB7) missense mutations. CMT2B was mapped to chromosome 3q13q22 (82) and results from mutations in the RAB7 gene (83). Mutations in RAB7 appear to be rare, and to date only the two missense mutations have been reported. The RAB7 protein is a member of the Rab family of ras-related GTPases. Members of this family are important regulators of vesicular transport and are located in speciﬁc intracellular compartments such as the late endosome and the late endocytic pathway. In addition, RAB7 has been shown to have a fundamental role in the cellular vacuolation induced by the cytotoxin VacA of Helicobacter pylori (84), and to be ubiquitously expressed (83). Interesting results have been produced with RAB7 knock-down models in various cell lines. Down regulation of RAB7 in HeLa cells by antisense RNA induces severe cell vacuolation that resembles the phenotype seen in ﬁbroblasts of patients with Chediak–Higashi syndrome (OMIM: 214500) (85). In growth factor deprived cells, Edinger et al. found that blocking RAB7 function prevented the clearance of glucose and amino acid transporters from the cell surface (86). In the same growth factor deprived–RAB7 inhibited cells, mitochondrial membrane potential was maintained and cells displayed prolonged growth-factor-independent, nutrient-dependent cell survival. The authors concluded that RAB7 functions as a proapoptotic protein by limiting cell-autonomous nutrient uptake. Interestingly, some comparisons can be made between the CMT2B and CMT1C. Both RAB7 and SIMPLE: (i) localize to the late endosome or lysosome/late endosome, respectively, (ii) cause CMT disease through AD inheritance of missense mutations, and (iii) are ubiquitously expressed.

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Further understanding of how these two proteins, which may have some functional overlap, can lead to rare forms of demyelinating or axonal CMT will be of interest and the subject of future functional studies. CMT2C: Causal Gene Unknown Further genetic heterogeneity within CMT2 is evidenced by the identiﬁcation of kindreds with the features of axonal neuropathy, weakness of the diaphragm, and vocal cord paralysis. Such pedigrees, which are rare, carry the designation CMT2C, and have been mapped to chromosome 12q23q24 (87). The onset of CMT2C is typically slow and insidious, with respiratory symptoms and alterations in voice. The muscles of the hand become weak and atrophic, but leg weakness may often be asymptomatic. EDX testing reveals an axonal sensorimotor peripheral neuropathy. NCVs are generally normal or only mildly slowed in patients. CMAP amplitudes are low or absent in the lower extremities and normal or low in the upper extremities. SNAPs are absent or low in the lower extremities. EMG testing shows mild active denervation (positive sharp waves and ﬁbrillation potentials) with chronic reinnervation including reduced recruitment of large, prolonged, polyphasic motor unit action potentials (88). Phrenic nerve stimulation reveals low or absent CMAP amplitudes in the majority of patients (88). Laryngeal EMG examination of one patient with CMT2C revealed ﬁbrillation potentials and reduced recruitment of large, polyphasic motor unit action potentials in some, but not all, laryngeal muscles (89). CMT2D: Glycyl-tRNA Synthetase CMT2D patients present with predominantly upper limb involvement. Onset has been documented in the second or third decade of life, with sensory deﬁcits reaching the same prevalence as motor impairment and the degree of progression seems to be mild (OMIM: 601472). The CMT2D locus has been mapped to chromosome 7p14 (90), and mutations in the glycyl tRNA synthetase gene (GARS) have been associated with CMT2D (91). Four mutations in GARS have been reported, and some families show evidence of distal hereditary motor neuropathy 5/distal spinal muscular atrophy V (HMN V/dSMA-V), proving these are allelic disorders (91). Aminoacyl-tRNA synthetases perform the essential function in protein synthesis of catalyzing the esteriﬁcation of an amino acid to its cognate tRNA. These enzymes are necessarily present in each cell. From their primary amino acid sequences, two classes of synthetases have been recognized, with similarity of certain structural features, amino acid attachment sites, and other properties between members of a class. The GARS gene seems to be another general housekeeping gene. Certain aminoacyl-tRNA synthetases are auto-antigens in patients with the idiopathic inﬂammatory myopathies, polymyositis, and dermatomyositis. On the surface, it may

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seem surprising that mutations of such genes are the cause of peripheral neuropathies. Further analysis will be required to uncover the pathogenetic mechanism in this disorder. CMT2E: Neurofilament-Light Gene A large Russian pedigree segregating an AD CMT2 axonopathy was mapped to chromosome 8p21 and later designated CMT2E (92). A nonconservative missense mutation (A998C; Q333P) in the neuroﬁlament light chain (NEFL) gene was found to cosegregate with the disease in all affected members of the family (93). The NEFL gene encodes a protein that is one of three major neuroﬁlament (NF) protein constituents. The NEFL Q333P mutation is located within the coil domain 2B of the NEFL protein, the last and largest of four coil domains that form the rod region. Supporting evidence of the single-point mutation found within the Russian pedigree comes from murine studies. A L394P mutation in the same coil domain in the mouse orthologue resulted in a severe peripheral neuropathy. Interestingly, NEFL null mice do not have a CMT-like phenotype. A second Slovenian family has been identiﬁed with a slowly progressive CMT with onset in the ﬁrst decade of life (94). Distal leg involvement precedes distal arm involvement, and all patients were ambulatory 20–30 years after disease onset. A P22S missense mutation was found to segregate with CMT2 disease. NEFL mutations have also been associated with CMT1F (not discussed in this review) with some patients in these pedigrees having near normal NCVs, suggesting that phenotypic overlap between CMT1 and CMT2 may occur with an identical mutation in a family (95,96). CMT2F: Heat-Shock 27-Kd Protein B1 A single Russian family was originally reported with typical CMT2 features. The age-of-onset in this large pedigree was the second or third decade of life. Initial lower limb involvement and slowly progressive disability, mild to moderate sensory deﬁcits, including pain and temperature, occur in the feet and hands. Linkage analysis identiﬁed a locus at chromosome 7q11-q21 for this form of CMT designated CMT2F (97). On EDX testing, the ﬁndings were consistent with an axonal sensorimotor peripheral neuropathy. Subsequent analysis in the original Russian pedigree identiﬁed a missense mutation in the Heat-Shock 27-kD B1 protein (HSPB1) gene (98). Screening for mutations in HSPB1 in 301 individuals with CMT and 115 individuals with distal hereditary motor neuropathies (dHMNs) conﬁrmed the previously observed mutation and identiﬁed four additional missense mutations. The additional HSPB1 mutations were found in four dHMN families and in one individual with CMT neuropathy (98).

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PROGNOSIS IN CMT The prognosis for many individuals with CMT is relatively favorable and it is very important to communicate this message to patients and to give them hope. Although occasional patients with CMT1 are conﬁned to a wheelchair, most can anticipate remaining ambulatory with the use of simple bracing and having only a mild to moderate degree of impairment in functional strength. Patients with CMT2 generally also remain ambulatory throughout their lifetime. Patients with DSS are often wheelchair bound in childhood or by adolescence. Patients with CMT and HNPP have a normal life expectancy, with the important exception of patients with CMT2C, with diaphragmatic and vocal cord involvement, who may have a shortened lifespan from complications related to respiratory and bulbar insufﬁciency. The prognosis in HNPP is relatively benign, although rare patients develop a progressive peripheral neuropathy.

SUMMARY As was stated in the opening sentences of this chapter, a large number of genes have been recently discovered that cause CMT. More than half of these genes have been identiﬁed in the past four to ﬁve years. This has necessarily precipitated a burst of information regarding peripheral nerve biology and pathologic mechanisms in CMT. It is likely that the next ﬁve years will see more genes discovered but possibly at a slower rate as more emphasis is placed on uncovering the pathogenic mechanisms leading to peripheral nerve disease. It is hoped that the next major phase in CMT research will include improvements in molecular-based diagnostics, rational approaches to molecular therapeutics, and the development of targeted drug therapies.

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7 Duchenne and Becker Muscular Dystrophies Leta S. Steffen Program in Genomics, Children’s Hospital Boston, and Department of Genetics, Harvard Medical School, Boston, Massachusetts, U.S.A.

Louis M. Kunkel Program in Genomics, Children’s Hospital Boston, Department of Genetics, Harvard Medical School, and Howard Hughes Medical Institute, Boston, Massachusetts, U.S.A.

INTRODUCTION Muscular dystrophies are a heterogeneous group of muscle disorders characterized by progressive muscle weakening due to degeneration of muscle ﬁbers and inﬁltration of connective and adipose tissue. To date, mutations in more than 20 genes have been identiﬁed in human muscular dystrophies (1,2). These genes encode structural proteins at the muscle ﬁber membrane and the contractile apparatus, as well as potential signaling and enzymatic molecules (see Fig. 1, reviewed in Refs. 2 and 3). Duchenne muscular dystrophy (DMD), a severe X-linked disease occurring in approximately 1 out of 3500 live male births, is the most common form of muscular dystrophy. Disease onset occurs in early childhood, causing bilateral weakness in the proximal muscles of the hip girdle and legs and progressing by age 11 to widespread weakening of the musculature and loss of ambulation. The average life expectancy of DMD patients is now in the third decade, with death most frequently resulting from respiratory failure

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Figure 1 The DAPC binds dystrophin at the muscle ﬁber sarcolemma, connecting the internal actin cytoskeleton to extracellular matrix components. The DAPC consists of the dystroglycans, the sarcospan–sarcoglycan complex, and dystrobrevin and syntrophin. Abbreviation: DAPC, dystrophin-associated protein complex.

or cardiac complications. Becker muscular dystrophy (BMD), affecting only 1 in 30,000 males, is a milder disease with onset after 5 years of age and a much broader variation in symptoms, rate of progression, and life expectancy. The gene mutated in both DMD and BMD was identiﬁed by Monaco et al. in 1986 using a revolutionary positional cloning method (4). Named dystrophin, the 2.4 Mb gene is the largest in the human genome, potentially explaining the high frequency of spontaneous disease-causing mutations (5). The 427 kDa dystrophin protein product was found to localize to the cytoplasmic face of the muscle membrane and co-purify with a large complex of proteins, called the dystrophin-associated protein complex (DAPC) (6–8). Subsequent research identiﬁed mutations in several genes encoding DAPC members that cause autosomal recessive limb girdle muscular dystrophies (2,3). Cloning of the dystrophin gene and subsequent protein studies have enabled signiﬁcant diagnostic and therapeutic advances for Duchenne and Becker muscular dystrophies. Analysis of dystrophin protein in muscle biopsy specimens can distinguish DMD and BMD from many other muscular dystrophies. Current polymerase chain reaction (PCR) and sequencing

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methods allow for the detection of most causative mutations, in many cases verifying diagnoses without invasive procedures and allowing accurate prenatal and carrier genetic testing. Most importantly, analysis of the genetic differences behind Duchenne and Becker muscular dystrophies has opened the door to several potential therapies, including cell and viral delivery of a functional truncated dystrophin gene and directed changes in dystrophin transcript splicing to alleviate the molecular and pathogenic characteristics of the disease. CLINICAL SYMPTOMS DMD affected boys are generally ﬁrst seen by a physician for a variety of muscular and/or cognitive complaints between one and ﬁve years of age (reviewed extensively in Refs. 9 and 10). The most common complaints are motor delays, abnormalities in gait, frequent tripping or falling, and/or difﬁculty climbing stairs. Patients often walk with a characteristic waddling gait or on their toes because of weakening hip girdle and tibialis anterior muscles, respectively. Affected boys also experience difﬁculty rising from the ﬂoor and eventually exhibit Gower’s sign, using the hands to push off the ﬂoor and walk up the thighs into a standing position. Weakness is usually accompanied by muscle pseudohypertrophy—apparent enlargement, typically of the calf muscles, which is caused by inﬁltration of adipose and connective tissue into the degenerating muscle. Muscle weakness is bilateral and symmetrical, initiating in the proximal muscles and progressing to more distal muscles (9,10). Weakness is usually apparent in lower limbs before upper limbs and will eventually affect all voluntary muscles. By the age of 11, most DMD patients experience loss of ambulation. As a result of weakening trunk muscles and inactivity, patients develop pronounced scoliosis and secondarily, lung atelectasis. The combination of atelectasis and weakened intercostal and diaphragm muscles leads to decreased lung capacity and function in the later stages of the disease. Eventually, DMD patients become hypercapnic and may have difﬁculty clearing respiratory secretions, increasing the risk of pneumonia and respiratory failure. DMD patients may live into their mid to late twenties with respiratory insufﬁciency being a common cause of death. Some female carriers may exhibit mild dystrophic symptoms with later onset, including weakness, muscle fatigue, and calf pseudohypertrophy (9). BMD was ﬁrst characterized as a distinct disease phenotype by Emil Becker in 1955 (11). Upon cloning of the dystrophin gene, it was found that BMD and DMD are allelic diseases. BMD exhibits many of the same symptoms as DMD, most strikingly the pattern of affected muscles. A subset of BMD patients also presents with symptoms of muscle pain and/or cramping (9). While DMD appears consistently severe and rapid in progression, BMD cases show later onset and slower progression but may vary in the severity of

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the clinical phenotype, making it difﬁcult to distinguish from many other muscular dystrophies without diagnostic tests (9). Cardiac muscle tissue is also affected in DMD/BMD (reviewed in Ref. 12). DMD patients often have abnormal electrocardiograms and exhibit cardiomyopathy, usually after 10 years of age. Similar abnormalities have been observed in BMD patients, though often less severe, consistent with the overall milder phenotype. However, a subset of BMD patients exhibits severe cardiomyopathy, occasionally without accompanying skeletal muscle deﬁciencies (10,12). Female carriers may also exhibit late life cardiomyopathies and should be monitored regularly. Both DMD and BMD are characterized by a nonprogressive decrease in mean IQ by one standard deviation (average 85) such that one-third of DMD/BMD patients are mildly mentally retarded (13,14). A variety of speciﬁc verbal and language deﬁciencies has been suggested, possibly accounting for the overall low IQ (15–18). No direct genotype/phenotype correlation has been made, though mutations affecting the distal portion of dystrophin, particularly the brain-speciﬁc Dp140 transcript, have been reported to correlate with decreased IQ (19–22). CLINICAL DIAGNOSIS Approximately 70% of DMD/BMD cases have affected relatives, while the remaining 30% are considered de novo mutations (23). A preliminary diagnosis may often be made on the basis of the patient’s family history, pattern of muscle involvement, age of onset, and presence of any of the abovementioned clinical symptoms (Fig. 2). In almost all cases, a blood sample will be tested for increased serum levels of muscle enzymes to distinguish DMD and BMD from other neuromuscular disorders. Serum testing reveals dramatically increased levels of creatine kinase (CK) in DMD/BMD patients, usually 20–200 times that of an unaffected child, and similarly increased levels in most female carriers (9). Serum aldolase, alanine amino transferase (ALT), aspartate amino transferase (AST), and lactate dehydrogenase (LDH) levels will also be increased in DMD/BMD but are expressed in non-muscle tissues as well and are therefore less speciﬁc (9). In certain cases that do not clearly ﬁt a dystrophic proﬁle, electromyograms may be performed to exclude the possibility of a primary neurogenic disease. DMD is caused by mutations that change the reading frame of the transcript, resulting in a premature stop codon and transcript instability. Approximately 60% of these mutations are large deletions in the gene, 5% are duplications, and the remaining 35% are point mutations or small insertions/deletions (24–26). BMD results from mutations maintaining the reading frame of the transcript, and is associated with a higher incidence of deletions than DMD (25). To conﬁrm a DMD/BMD diagnosis, multiplex PCR testing is performed across the deletional ‘‘hot spots’’ of the dystrophin

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Figure 2 Current diagnostic procedure and a predicted future diagnostic procedure for Duchenne and Becker muscular dystrophies. The establishment of universal ampliﬁcation techniques such as SCAIP for full, automated coverage of entire genes using single condition PCR may allow for identiﬁcation of nearly all dystophin mutations. Abbreviation: SCAIP, single condition ampliﬁcation/internal primers.

gene (Fig. 2). PCR across 18 exons (out of 79 total) detects 98% of deletional mutations (approximately 59% of total DMD/BMD) and can be used as an inexpensive primary diagnostic test for suspected DMD/BMD cases (27). If PCR screening does not identify an obvious mutation in dystrophin and there is no previous family history of the disease, a muscle biopsy is often performed to conﬁrm the diagnosis. Histological examination of dystrophic muscle reveals variation in muscle ﬁber size, necrosis and phagocytosis, regenerating ﬁbers, and connective and adipose tissue inﬁltration. Protein studies using antibodies against portions of the dystrophin protein may be used to distinguish DMD (and often BMD) from other muscular dystrophies. Immunohistochemistry on muscle biopsy sections shows nearly complete absence of dystrophin at the muscle ﬁber sarcolemma in DMD, muscle and often decreased dystrophin in BMD muscle. On western blot of muscle proteins, DMD samples exhibit little or no dystrophin protein (<3%) (28). BMD samples most commonly display decreased dystrophin size and/or abundance (28). Sequencing of the entire coding region of dystrophin may be needed to identify a mutation and conﬁrm a diagnosis. Knowledge of the exact

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mutation also may be desired for entry into clinical trials, for prenatal testing or genetic testing of other family members, or for future tailoring of potential therapies. However, due to the enormous size of the dystrophin gene (79 exons over 2.4 kb), full sequencing to detect point mutations and small deletions/ duplications has been prohibitively costly and laborious until recently. Three main techniques are now employed by diagnostic laboratories to screen for non-deletional mutations not detected by multiplex PCR. Denaturing gradient gel electrophoresis (DDGE), denaturing high performance liquid chromatography (DHPLC), and ‘‘detection of virtually all mutations’’ single strand conformation polymorphism (DOVAM-SSCP) provide methods of screening dystrophin exons for deletions and small mutations, limiting the number of exons ultimately sequenced (29–31). If no deletional or point mutations are detected, duplications may be identiﬁed by multiplex ampliﬁable probe hybridization (MAPH), requiring a lengthy process of hybridizations and PCR ampliﬁcations for each patient (32). Recently, however, Flanagan et al. have established the technique SCAIP (single condition ampliﬁcation/internal primers), allowing PCR ampliﬁcation of the entire dystrophin gene and promoter regions under a single set of conditions to screen for large deletions (33). If no deletion is found, PCR products can be robotically puriﬁed and sequenced within a day, reducing the labor, time, and cost associated with traditional direct sequencing. Such universal techniques should eventually lead to accurate molecular diagnosis of all dystrophy mutations. Identiﬁcation of dystrophin mutations in female carriers presents a unique problem: the technique must be sensitive enough to detect a mutation under heterozygous conditions. Southern blotting, FISH, and MAPH can be used to detect deletions and duplications, but are time consuming. Instead, a quantitative real-time PCR approach has recently been shown to reveal heterozygosity in female patients, allowing detection of deletions and duplications in carriers (34). This method can be theoretically extended to cover the entire dystrophin gene, allowing for PCR assessment of all carriers and duplications. CLINICAL TREATMENT Curative therapies do not yet exist for DMD/BMD, so current treatments are intended to maintain muscle ﬂexibility and strength or provide mechanical or respiratory support (reviewed in Ref. 9). Early treatment involves physical therapy and orthotics while patients are still ambulatory. When joint contractures of the ankles, knees, and hips begin to interfere with walking, single or multiple surgical releases of the joints may be recommended (though no agreement has yet been reached about when and on whom such treatment should be used). At ﬁve to eight years of age, glucocorticoid steroids are often prescribed to boost muscle mass and forestall wheelchair

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reliance. After loss of ambulation and onset of scoliosis, patients may undergo spinal surgery, inserting rods and hooks into the spine to straighten and support the trunk. The average lifespan of DMD patients has increased from 14.4 years in the 1960s to 25.3 years in ventilated patients in the 1990s, primarily due to advances in respiratory assistance and careful monitoring of respiratory function (35). As the respiratory muscles weaken and lung capacity diminishes, nocturnal breathing will decrease as well. The availability of portable nasal ventilators allows for assisted nighttime ventilation (and eventually daytime support) and is recommended to prevent nocturnal hypoventilation when respiratory function declines (36). The use of portable ventilators has greatly increased the quality and length of life. Vaccinations against inﬂuenza and pneumonia may also contribute to the lifespan extension by preventing acute respiratory infections leading to respiratory failure (35). Development of cardiomyopathy is frequent in DMD patients and to a lesser extent in BMD patients and female carriers. All affected individuals and carriers should have regular electrocardiograms, and angiotensin-converting enzyme (ACE) inhibitor treatment should be initiated at signs of cardomyopathic progression (37). Later stage DMD patients may also beneﬁt from beta-blockers while a subset of BMD and female carrier patients ultimately require cardiac transplantation (37). Although the beneﬁts of glucocorticoid steroid use in DMD have been well documented, there is only emerging consensus on the best dose, dosage regimen, and age at which treatment should be initiated. Prednisone is the most commonly prescribed in the United States, followed by its derivatives prednisolone and deﬂazacort (not available in the United States). All have been reported to improve muscular dystrophy symptoms in humans and animal models, potentially through immunosuppression, anti-inﬂammatory effects, stabilization of the sarcolemma, protection from Ca2þ inﬂux, or utrophin protein upregulation (reviewed in Refs. 38–46). Signiﬁcant side effects include weight gain, behavioral changes, cushingoid appearance, stunting of growth, loss of bone density, and cataracts (38). Clinical trials have recently demonstrated that alternative dosing regimens (e.g., weekend only or 10-days on/10-days off schedules) may offer all of the beneﬁts of glucocorticoid use in DMD patients without many side effects (47,48). In addition, evidence suggests that glucocorticoid treatment of very young DMD patients may elicit an even greater response (49,50). In addition to traditional medical and surgical procedures, the potential of several nutritional supplements to affect muscle or muscular dystrophies has initiated a series of clinical trials. Creatine in particular has been most closely studied and, in a small DMD group, demonstrated mild increases in arm muscle strength and time until fatigue (51). Work in the mdx mouse, a model for DMD, also demonstrated decreased skeletal muscle necrosis in response to creatine (52). At the time of writing, clinical trials for

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creatine, glutamine, and CoenzymeQ for maintenance of muscle strength in DMD were underway. HISTORY OF THE DYSTROPHIN DISCOVERY Many genetic diseases have been described, but as of the mid-1980s, very few of the causative genes had been isolated. Identiﬁcation of the dystrophin gene represented an innovative step forward, allowing researchers to isolate the causative mutation for a human disease. Since then, similar methods have been used to identify mutations causing over 2000 different human genetic disorders, including cystic ﬁbrosis, familial breast cancer, and Huntington’s disease (53–55). Even as the dystrophin gene location was being mapped, knowledge of co-localizing markers allowed for early genetic diagnosis and prenatal screening (56). Since the 1900s, Duchenne and others had noticed that DMD was inherited as a sex-linked trait affecting almost solely male children. In the mid-1980s, restriction fragment length polymorphisms (RFLPs) of DMD patients and DNA from three key patients positioned the mutation on the short arm of the X chromosome: two rare female DMD patients exhibited X:autosome translocations with breakpoints in Xp21, and a male patient (B.B.) with DMD and three other X-linked diseases showed a macroscopic deletion in Xp21 (57–60). Ronald Worton’s group pursued the female X:autosome translocations, using one patient with an autosomal breakpoint in the 28S ribosomal RNA gene. Clones from the normal 28S rRNA gene were used to isolate an altered genomic restriction digest fragment of the patient’s DNA, presumably spanning the breakpoint region. A genomic phage library of the patient was scanned with the breakpoint clone to identify the corresponding phage that was then restriction enzyme mapped to locate the exact X chromosome breakpoint (61). Concurrently, our laboratory at Children’s Hospital, Boston was focusing on Xp21 using the male patient BB. Recognizing that BB’s DNA represented a void in the desired region, it was used in a subtractive cloning method with DNA from an XXXXY human cell line (62). An excess of sheared BB DNA was incubated with limited MboI-cleaved XXXXY DNA and allowed to hybridize to one another. The resulting fragments were ligated into a vector with MboI ends, thus cloning only XXXXY self-hybridizing fragments missing from the BB region. This library of Xp21 clones was used to scan other unrelated DMD patients identifying a single clone, pERT87, that was missing from 5 out of 57 affected boys (57). The pERT87 clone was used to initiate a series of chromosomal walks. Non-repetitive sequences isolated in the walk were subcloned and used to identify RFLPs for a large collaborative study (56). A 5% to 6% recombination frequency in several diagnostic RFLP sites plus mapping of patient’s

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deletion breakpoints suggested that the DMD gene was very large (63). To locate coding exons, the subcloned fragments were hybridized to Southern blots of several mammalian species to identify conserved, potentially exon-coding stretches (4). Two putative exons were identiﬁed in humans and their counterparts cloned from a mouse phage library. One of the exons hybridized with an approximately 16 kb polyAþ mRNA from human fetal skeletal muscle and was used to screen a cDNA library, initiating a ‘‘walk’’ that identiﬁed cDNA clones covering the entire dystrophin transcript (4,5). The cloned cDNA fragments were hybridized with genomic DNA in a Southern blot, identifying approximately 60 exons with large intronic sequences (4,5). Later mapping and sequencing of the region demonstrated that the gene, now called dystrophin, consists of 79 exons and spans 2.4 Mb, constituting the largest gene in the human genome. DYSTROPHIN—MOLECULAR CHARACTERIZATION The longest transcript from the dystrophin locus is 14 kb, resulting in a 427 kDa protein (6). To date, seven tissue-speciﬁc promoter regions have been identiﬁed: three yield full-length transcripts differing only in the ﬁrst exon (50 UTR plus the ﬁrst few amino acids). Other promoters lie in intronic regions and share the C0 terminus (reviewed in Ref. 64). Function of the shorter transcripts (along with several identiﬁed splice variants) is not yet clear, but some, like the Dp140 transcript in neural tissues, may contribute to pathogenesis of the disease in non-muscle tissue (22). The dystrophin protein contains four discrete regions: an amino terminus actin-binding sequence, an extended rod domain of 24 spectrin-like repeats interrupted by four hinge regions, a cysteine-rich domain required for b-dystroglycan binding and dystrophin stability, and a unique carboxy terminus domain (6). Early studies using antibodies to dystrophin showed localization to the cytoplasmic face of the muscle ﬁber sarcolemma and co-immunoprecipitation with a group of proteins, called the DAPC (7,65,66). Localization of the DAPC to the sarcolemma requires dystrophin presence and is signiﬁcantly decreased in DMD membranes. Thus, it has been hypothesized that one function of dystrophin is to direct localization of the DAPC. The DAPC is composed of three groups of proteins: the dystroglycans, the sarcoglycans and sarcospan, and dystrobrevins and syntrophins (Fig. 1, reviewed in Ref. 2). Dystrophin binds the integral membrane b-dystroglycan that associates with a-dystroglycan, a peripheral membrane protein binding extracellular matrix components (Fig. 1). Dystrophin’s primary function may thus be structural, connecting the actin cytoskeleton to the extracellular matrix and stabilizing the sarcolemma against repeated cycles of muscle contraction and relaxation. In support, sarcolemmal lesions and increased permeability to dyes have been observed in both DMD and mdx (the mouse

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model for DMD) muscle (67). Several lines of research have suggested that dystrophin may also have a role in signaling, as the molecules of the DAPC have been shown to interact with potential signaling molecules, such as nNOS, g-ﬁlamin, and the Grb2-Sos-Rac1 pathway (68–71). In muscle lacking dystrophin, localization of these molecules is altered and may contribute to the disease phenotype. Calcium concentrations in muscle are ﬁnely controlled and play an important role in the contractile signal. It has been hypothesized that improper calcium homeostasis, secondary to dystrophin absence and sarcolemmal instability, may account for the muscle ﬁber necrosis in DMD and mdx muscle. In support, cellular increases in calcium concentration have been reported in DMD patients and several animal models (72,73). An increase in calcium-leak channels has been seen in dystrophic muscle during normal membrane sealing, increasing the sub-sarcolemmal level of Ca2þ (73,74). In addition, several calcium sequestering and buffering proteins exhibit decreased expression and/or function, suggesting that calcium handling is improperly controlled in dystrophic muscle (75). ANIMAL MODELS OF DMD Several animal models of DMD have allowed signiﬁcant research into dystrophin function and interactions, disease pathophysiology, and response to pharmacological and therapeutic agents (reviewed in Ref. 76). In particular, research on murine models may be performed on a scale available to most laboratories and allows experimentation not possible in humans. Five strains of mice with dystrophin mutations have been used to model DMD, the most common being the naturally occurring mdx mouse identiﬁed by its dramatically increased serum creatine kinase levels (77,78). Mdx carries a T to A point mutation in exon 23, resulting in a nonsense codon (79). Despite almost complete absence of the protein, the mdx phenotype is far milder than human DMD, showing very little functional deﬁciency, progressive muscle wasting, or connective tissue inﬁltration and only mild cardiac involvement. Many muscles, however, exhibit variation in ﬁber size, necrosis and phagocytosis, and centrally nucleated regenerating ﬁbers. More recently, mdx mice also carrying a mutation in the dystrophin homolog, utrophin (mdx/; utr/) have demonstrated disease progression and pathology more similar to human DMD and may become a more suitable model for certain therapeutic studies (80). The canine X-linked muscular dystrophy (CXMD) golden retrievers are perhaps the best physiological model for DMD (81). Affected dogs show early onset of severe progressive muscle weakening and early death. Histology in these animals is very similar to humans, including ﬁbrotic inﬁltration of degenerating muscles. However, size, litter-to-litter variation, and ethical considerations have limited the number of studies in these animals. A feline

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DMD model also exists but exhibits muscle hypertrophy instead of muscle wasting and is not commonly used in DMD research (76,82). Two newer models of DMD may enable valuable research into pharmacological means of treating the disease. Deletion of the dystrophin-like gene (dys-1) coupled with a mild mutation in the muscle transcription factor MyoD results in a muscle degeneration/regeneration phenotype in the nematode C. elegans. This model has recently been used in a proof of principal drug screen that isolated the glucocorticoid prednisone, currently used in DMD patients (83). In zebraﬁsh, an ENU mutagenesis screen has identiﬁed several potential dystrophic ﬁsh including sapje, a strain that has been mapped to a mutation in the dystrophin gene (84). While neither model precisely phenocopies human DMD, they represent similar genetic mutations that may be screened in large numbers for drug responsiveness in a whole organism approach. CURRENT RESEARCH FOR DMD/BMD THERAPIES Because the rate of spontaneous mutations is high, DMD and BMD will never be preventable. The diseases also present unique difﬁculties for therapeutic research, including the large size of the dystrophin gene, the variety of muscles affected, and the need for nearly systemic targeting. However, studies in mice suggest that achieving 20% to 30% of normal dystrophin expression is enough to correct the disease phenotype (85). Currently, cell-based and viral based therapies are being developed to deliver functional dystrophin to degenerating muscles. Pharmacological and antisense models are also being developed to induce translational or splicing inﬁdelity, resulting in at least semifunctional dystrophin. Finally, a number of genes have been identiﬁed that ameliorate the dystrophic phenotype and may offer non-immune reactive targets. Cell-Based Therapy Introduction of cells bearing a functional dystrophin gene is an attractive method for muscular dystrophy therapies. Mature muscle is composed of syncytial ﬁbers, created and maintained by proliferation and fusion of mononuclear myogenic precursors called satellite cells. Early studies in mice demonstrated that normal neonatal mouse or human myoblasts could fuse with mdx ﬁbers in vitro and in vivo, generating expression of the normal dystrophin gene at the sarcolemma in a small percentage of muscle (86,87). Several clinical trials of intramuscular myoblast transfer in human DMD and BMD patients were initiated, but none were able to demonstrate functional strength improvements or more than low, transient dystrophin expression (reviewed in Ref. 88). Since then, research has focused on use of less differentiated myogenic populations delivered via intravenous or intramuscular injection. Side

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population (SP) cells, so-called because of their low staining with the vital dye Hoechst 33342 using a ﬂuorescence-activated cell sorter (FACS), were ﬁrst isolated from mouse bone marrow in 1996 and shown to have potent hematopoietic stem cell properties (89). Using a similar procedure, SP cells were isolated from adult mouse muscle and were shown to exhibit myogenic properties in vitro when co-cultured with muscle cell lines (90). Intravenous injection of muscle-derived SP cells in mdx mice demonstrated homing of donor cells to the damaged muscle and fusion into and dystrophin expression in host muscle ﬁbers, albeit at low percentages. In addition, mononuclear donor cells were identiﬁed in the position of satellite cells, suggesting that cell transfer may provide a semipermanent progenitor population (90). Recent work has demonstrated the successful potential of autologous transplantation, isolating muscle SP from an mdx5cv mouse, transducing in vitro with a lentiviral vector carrying the human micro-dystrophin gene, and intravenously injecting corrected cells back into an mdx5cv mouse (91). Other muscle-derived cell types have shown some success in muscle engraftment, including non-adherent cell populations such as muscle-derived stem cells (MDSC) (92–95). Cells from bone marrow, liver, and skin have also demonstrated low efﬁciency integration into dystrophic muscle and delivery of normal gene products (90,95–98). In order to achieve successful systemic delivery of donor cells, more recent research has employed intraarterial delivery, potentially because cells are immediately delivered to the capillary bed of affected muscles. The highest level of engraftment to date has been reported using intraarterial injection of a fetal dorsal aorta-derived cell population, named mesangioblasts (99). These cells are capable of differentiating into several mesodermal cell types in vivo and could be expanded in vitro indeﬁnitely. Intraarterial delivery of mouse wild type or autologous lentiviral-corrected mesangioblasts into a-sarcoglycan null mice (a model for LGMD 2D) resulted in expression of a-sarcoglycan and correct localization in wide swaths of all downstream muscles (100). Serial injection showed functional and histological improvements and in one muscle tested, it is reported that >50% of ﬁbers were a-sarcoglycan positive. Intraarterial delivery has demonstrated the potential application of this method to the dystrophin-deﬁcient mdx model as well. Sca-1/CD-34 double positive cells isolated from newborn mouse muscle and delivered intraarterially were able to localize to and express dystrophin in regenerating muscle (101). Preliminary experiments using intraarterial delivery of muscle SP cells have recently yielded better results, with engraftment of 5% to 8% after a single injection in the mdx mouse (Fig. 3) (E. Bachrach, unpublished data, 2004). These studies hold great therapeutic promise, identifying a replicating pool of cells that may deliver corrected genes as well as developing a technique for multimuscle engraftment with a single injection. Descriptions of

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Figure 3 Intravenous and intraarterial delivery of muscle SP cells results in engraftment of SP cells into mdx muscle ﬁbers and expression of dystrophin. Muscle SP cells isolated by FACS were transduced with a lentiviral vector carrying the human microdystrophin gene and injected either (A) intravenously or (B) intraarterially into mdx mice. Four to seven weeks later, the quadriceps muscles were sectioned transversely and immunohistochemistry was performed using a human-speciﬁc dystrophin antibody (positive ﬁbers labeled with an asterisk). Preliminary studies demonstrate that 1% of ﬁbers express human dystrophin at the muscle membrane upon intravenous injection (A) while 5% to 8% of ﬁbers express human dystrophin upon intraarterial injection (B) (E. Bachrach, unpublished data). Abbreviations: FACS, ﬂourescenceactivated cell sorter; SP, side population.

donor cell contributions to the in vivo satellite cell pool yields hope that cellbased correction may also be long lived (90,100). Current studies are focused on translating these results to humans and to comparison with more easily isolated cell populations (e.g., autologous skin, bone marrow, and muscle) delivered in the same method. The ultimate goal would be isolation of a

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progenitor cell population from the affected individual, in vitro expansion and correction of the mutated gene, and delivery and engraftment of the autologous cells for therapeutic amelioration of the disease. Viral Therapies Over the last decade, many studies have also introduced viral delivery of functional genes that can incorporate into the host DNA for permanent expression. The 14 kb size of the dystrophin transcript initially prevented its use in traditional vectors, but newer vector backbones and development of mini- and micro-dystrophin genes has enabled several promising avenues of experimentation. Retroviruses showed the ﬁrst in vitro and in vivo delivery of a 6.3 kb mini-dystrophin gene carrying deletions in the rod domain, similar to a deletion mutation in very mild BMD (102,103). Intramuscular injection of the vector resulted in approximately 6% of ﬁbers expressing dystrophin, and relocalized the DAPC to the sarcolemma in mouse models of DMD (103). However, retrovirus use is limited in humans due to their high immunogenicity and inability to infect nondividing cells. Newer lentiviral vectors have been employed for gene delivery and can encode up to 9 kb of sequence. These viruses have greatly decreased immunoreactivity and can transduce nondividing cells, making them ideal vectors for therapy (104). In vitro infection of primary myogenic cells has demonstrated highly efﬁcient gene transduction, which is maintained upon cell injection and fusion into muscle in vivo (91). The adenovirus backbone has also been used to introduce the dystrophin gene. Early adenoviral vectors and adeno-associated viral vectors (AAV) have been used to introduce the dystrophin mini-gene and microgene (3.6–4.2 kb) (105–107). Later generation adenoviruses consist of an almost entirely gutted backbone and can hold the full-length dystrophin gene. Intramuscular injection of these viruses with dystrophin under a muscle-speciﬁc promoter resulted in 25% to 30% of ﬁbers expressing the protein and improvement of contraction-induced injury to the sarcolemma one month after injection (108). A similar study using tandem dystrophin genes showed similar infection rates and retention of dystrophin expression for up to six months (109). Aminoglycoside Antibiotic Read-Through of Stop Codons It has long been known that aminoglycoside antibiotics can induce translational inﬁdelity in bacteria and yeast (110,111). These antibiotics bind a site on the small subunit rRNA to allow improper tRNA-codon recognition and amino acid substitution for intra-exon stop codons (112), possibly also stabilizing transcripts against nonsense-mediated decay (NMD). In the mid-1990s, it was shown that treatment of mammalian cells with the aminoglycoside gentamicin partially restored expression of the gene involved in cystic ﬁbrosis

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from a gene with a premature stop codon mutation (113,114). It was therefore hypothesized that antibiotic treatment might also be of use in allowing translational read-through in the 5–10% of DMD patients with intra-exon stop codons, allowing expression of full length dystrophin (115). Barton-Davis et al. published the ﬁrst demonstration both in vitro and in vivo of gentamicin-induced dystrophin restoration in the mdx mouse, showing that dystrophin was expressed and localized with the DAPC to the sarcolemma in 10% to 20% of muscle ﬁbers (116). Gentamicin offers the beneﬁt of already being approved for use in humans, but follow-up studies and clinical trials have returned mixed results for its efﬁcacy in the treatment of DMD (117,118). Additional work in mdx mice also failed to reproduce the original results (119). Clinical trials are now underway to study gentamicin treatment in DMD patients with nonsense mutations on a broader scale. The variability of gentamicin effectiveness suggests that not all DMD patients with nonsense mutations may beneﬁt from antibiotic treatment. Work in yeast demonstrated that certain stop codons are more stringent, and that surrounding sequences also affect translational ﬁdelity, suggesting that only certain mutations may beneﬁt from gentamicin (120–122). Therefore, future research may be directed toward the identiﬁcation of other pharmacological means to target translational ﬁdelity with wider sequence speciﬁcity. Induced Exon-Skipping to Yield a BMD Phenotype from a DMD Mutation While the great majority of DMD/BMD cases adhere to the ‘‘frame-shift hypothesis,’’ approximately 8% of patients exhibit mutations inconsistent with the expected reading frame of the clinical diagnosis (123). Recent research suggests that these outliers may result from mutations that alter the normal splicing pattern, causing occasional skipping of the mutated exon(s) and restoration of the transcript reading frame (124,125). Thus, induced exon-skipping to correct the transcript reading frame may offer an alternative to cell-based and viral-based therapies, speciﬁcally for the purposes of reducing immune response. Mann et al. ﬁrst demonstrated in vivo induction of dystrophin exon skipping by using antisense oligoribonucleotides (AOs) directed against the splice site of the affected intron in mdx mice (126). Similar techniques have achieved 15% to 20% dystrophin positive ﬁbers in the injected muscle and moderate strength improvement with detectable dystrophin remaining for up to three months (127). In addition to splice site targeting, success has been achieved with AOs directed against exon-recognition sequences (ERS) within the exons (128). Currently, AOs have been developed to target at least 20 exons, covering over 75% of known human DMD mutations (128–130). Work with other types of antisense molecules, including

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morpholinos and altered snRNAs, has also demonstrated the ability to alter dystrophin splicing (131,132). Current antisense methods only transiently affect dystrophin transcripts and would require repeated injection into all affected muscles. An alternative method, however, has recently shown some success achieving permanent induction of exon-skipped dystrophin species in vitro using chimeraplasts. These chimeric DNA/RNA oligomers use cellular mismatch repair mechanisms to induce base pair changes in the genome (133). Chimeraplast targeting of the mdx point mutation demonstrated gene correction in mdx cell culture while targeting to mask exon splice sites induced exonskipping to correct the transcript frame shift in vivo (133,134). Though currently highly inefﬁcient, chimeraplast treatment may hold the potential to affect permanent dystrophin changes without the immune difﬁculties of cellular and viral treatments, nor the limitation of transient expression seen with antisense treatments. Regulation of Other Genes to Ameliorate the DMD/BMD Phenotype Human, mouse, and canine models of DMD have shown sarcolemmal upregulation of utrophin, a dystrophin homolog that is expressed at the sarcolemma during development and is maintained at the neuromuscular junction (NMJ) in the adult (135–137). At the NMJ, utrophin complexes with the DAPC and may provide a similar link between the cytoskeleton and extracellular matrix. Data from the mdx-utrophin murine mutant showing increased severity suggests that utrophin may be partially compensating for dystrophin loss in the mdx model (80). Transgenic mdx mice expressing a mini-utrophin gene showed disease amelioration while highexpressing full length utrophin transgenics demonstrated almost complete prevention of dystrophy (138,139). Transgenic mdx mice overexpressing ADAM12 (140) or calcineurin (141) also showed upregulation of endogenous utrophin and pathological improvement, suggesting that methods to increase endogenous levels or introduce exogenous utrophin may provide a therapy for DMD. Myostatin, a TGF-beta family member, has been shown to act as a negative regulator of muscle growth. Myostatin knockout mice exhibit increased muscle mass due to hyperplasia and some hypertrophy, a phenotype indicating a potential applicability to therapeutic targeting in DMD/ BMD (142). Weekly injection of anti-myostatin antibodies in mdx mice elicits highly effective strength and histological improvements, and nearly normal serum creatine kinase levels (143). Research has also demonstrated functional improvements following injection into adult mdx mice (144), suggesting that inhibition of myostatin may beneﬁt patients at all stages of DMD/BMD.

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CONCLUSION Advances in ventilation, surgical, and pharmacological methods for alleviating DMD/BMD symptoms have greatly increased patient lifespans in the last decade. New techniques in automated dystrophin ampliﬁcation and sequencing have also established faster ways of mutation identiﬁcation for the purposes of diagnosis, genetic testing, and in the future, therapeutic tailoring. Current research is now focused on understanding the function of dystrophin and methods of dystrophin replacement for long-term therapeutic treatment of Duchenne and Becker muscular dystrophies. Intraarterial delivery of cells carrying functional dystrophin offers a particularly exciting possibility for systemic replacement of muscle ﬁber dystrophin, while the identiﬁcation of drugs and molecules that can induce frame-shifting, translational read-through, or alteration of compensating endogenous proteins may offer pharmacological methods of alleviating disease symptoms.

ACKNOWLEDGMENTS We would like to thank members of the Kunkel lab for critical reading of this manuscript, especially Kaliopi Liadaki and Richard Bennett. L.M.K is an investigator of the Howard Hughes Medical Institute. Funding support was provided by the Bernard F. and Alva B. Gimbel Foundation and the Muscular Dystrophy Association (L.M.K.). L.S.S. is supported by a Graduate Research Fellowship from the National Science Foundation.

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8 The Congenital and Limb-Girdle Muscular Dystrophies Janbernd Kirschner Division of Neuropediatrics and Muscle Disorders, University Children’s Hospital, Freiburg, Germany

Carsten G. Bo¨nnemann Division of Neurology, The Children’s Hospital of Philadelphia, Abramson Research Center, Philadelphia, Pennsylvania, U.S.A.

INTRODUCTION Muscular dystrophies as a group are common but genetically very heterogeneous. As a clinical entity they were ﬁrst recognized with the detailed description of the clinical presentation of Duchenne muscular dystrophy (DMD) in 1852 and thereafter (1,2). About 50 years later Batten (3) published the ﬁrst cases of a congenital form of muscular dystrophy (CMD). In contrast to DMD and related conditions, patients with CMD typically have weakness and dystrophic changes in the muscle biopsy at birth, but the course is frequently less progressive compared to that of patients with the Duchenne form. During the following years other clinical groups of muscular dystrophy were delineated as distinct entities on a clinical basis. These now include Emery-Dreifuss muscular dystrophy (EDMD), fascioscapulohumeral dystrophy (FSHD), myotonic dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophies, and limb-girdle muscular dystrophies.

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The term limb-girdle muscular dystrophy (LGMD) was introduced in the middle of the 20th century when it became obvious that there was an additional major group of noncongenital muscular dystrophies in addition to DMD and FSHD (4). The term LGMD is now used for clinical phenotypes with progressive weakness, onset in the limb girdle muscles, and histological evidence for a dystrophic process in muscle. Other causes for a limb girdle distribution of weakness also must have been excluded on the basis of the muscle biopsy. The age of onset of LGMD as a group may range from early childhood to late adulthood (5). Even though possible on the basis of this deﬁnition, DMD and Becker muscular dystrophies (BMD) are not customarily subsumed under this group. During recent years exciting progress has been made in the ﬁelds of CMD and LGMD emphasizing differences as well as commonalities between the two groups. Careful clinical delineation of phenotypes within CMD and LGMD combined with advances in genetic and histochemical characterization has led to the identiﬁcation of numerous molecularly deﬁned subtypes of muscular dystrophy. This has brought individual clinical entities into sharper focus, but has also blurred the boundaries between the traditional categories of muscular dystrophy. For example, it has now been recognized that mutations in the same protein can give rise to very different phenotypes. Fukutin-related protein (FKRP) mutations can manifest with variable severity ranging from a severe CMD [MDC1C (6), including variants of muscle–eye–brain disease, or Walker–Warburg syndrome (7)] to juvenile and a milder adult onset LGMD (type 2I) (8–10). Dysferlinopathies can present either as a classical LGMD phenotype (type 2B), as a distal muscular dystrophy (Miyoshi type) or as mixed phenotypes (10,11). Mutations in lamin A/C have an even wider spectrum of phenotypes associated with them, extending even to include disorders such as lipodystrophy, progeria, and types of Charcot-Marie-Tooth neuropathy (12–18). Table 1 gives an overview of genetically recognized forms of CMD and LGMD organized according to the different groups of proteins involved in their pathogenesis. In this chapter, we will focus on entities within the scope of congenital and limb girdle muscular dystrophies. After some general comments on the approach in the differential diagnosis of patients with possible muscular dystrophies, we will give some more details on the clinical and genetic characteristics of the more common subtypes of congenital and limb girdle muscular dystrophies. CONGENITAL MUSCULAR DYSTROPHIES The congenital muscular dystrophies (CMDs) are a group of genetic disorders in which weakness and abnormal muscle histology typically are present at birth. Muscle weakness is variable but is usually stable, depending on the individual disease, whereas complications of the disease can become

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Protein group Dystrophin associated and membrane based

Extracellular matrix

Contractile apparatus

Putative glycosyltransferases (posttranslational modiﬁcation of a-dystroglycan)

Protein

Gene symbol

Associated muscular dystrophy

Dysferlin

SGCA, SGCB, SGCD, SGCG DYSF

LGMD2B

Calveolin-3

CAV3

LGMD1C

Laminin a2 (Merosin) Collagen VI

LAMA2 COL6A1, COL6A2, COL6A3 ITGA7 TCAP TTID TTN FLNC

MDC1A Ullrich CMD

a-, b-, g-, d-Sarcoglycan

Integrin a7 Telethonin, Titin-Cap Myotilin Titin Filamin C O-mannose beta-1, 2-N-acetylglucosaminyltransferase Protein O-mannosyltransferases

POMGNT1 POMT1, POMT2

Other allelic phenotypes

LGMD2C-F

CMD LGMD2G LGMD1A LGMD2J Limb gridle myopathy Muscle-eye-brain disease Walker–Warburg LGMD2K

Miyoshi myopathy, anterior distal myopathy Rippling muscle disease, hypertrophic cardiomyopathy Bethlem myopathy

Dilated cardiomyopathy Hypertrophic and dilated cardiomyopathy

159

(Continued)

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The Congenital and Limb-Girdle Muscular Dystrophies

Table 1 Different Groups of Proteins Associated with Muscular Dystrophies and Selected Examples of Associated Allelic Variants

Protein group

Protein

Gene symbol

Associated muscular dystrophy

FCMD FKRP

Other enzymes

LARGE Selenoprotein 1

LARGE SEPN1

CAPN3 TRIM32

LGMD2A LGMD2H

Nuclear membrane

Calpain 3 Tripartite motif-containing protein 32 Lamin A/C

LMNA

LGMD1B, EmeryDreifuss muscular dystrophy

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Fukuyama CMD LGMD2I, MDC1C MDC1D Rigid spine syndrome

Other allelic phenotypes

Multiminicore myopathy, Desminrelated myopathy with Mallory-like inclusions Sarcotubular myopathy Progerias, lipodystrophies, cardiomyopathies, mandibuloacral dysplasia, CharcotMarie-Tooth neuropathy

Kirschner and Bo¨nnemann

Fukutin Fukutin related protein

160

Table 1 Different Groups of Proteins Associated with Muscular Dystrophies and Selected Examples of Associated Allelic Variants (Continued )

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more prominent over the time. In order to establish a diagnosis of CMD, a muscle biopsy is required in addition to the clinical examination. Pathological ﬁndings include variation in ﬁber size, internal nuclei, and increased endomysial and fatty tissue (19). Signs of active degeneration and regeneration often are less prominent compared with later onset muscular dystrophies (e.g., DMD/BMD or sarcoglycanopathies). Elevations in serum creatine kinase concentrations are variable; they are elevated in forms affecting dystroglycan and merosin but can be normal in other forms. The mode of inheritance for most CMD is autosomal recessive with signiﬁcant genetic heterogeneity. The worldwide incidence of CMDs is not known, but for northeast Italy it has been estimated at 4.7/100,000 (20). For a useful clinical approach CMD can be segregated in subgroups with normal mental development on the one hand and with mental retardation on the other hand. Magnetic resonance imaging (MRI) of the brain is an indispensable tool in the clinical approach to CMD as it may show abnormalities of brain formation and neuronal migration, changes of the white matter, or be completely normal even in the presence of mental retardation. Although this clinical approach does not completely coincide with a classiﬁcation according to the involved proteins, most of the forms with abnormalities of brain formation that have been deﬁned on a molecular level show abnormalities of posttranslational processing of a-dystroglycan, while mutations of laminin a2 (part of laminin-2/merosin) and other proteins of the extracellular matrix generally allow normal mental development, even though abnormalities of the white matter are seen in laminin a2 mutations. CMD with Normal Mental Development An important subgroup in this category consists of patients with a primary laminin-2 (merosin) deﬁciency due to mutations in the laminin-a2 chain (MDC1A) (21–24). These patients mostly present at birth or during the ﬁrst month of life with muscular hypotonia, contractures, and respiratory and feeding problems. Facial weakness is often prominent and motor development is markedly delayed precluding independent ambulation in patients with complete deﬁciency. Most patients develop respiratory insufﬁciency requiring ventilatory support during the ﬁrst decade of life. Although cognitive function is usually normal, T2 weighted magnetic resonance imaging of the brain by six months of age almost always shows abnormalities of the white matter. A small minority of patients shows structural brain changes in the form of occipital pachy- or agyria, which is associated with mental retardation and epilepsy. However, epilepsy is also found in about one-third of patients without such structural brain abnormalities (22). Most children will have a demyelinating or hypo-myelinating motor neuropathy since laminin-a2 normally is also expressed in Schwann cells. Partial deﬁciency of laminin-a2 can lead to milder phenotypes including an LGMD-like

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presentation as well as contractures akin to EDMD. However, white matter abnormalities and a mild demyelinating or hypo-myelinating peripheral neuropathy are features even in mild cases of lamininopathy (25). A related phenotype is shared by some patients with severe mutations in the FKRP gene, encoding a putative glycosyltransferase involved in the posttranslational O-mannose linked glycosylation of a-dystroglycan, leading to a variable secondary deﬁciency of laminin-2/merosin (MDC1C) (6,10). Useful clinical features that are divergent from MDC1A include the presence of pseudohypertrophy of leg muscles and evidence of left ventricular dilative cardiomyopathy in many patients later in the course as well as the absence of the white matter abnormalities and the peripheral neuropathy seen in MDC1A (6,10). Very recently, the phenotypic spectrum of this entity has been widened with the identiﬁcation of homozygous FKRP mutations in two patients with the severe phenotypes of Walker–Warburg syndrome and muscle–eye–brain disease (7). As described later, mutations in the FKRP gene more commonly give rise to a type of LGMD (LGMD2I). Ullrich muscular dystrophy (UCMD) deﬁnes a distinct group of CMD with normal brain development. Clinically, it is characterized by generalized muscle weakness and striking hypermobility of distal joints in conjunction with contractures of more proximal joints, which may be present early (torticollis) but may also develop over time (26). Additional ﬁndings may include kyphoscoliosis, protruded calcaneus, and early respiratory failure. Although in the classical presentation the disease is severe precluding or leading to early loss of ambulation, considerably milder presentations have now also been recognized (27). A number of patients with UCMD are deﬁcient in collagen type VI on the basis of autosomal recessive mutations in one of the three a-chain genes (27–31). Autosomal dominant mutations of collagen VI are known to cause the milder phenotype of Bethlem myopathy (32). However, recently it has been shown that de novo dominant mutations are also a common cause of severe UCMD so that mutation detection becomes critical for accurate genetic counseling in this disorder (30,31,33). Mildly affected patients with UCMD have only mild muscle weakness with marked joint hypermobility, and there is ultrastructural evidence to suggest pathophysiological overlap with the hypermobility type of Ehlers–Danlos syndrome (34). Even though the majority of patients with phenotypic UCMD in Western countries will have mutations in the collagen VI genes, there is evidence of further genetic heterogeneity underlying the phenotype of UCMD as involvement of collagen VI was excluded by immunohistochemistry or linkage analysis in a number of patients with the clinical characteristics of UCMD (35). The group of CMD with rigid spine syndrome (RSS) is characterized by early rigidity of the spine and frequently associated with a restrictive respiratory syndrome. These signs are often preceded by hypotonia in the ﬁrst months of life and predominantly axial muscle weakness. However,

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independent ambulation is normally achieved before 18 months of age. Many patients with this phenotype have mutations of selenoprotein N (SEPN1) (27). Selenoprotein N mutations have now also been described in patients with the classical form of multi-minicore disease (37), and with a desmin-related myopathy with Mallory body-like inclusions (38) indicating signiﬁcant overlap with other congenital myopathies. Not surprisingly, corelike structures can sometimes also be seen in biopsies from patients with RSS CMD caused by mutations in SEPN1 (37). Overall the clinical phenotype in this group of disorders is more consistent than the morphology. CMD with Abnormal Brain Development and Mental Retardation The main phenotypes in this group have been delineated on clinical grounds and include Fukuyama CMD (39), muscle-eye-brain disease (40,41), and Walker–Warburg syndrome (42). All three of these syndromes can be caused by mutations in distinct glycosyltransferases or putative glycosyltransferases involved in the posttranslational modiﬁcation of a-dystroglycan (Table 1). Common characteristics include severe muscular dystrophy, neuronal migration defects including lissencephaly type II (cobblestone complex), pachygyria, cerebellar dysplasia, and variable ocular anomalies. Fukuyama CMD is mainly found in the Japanese population and characterized by congenital weakness, profound delay of motor development (generally precluding independent ambulation), and severe mental retardation. Typical ﬁndings on cerebral imaging include abnormal gyral formation as outlined above, a ﬂat brainstem, and cerebellar hypoplasia (39). The mutation in Japan is a retrotransposon insertion causing a hypomorphic allele (43); a homozygous null mutation has been described in a case of Walker–Walker syndrome (44). Isolated cases of Fukuyama CMD in non-Japanese have been reported in other countries (45). Muscle–eye–brain disease was ﬁrst described in Finland, where it still remains most prevalent. In addition to severe CMD and brain malformations such as lissencephaly II/pachygyria and cerebello-pontine hypoplasia, this syndrome presents with more severe ocular abnormalities including severe congenital myopia, congenital glaucoma, pallor of the optic discs, and retinal hypoplasia (40,41). Walker–Warburg syndrome generally presents with the most severe brain involvement and frequently is lethal either prenatally or within the ﬁrst years of life. The morphological features are altogether more severe than what is found in muscle–eye–brain disease; in addition to the features seen in that condition, affected children may have congenital cataracts, microophthalmia, hydrocephalus, occipital encephalocele, fusion of the hemispheres, and absence of the corpus callosum (42). However, with the identiﬁcation of the genes involved in these three syndromes (Table 2) it has also been recognized that the phenotypic spectrum and the regional

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Disease entity

Locus protein product

Helpful clinical features

164

Table 2 Congenital Muscular Dystrophies (CMD) with Identiﬁed Gene Defects CNS involvement

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Mostly complete laminin-a2 deﬁciency on IH/WB, secondary reduction of integrin a7 possible, mutation analysisa a-DG with diminished MW on WB, or reduction of IH using antibodies against glycosylated isotopes, secondary reductions in laminin-a2 on IH/WB, mutation analysis IH/WB comparable to MDC1C, mutation analysisa

IH for collagen VI with severe to mild deﬁciency, mutation analysisa

Normal expression of laminin a2, mutation analysis

Kirschner and Bo¨nnemann

Sitting and standing with support as Abnormal white matter signal (T2 maximal motor ability if complete MRI), 5% occipital deﬁciency, neuropathy, epilepsy in pachy- or agyria about 30%, possible subclinical cardiomyopathy, generally normal mental development Often reminiscent of MDC 1A, but Generally normal, Fukutin related 19q13.3 Fukutin structural severity more variable, from severe proteinopathy related protein abnormalities with CMD to LGMD (see there), (MDC1C) (FKRP, putative cerebellar cysts generally normal mental phospholigand possible development, rare cases with transferase) structural brain involvement and mental retardation LARGE related 22q12.3 LARGE So far only one patient described. White matter changes, CMD (MDC1D) (putative Congenital muscular dystrophy hypoplastic glycosyltransferase) with profound mental retardation brainstem, mild pachygyria No Distal joint hyperextensibility, Ullrich CMD 21q22.3 and 2q37 proximal contractures, motor (UCMD) a1/2 and a3 abilities variable, precludes collagen VI independent ambulation in severe cases, soft palmar skin No CMD with early Delayed walking, predominantly 1p36–p35 spine rigidity axial weakness with early Selenoprotein Nb (RSMD) development of rigidity of the spine, restrictive respiratory syndrome 6q22–q23 CMD with Laminin-a2 primary laminin2 (merosin) deﬁciency (MDC1A)

Laboratory testing

Muscle-eye-brain disease (MEB)

Walker–Warburg syndrome (WWS)

Integrin a7 (1)

IH/WB comparable to 9q31 Fukutin Frequent in Japanese population, Lissencephaly type MDC1C, mutation analysis (putative never walk, mental retardation, II/pachygyria, phospholigand epilepsy common cerebellar transferase)b abnormalities IH/WB comparable to Lissencephaly type Severe weakness and mental 1q32–q34 ProteinMDC1C, mutation analysis II/pachygyria, eye retardation, large head, prominent O-linked mannose malformations, forehead, ﬂat midface, walking b1, 2-N-acetylgluco brain stem and rarely achieved, ocular saminyltranferase 1 cerebellar involvement (e.g. severe myopia, (POMGnT1)c abnormalities retinal hypoplasia), deterioration because of spasticity Severe, lethal within ﬁrst years of life Lissencephaly type II, IH/WB comparable to 9q34.1 and 14q24.3 MDC1C, same phenotype pachygyria, hydrobecause of severe CNS protein-O-mannocan also be caused by cephalus, involvement syltransferases Fukutin or FKRP encephalocele, eye (POMT1 and mutations, mutation malformations POMT2)d analysis 12q13 Integrin a7 Rare, delayed motor milestones, No Absence of integrin a7 on IH walking with 2–3 years (secondary reduction possible), mutation analysisa

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Fukuyama CMD (FCMD)

a

Currently not available as diagnostic testing, only performed on a research basis. Marked genetic heterogeneity for this phenotype. c Genetic heterogeneity for MEB includes FKRP. d Genetic heterogeneity for WWS includes FKRP and Fukutin. Abbreviations: CK, creatine kinase; DG, dystroglycan; IH, immunohistochemistry; MW, molecular weight; WB, Westernblot. Source: References for CMD entities not mentioned in the text: (1) Hayashi YK, et al. Nat Genet 1998; 19(1):94–97. b

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Disease entity

Helpful clinical features

CMD with partial merosin deﬁciency (MDC1B) (1)

Rare, variety of severity, delayed onset possible, proximal girdle weakness, generalized muscle hypertrophy, early respiratory failure possible

CMD with microcephaly/calf hypertrophy (3) CMD with adducted thumbs (4)

Joint contractures associated, severe psychomotor retardation, no walking, striking enlargement of the calf and quadriceps muscles, CK grossly elevated Rare, adducted thumbs, toe contractures, generalized weakness, delayed walking, ptosis, external ophthalmoplegia, mild mental retardation Microcephaly, delayed psychomotor development, generalized muscular wasting and weakness with mild facial involvement, calf pseudohypertrophy, joint contractures, severe mental retardation Delayed motor milestones, mild intellectual impairment

CMD with mental retardation and microcephaly (5)

CMD with short stature, proximal contractures and distal laxitiy (7)

Generalized weakness, more proximal, no independent ambulation, short stature, rigidity of the spine, proximal contractures, distal laxity, early respiratory failure, mild to moderate mental retardation

Laboratory testing

Abnormal white matter and Partial deﬁciency of laminin-a2 on IH/ structural changes WB, a-DG signipossible ﬁcantly reduced on IH, phenotype has been linked to 1q42 Megacisterna magna, Mild to moderate cerebellar hypoplasia, partial deﬁciency of white matter changes laminin a2 on IH Mild cerebellar hypoplasia Normal expression of laminin a2 and aDG on IH Pontocerebellar hypoplasia, Normal expression of laminin a2 focal cortical dysplasia, white matter changes, cerebellar cysts Normal expression of Moderate to severe laminin a2 cerebellar hypoplasia, no white matter abnormalities Normal MRI in most cases, Normal expression of collagen VI patchy dysmyelination in one case

Abbreviations: DG, dystroglycan; IH, immunohistochemistry; MW, molecular weight; WB, Westernblot. Source: References for CMD entities not mentioned in the text: (1) Brockington M, et al. Am J Hum Genet 2000; 66:428–435, (2) Hayashi YK, et al. Nat Genet 1998 May; 19(1):94–97, (3) Villanova M, et al. Neuromuscul Disord 2000; 10:541–547, (4) Voit T, et al. Neuromuscul Disord 2002; 12:623–630, (5) Ruggieri V, et al. Neuromuscul Disord 2001; 11: 570–578, (6) Echenne B, et al. Neurol 1998; 50:1477–1480, (7) Mercuri E, et al. Neuropediatrics 2004; 35:224–229. Copyright 2006 by Taylor & Francis Group, LLC

Kirschner and Bo¨nnemann

CMD with cerebellar atrophy (6)

CNS involvement

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Table 3 Congenital Muscular Dystrophies (CMD) without Identiﬁed Gene Defects

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167

distribution for individual genetic defects is probably wider than previously assumed, thus blurring the boundaries between the clinically deﬁned entities (46). LIMB-GIRDLE MUSCULAR DYSTROPHIES The term LGMD is used for all noncongenital muscular dystrophies with progressive proximal weakness and spared facial muscles that are not caused by the dystrophin-related muscular dystrophies DMD and BMD. Onset, progression, and distribution of weakness vary considerably among patients and genetic subtypes (5). As in CMD, most of the time a muscle biopsy will be necessary to conﬁrm the presence of dystrophic changes including evidence for degeneration and regeneration and also to differentiate LGMD from other causes of progressive proximal weakness that do not qualify as muscular dystrophies. To keep track of an increasing number of distinct forms of LGMD, a purely genetic nomenclature was introduced (47). This nomenclature chronologically assigned LGMD 1A, 1B, 1C and so on to the autosomal dominant forms and 2A, 2B, 2C and so on to autosomal recessive forms. Autosomal recessive forms are about 10 times more common than dominant forms (48). Within autosomal recessive LGMD, calpainopathy, sarcoglycanopathies, dysferlinopathy, and FKRP mutations are the most important entities. In addition, a number of other disorders not strictly recognized as LGMD in this classiﬁcation may present with LGMD-like phenotypes and therefore have to be considered in the differential diagnosis. Examples include FSHD, partial deﬁciency of laminin a2, and myoﬁbrillar myopathies, X-linked EDMD, or Bethlem myopathy. In developing a clinical approach, careful evaluation of the family history, the age of onset and progression, and the pattern of weakness and contractures are important clues to narrow the differential diagnostic possibilities and to guide further histological and genetic workup. Even in the absence of other affected family members, a careful analysis of the family structure is important to see if haplotype analysis would be feasible within the family to narrow down diagnostic possibilities. Some characteristic clinical features can direct the clinician to individual disorders, though there is considerable clinical heterogeneity and overlap among these disorders. In characterization of large numbers of patients with LGMD, there seems to be a gradation according to the age of onset and clinical severity based on genotype. While early onset Duchenne-like phenotypes tend to be caused by either sarcoglycan or FKRP mutations, calpain mutations more commonly present with juvenile onset, and dysferlin mutations in early adulthood (49,50). The autosomal-dominant types often show milder phenotypes, with onset in the later teens or adulthood. However, late onset is also common in FKRP mutations and can indeed occur in any type of LGMD.

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Calpainopathy (LGMD 2A) In this relatively frequent type of LGMD the age of onset is in childhood or adolescence for most patients, with a range from about 2 to 40 years, and a peak onset around 16–18 years. An initial scapular–humeral–pelvic distribution of muscle weakness and atrophy with preserved strength of the adductor muscles is characteristic and allows for its clinical recognition in perhaps two–thirds of the cases. In general, calpainopathy is a more atrophic muscular dystrophy with early development of contractures, so that toe walking can be one of the ﬁrst symptoms. Contractures can be so extensive as to mimic EDMD. The course is progressive with loss of ambulation mostly in the second or third decade, or 20 years after onset (4–44 years) (51). Life expectancy, however, is close to normal (52). Muscle biopsies from patients with calpain-3 mutations often show so-called ‘‘lobulated’’ ﬁbers, in particular on the histochemical type I stain. However, these ﬁbers can occur later in the course of the disease and are not speciﬁc for this disorder. In addition, while a clear reduction of calpain-3 on immunoblot analysis is indicative of a calpainopathy, it is not speciﬁc (e.g., secondary reduction in dysferlinopathies) and a normal protein level does not exclude the diagnosis (51,53). Dysferlinopathy (LGMD 2B, Miyoshi Myopathy) Although the initial clinical presentation can be of various types, the time of onset in dysferlinopathies clusters around 20 years of age (54,55). Possible patterns of muscle involvement include a limb-girdle phenotype, a posterior distal presentation (gastrocnemius) as Miyoshi myopathy, an anterior distal presentation and mixed presentations even with identical mutations (56). Various forms of onset can be observed within the same family. In the LGMD phenotype, the periscapular muscles are relatively spared in the early course compared with other types of LGMD (e.g., sarcoglycanopathies). Even in the LGMD presentation there is often characteristic early involvement of the gastrocnemius and soleus muscle, which can lead to wasting and difﬁculties in walking on the toes, a diagnostically helpful feature (40,54). The weakness is slowly progressive and loss of ambulation may occur in the fourth decade. Serum levels of creatine kinase are very high, at least in early stages of the disease. Immunohistochemistry and immunoblot of muscle biopsy show absence of dysferlin in most patients and are very helpful in the diagnostic work up, as mutation analysis is very laborious due to the size of the dysferlin gene (44 exons). Sarcoglycanopathies This group comprises defects of g-, a-, b-, and d-sarcoglycan (LGMD2C, D, E, F) and is more frequently found in the severe forms of LGMD as opposed to milder or later onset presentations (41,57–62). The clinical picture

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(although variable) resembles that of DMD in many aspects. The course is invariably progressive with loss of ambulation often occurring during the second decade of life. However, especially in cases with later onset, ambulation may be preserved until adult life. Unlike DMD there is no cognitive involvement and overt cardiomyopathy is much less common. However, in about 30% of patients subclinical ﬁndings on ECG or echocardiography indicate dilative cardiomyopathy (63). Patients with b- and d-sarcoglycan mutations may be at higher risk for cardiac manifestations that are clinically relevant. Immunohistochemical analysis of the muscle biopsy may help to prioritize genetic analysis within the four sarcoglycan genes. Fukutin-Related Proteinopathy (LGMD2I) Mutations of the enzyme FKRP cause defects of the posttranslational glycosylation of a-dystroglycan with phenotypic presentations ranging from severe CMD with or without brain malformation to mild forms of LGMD in late adulthood (6,8,10,11). Characteristic features include early weakness of the upper arms, shoulders, and neck ﬂexors. Muscle hypertrophy is relatively common and muscle MRI might be helpful to recognize a typical pattern of muscle involvement (64). There may be mild facial weakness especially in early onset cases. Intelligence and MRI of the brain are normal, but dilative cardiomyopathy and respiratory failure are common and occur in more than one-third of patients (8,10,11). The risk of respiratory failure increases in particular after loss of ambulation. In clinical appearance and because of the cardiac involvement, there are considerable clinical similarities to DMD and BMD. Immunohistochemistry can show a mild reduction of a-dystroglycan but can also be normal, so that mutation analysis of FKRP is often necessary to conﬁrm or rule out the diagnosis of LGMD2I. Other Autosomal Recessive LGMD These rare disorders include telethoninopathy (LGMD2G) (65,66), TRIM32related dystrophy (LGMD2H) (67,68) and titinopathy (LGMD2J) (69,70) and have so far been conﬁned to speciﬁc pedigrees or populations. Autosomal Dominant LGMD with Cardiac Involvement LGMD1B (laminopathy) is allelic with autosomal dominant EDMD caused by lamin A/C mutations (71). In addition, this gene has been associated with a striking number of clinical phenotypes including cardiomyopathies, progeria, lipodystrophies, and neuropathies (72). Recently, a number of combinations of these phenotypes have been described and some of these also include myopathy (73,74). The clinical spectrum of myopathies associated with lamin A/C mutations ranges from early onset myopathies with severe contractures (EDMD phenotype) to a late onset limb girdle phenotype

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Table 4 The Limb-Girdle Muscular Dystrophies (LGMD) Locus protein product

Disease entity

15q15 Calpain-3

LGMD2B Dysferlinopathy

2p13 Dysferlin

LGMD2C g-SGpathy, LGMD2D a-SGpathy, LGMD2E b-SGpathy LGMD2F d-SGpathy LGMD2G Telethoninopathy

13q12 g-sarcoglycan 17q21 a-sarcoglycan 4q12 b-sarcoglycan 5q33–34 d-sarcoglycan 17q11–12 Telethonin (Titin-cap)

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Laboratory testing

Onset around 10 years of age, but variable, early periscapular and humeral weakness, in the lower extremity, hip abductors relatively spared and posterior compartment of the thigh prominently affected Onset around 18 years of age with proximal, proximal-distal or distal (Miyoshi) weakness, periscapular muscles relatively spared, distal biceps involved, gastrocnemius involved early Onset around six to eight years of age, but great variability with much later onset, distribution of weakness reminiscent of DMD/BMD, but sometimes earlier scapular involvement, calf hypertrophy very common, mental development normal, signiﬁcant cardiomyopathy possible in a subset Variable clinical picture, may have initial anterior tibial weakness with foot drop, but may also present as pure LGMD picture

Imaging to conﬁrm selective posterior involvement, lobulated type I ﬁbers, calpain-3 reduction only on WB, mutation analysis CK excessively elevated, deﬁciency of dysferlin in IH/WB, secondary reduction of calpain-3 on WB possible, mutation analysis possible but laborious (55 exons), dysferlin expression in lymphocytesa CK elevated to very high, IH pattern of primary and/or secondary reduction of all four SG antibodies helpful to direct mutation analysis, secondary reduction in IH of dystrophin possible but normal size on WB, mutation analysis mostly necessary for deﬁnitive diagnosis Rimmed vacuoles in some patients, reduction in IH/WB for telethonin, mutation analysis (2 exons)a

Kirschner and Bo¨nnemann

LGMD2A Calpainopathy

Helpful clinical features

LGMD2I FKRPpathy

LGMD2J Titinopathy LGMD2K(1)

LGMD1A Myotilinopathy

LGMD1B Laminopathy LGMD1C Caveolinopathy

9q33.2 TRIM32

Onset usually mid 20s, somewhat slower progression, back pain, some evidence for cardiac involvement on ECG, so far only in Hutterites 19q13.3 Fukutin Wide spectrum of possible age of onset and related protein severity, from Duchenne-like severity to (FKRP) mild late onset presentation, often reminiscent of dystrophinopathies, including muscle pseudohypertrophy and cardiomyopathy, more weakness in the upper extremity/shoulders relative to the lower extremities 2q24.3 Titin Heterozygous parents might present with tibial muscular dystrophy, proximal weakness before 10 years of age 9q34.1 POMT1 Onset during childhood, mild proximal weakness, mental retardation, elevated CK levels 5q22–q34 Onset in young adulthood, slowly progressive, Myotilin nasal quality of speech, mildly raised CK values, no cardiac involvement, only described in two families 1q21 Lamin A/C Onset in late teens or early adulthood, cardiac manifestation with conduction system disease, overlap with AD-EDMD 3p25 Caveolin-3 Onset in childhood, muscle cramping and calf hypertrophy possible, no cardiac involvement

CK levels 5- to 50-fold elevated, dystrophic picture on biopsy, mutation analysisa

a-dystroglycan with diminished MW on WB, or reduction of IH using antibodies against glycosylated isotopes, secondary reduction in laminin-a2 on IH/WB possible, mutation analysis

Possibly secondary deﬁciency of calpain-3 on WB, mutation analysisa Reduced glycosylation of L-dystroglycan

Biopsy with autophagic vacuoles and nemaline rod like structures, IH normal, mutation analysisa Antibody studies for lamin A/C normal in heterozygous mutations, mutation analysis Reduction in IH for caveolin 3, mutation analysis (2 exons)

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(Continued) Copyright 2006 by Taylor & Francis Group, LLC

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LGMD2H

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Table 4

The Limb-Girdle Muscular Dystrophies (LGMD) (Continued )

Disease entity

Locus protein product

LGMD1D (2)b

7q

LGMD1E (3)b

6q23

LGMD1F (4)

7q32 (distinct from LGMD1D locus) 4p21

LGMD1G (5)

Helpful clinical features

Laboratory testing

Onset in late adulthood, dysphagia possible, Only by linkage analysisa no cardiac involvement, 2 families Onset in early adulthood, slowly progressive, Only by linkage analysisa cardiomyopathy, 1 family Progressive weakness mainly affecting Only by linkage analysisa proximal muscle, age of onset ranging from less than 1 year to 58 years, 1 family One Brazilian family with adult onset weakness and progressive ﬁnger and toe ﬂexion contractures

Only by linkage analysisa

a

Currently not available as diagnostic testing, research only. Confused terminology between these two disorders in the literature. Abbreviations: CK, creatine kinase; IH, immunohistochemistry; WB, Westernblot. Source: References for LGMD entities not mentioned in the text: Balci B, et al. Neuromuscul Disord 2005; 15:271–275, (2) Speer MC, et al. Am J Hum Genet 1999; 64:556–562, (3) Messina DN, et al. Am J Hum Genet 1997; 61:909–917, (4) Palenzuela L, et al. Neurology 2003; 61:404–406, (5) Starling A, et al. Europ J Hum Genet 2004; 12:1033–1040. b

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without contractures; there seems to be a continuum between these disorders. The possible cardiac involvement with various degrees of conduction block is comparable to the EDMD phenotype and necessitates early diagnoses and close follow up. LGMD1E is very similar to LGMD1B with respect to the nature of the cardiac involvement; however a gene has not yet been isolated for that type (75).

Autosomal Dominant LGMD Without Cardiac Involvement LGMD1A is a slowly progressive form of LGMD in which some affected members have characteristic nasal speech. Mutations in myotilin have been identiﬁed in two families. Mutations in myotilin are also a cause of myoﬁbrillar myopathy, indicating overlap between these phenotypes (76). The spectrum of clinical phenotypes associated with caveolin-3 mutations has recently broadened considerably. In addition to the rare LGMD phenotype (1C), possible presentations include asymptomatic hyperCKemia (77), myalgias (78), an autosomal dominant and recessive form of rippling muscle disease (79,80), and distal myopathy (81). CK levels tend to be higher than in other autosomal dominant LGMDs and immunohistochemistry and immunoblot can show signiﬁcant reduction of caveolin-3 in the muscle biopsy. REFERENCES 1. Meryon E. On granular and fatty degeneration of the voluntary muscles. Med Chir Trans 1852; 35:73–84. 2. Duchenne G. Recherches sur la paralysie musculaire pseudohypertrophique on paralysie myosclerosique. Arch Gen Med 1868; 11:5–25. 3. Batten F. Three cases of myopathy, infantile type. Brain 1903; 26:147–148. 4. Bell J. On pseudohypertrophic and allied types of progressive muscular dystrophy. In: Fischer RA, ed. The Treasury of Human Inheritance. London: Cambridge University Press, 1943:283–342. 5. Bushby KMD. Diagnostic criteria for the limb-girdle muscular dystrophies: Report of the ENMC consortium on limb-girdle dystrophies. Neuromuscul Disord 1995; 5:71–74. 6. Brockington M, Blake DJ, Prandini P, Brown SC, Torelli S, Benson MA. Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deﬁciency and abnormal glycosylation of alpha-dystroglycan. Am J Hum Genet 2001; 69(6):1198–1209. 7. Beltran-Valero DB, Voit T, Longman C, Steinbrecher A, Straub V, Yuva Y. Mutations in the FKRP gene can cause muscle-eye-brain disease and WalkerWarburg syndrome. J Med Genet 2004; 41(5):e61. 8. Brockington M, Yuva Y, Prandini P, Brown SC, Torelli S, Benson MA. Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C. Hum Mol Genet 2001; 10(25):2851–2859.

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55. Saenz A, Leturcq F, Cobo AM, Poza JJ, Ferrer X, Otaegui D, et al. LGMD2A: genotype-phenotype correlations based on a large mutational survey on the calpain 3 gene. Brain 2005. 56. Fardeau M, Hillaire D, Mignard C, Feingold N, Feingold J, Mignard D, et al. Juvenile limb-girdle muscular dystrophy: Clinical, histopathological and genetic data from a small community living in the Reunion Island. Brain 1996; 119:295–308. 57. Fanin M, Fulizio L, Nascimbeni AC, Spinazzi M, Piluso G, Ventriglia VM, et al. Molecular diagnosis in LGMD2A: mutation analysis or protein testing? Hum Mutat 2004; 24(1):52–62. 58. Argov Z, Sadeh M, Mazor K, Soffer D, Kahana E, Eisenberg I, et al. Muscular dystrophy due to dysferlin deﬁciency in Libyan Jews. Clinical and genetic features. Brain 2000; 123(Pt 6):1229–1237. 59. Linssen WH, Notermans NC, Van der Graaf Y, Wokke JH, Van Doorn PA, Howeler CJ, et al. Miyoshi-type distal muscular dystrophy: Clincal spectrum in 24 Dutch patients. Brain 1997; 120:1989–1996. 60. Weiler T, Bashir R, Anderson LV, Davison K, Moss JA, Britton S, et al. Identical mutation in patients with limb girdle muscular dystrophy type 2B or miyoshi myopathy suggests a role for modiﬁer gene(s). Hum Mol Genet 1999; 8(5):871–877. 61. Bo¨nnemann CG, Modi R, Noguchi S, Mizuno Y, Yoshida M, Gussoni E, et al. b-sarcoglycan (A3b) mutations cause autosomal recessive muscular dystrophy with loss of the sarcoglycan complex. Nat Genet 1995; 11(3):266–273. 62. Fanin M, Duggan DJ, Mostacciuolo ML, Martinello F, Freda MP, Soraru G, et al. Genetic epidemiology of muscular dystrophies resulting from sarcoglycan gene mutations. J Med Genet 1997; 34(12):973–977. 63. Lim LE, Duclos F, Broux O, Bourg N, Sunada Y, Allamand V, et al. b-sarcoglycan (43 DAG): Characterization and involvement in a recessive form of limb-girdle muscular dystrophy linked to chromosome 4q12. Nat Genet 1995; 11:257–265. 64. Nigro V, de Sa Moriera E, Piluso G, Vainzof M, Belsito A, Politano L, et al. The 5q autosomal recessive limb-girdle muscular dystrophy (LGMD 2F) is caused by a mutation in the d-sarcoglycan gene. Nature Genetics 1996; 14(2):195–198. 65. Noguchi S, McNally EM, Ben Othmane K, Hagiwara Y, Mizuno Y, Yoshida M, et al. Mutations in the dystrophin-associated protein g sarcoglycan in chromosome 13 muscular dystrophy. Science 1995; 270:819–822. 66. Roberds SL, Leturcq F, Allamand V, Piccolo F, Jeanpierre M, Anderson RD, et al. Missense mutations in the adhalin gene linked to autosomal recessive muscular dystrophy. Cell 1994; 78:625–633. 67. Melacini P, Fanin M, Duggan DJ, Freda MP, Berardinelli A, Danieli GA, et al. Heart involvement in muscular dystrophies due to sarcoglycan gene mutations. Muscle Nerve 1999; 22(4):473–479. 68. Fischer D, Walter MC, Kesper K, Petersen JA, Aurino S, Nigro V, et al. Diagnostic value of muscle MRI in differentiating LGMD2I from other LGMDs. J Neurol 2005. 69. Moreira ES, Vainzof M, Marie SK, Sertie AL, Zatz M, Passos-Bueno MR. The seventh form of autosomal recessive limb-girdle muscular dystrophy is mapped to 17q11–12. Am J Hum Genet 1997; 61(1):151–159. 70. Moreira ES, Wiltshire TJ, Faulkner G, Nilforoushan A, Vainzof M, Suzuki OT, et al. Limb-girdle muscular dystrophy type 2G is caused by mutations in the gene encoding the sarcomeric protein telethonin. Nat Genet 2000; 24(2):163–166. Copyright 2006 by Taylor & Francis Group, LLC

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71. Frosk P, Weiler T, Nylen E, Sudha T, Greenberg CR, Morgan K, et al. Limbgirdle muscular dystrophy type 2H associated with mutation in TRIM32, a Putative E3-Ubiquitin-Ligase Gene. Am J Hum Genet 2002; 70(3):663–672. 72. Weiler T, Greenberg CR, Zelinski T, Nylen E, Coghlan G, Crumley MJ, et al. A gene for autosomal recessive limb-girdle muscular dystrophy in manitoba hutterites maps to chromosome region 9q31-q33: evidence for another limbgirdle muscular dystrophy locus. Am J Hum Genet 1998; 63(1):140–147. 73. Hackman P, Vihola A, Haravuori H, Marchand S, Sarparanta J, De Seze J, et al. Tibial muscular dystrophy is a titinopathy caused by mutations in TTN, the gene encoding the giant skeletal-muscle protein titin. Am J Hum Genet 2002; 71(3):492–500. 74. Udd B. Limb-girdle type muscular dystrophy in a large family with distal myopathy: homozygous manifestation of a dominant gene? J Med Genet ; 29(6):383–389. 75. van der Kooi AJ, Ledderhof TM, de Voogt WG, Res CJ, Bouwsma G, Troost D, et al. A newly recognized autosomal dominant limb girdle muscular dystrophy with cardiac involvement. Ann Neurol 1996; 39(5):636–642. 76. Maraldi NM, Squarzoni S, Sabatelli P, Capanni C, Mattioli E, Ognibene A, et al. Laminopathies: Involvement of structural nuclear proteins in the pathogenesis of an increasing number of human diseases. J Cell Physiol 2004. 77. Walter MC, Witt TN, Weigel BS, Reilich P, Richard P, Pongratz D, et al. Deletion of the LMNA initiator codon leading to a neurogenic variant of autosomal dominant Emery-Dreifuss muscular dystrophy. Neuromuscul Disord 2005; 15(1):40–44. 78. Kirschner J, Brune T, Wehnert M, Denecke J, Wasner C, Feuer A, et al. p.S143F mutation in lamin A/C: a new phenotype combining myopathy and progeria. Ann Neurol 2005; 57(1):148–151. 79. Messina DN, Speer MC, Pericak-Vance MA, McNally EM. Linkage of familial dilated cardiomyopathy with conduction defect and muscular dystrophy to chromosome 6q23. Am J Hum Genet 1997; 61(4):909–917. 80. Selcen D, Engel AG. Mutations in myotilin cause myoﬁbrillar myopathy. Neurology 2004; 62(8):1363–1371. 81. Carbone I, Bruno C, Sotgia F, Bado M, Broda P, Masetti E, et al. Mutation in the CAV3 gene causes partial caveolin-3 deﬁciency and hyperCKemia. Neurology 2000; 54(6):1373–1376. 82. Herrmann R, Straub V, Blank M, Kutzick C, Franke N, Jacob EN, et al. Dissociation of the dystroglycan complex in caveolin-3-deﬁcient limb girdle muscular dystrophy. Hum Mol Genet 2000; 9(15):2335–2340. 83. Kubisch C, Ketelsen UP, Goebel I, Omran H. Autosomal recessive rippling muscle disease with homozygous CAV3 mutations. Ann Neurol 2005; 57(2): 303–304. 84. Betz RC, Schoser BG, Kasper D, Ricker K, Ramirez A, Stein V, et al. Mutations in CAV3 cause mechanical hyperirritability of skeletal muscle in rippling muscle disease. Nat Genet 2001; 28(3):218–219. 85. Tateyama M, Aoki M, Nishino I, Hayashi YK, Sekiguchi S, Shiga Y, et al. Mutation in the caveolin-3 gene causes a peculiar form of distal myopathy. Neurology 2002; 58(2):323–325.

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9 Non-dystrophic Myotonias and Periodic Paralyses Arie Struyk and Stephen Cannon Department of Neurology, University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A.

INTRODUCTION One of the intriguing developments in the biology of excitable membranes has been the discovery that several heritable disorders are due to mutations of ion channel genes. The integrated function of many ion channels is required for the generation and propagation of action potentials over the skeletal muscle sarcolemma and into the transverse tubules. Derangements in muscle electrical excitability cause myotonia or periodic paralysis. This chapter reviews the molecular genetic origins of these disorders and outlines the pathophysiological link between altered behavior of mutant channels and clinical phenotype. Disordered Sarcolemmal Excitability Is a Spectrum of Phenotypes The phenotypic manifestations of disordered sarcolemmal excitability cover a spectrum between transient loss of excitability (paralytic attacks) and enhanced excitability manifest as involuntary tonic muscle activity lasting several seconds, preventing muscle relaxation after voluntary contraction (myotonic stiffness). The electrophysiological basis of this delayed relaxation is a self-sustaining train of action potentials, which ﬂuctuates in amplitude and frequency over several seconds (Fig. 1) and produces a characteristic

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Figure 1 Sustained discharges recorded from a myotonia congenita patient. Myotonia waxes and wanes for several seconds in the top trace, while decay of a myotonic discharge is demonstrated in the lower trace. Voltage and time scales are noted for each trace. (Courtesy of Gil Wolfe, M.D., Department of Neurology UTSouthwestern Medical Center.

‘‘dive-bomber’’ sound on the audio monitor during needle ‘‘compound muscle action potential’’ (CMAP) (1). Attacks of weakness and periodic paralysis are characterized by loss of muscle contractility, often with reduced tendon reﬂexes and tone. The EMG during paralytic episodes shows a reduced interference pattern and reduced amplitude of the compound muscle action potential (CMAP). Remarkably, some affected individuals may have both myotonia and periodic paralysis. Physiological Basis of the Sarcolemmal Excitability The physiological underpinnings of muscle excitability have been an important guide in the identiﬁcation of disease genes and conceptual understanding of disease mechanisms. Microelectrode studies of muscle biopsied from patients with myotonia or periodic paralysis revealed the alterations in speciﬁc ionic currents, which then implicated candidate disease genes in these disorders. Once the genetic basis of these disorders was identiﬁed, the identiﬁcation of speciﬁc mutations has led to a consideration of genotype– phenotype correlations and to both theoretical and biological models of how functional alterations of mutant channels account for the spectrum of phenotypes in these disorders. A brief review of this physiology will help put descriptions of these disorders in context. Electrical excitability arises from two fundamental cellular properties: the concentration gradient of diffusible ions and the selective permeability of the membrane. Steep concentration gradients of Naþ and Kþ ions are

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maintained across the sarcolemma through the continuous activity of ion pumps. The electrochemical gradients thus established deﬁne equilibrium potentials for each of these ions, denoted ENaþ and EKþ where the net inward movement balances the outward ﬂux of these ions. In muscle, EKþ is about 95 mV and ENa is þ66 mV. The importance of these values lies in the fact that the membrane potential is determined by a weighted average of the equilibrium potentials, in proportion to their relative contribution to the total membrane conductance. At rest, the sarcolemmal permeability to Kþ, although small, nevertheless predominates, setting the resting membrane potential near EKþ of 95 mV. In response to the small local depolarization caused by acetylcholine released from the nerve terminal binding to its postsynaptic receptor, an action potential is initiated and propagated by the opening of Naþ channels, which depolarizes the membrane potential toward ENaþ. An unusual feature of skeletal muscle is the large number of chloride channels open at the resting membrane potential (2). The resting Cl conductance is 2.5 times larger than the potassium conductance that sets the resting membrane potential. In contrast to Naþ and Kþ, however, Cl ions are not distributed by an active pump mechanism, and the passive equilibration of the Cl across the membrane adjusts the intracellular concentration until ECl equals the resting potential. The importance of the Cl conductance is revealed whenever the membrane potential deviates from its resting value. In response to this shift, a large Cl current will ﬂow that tends to return the membrane potential to its previously established resting value. In effect, the Cl conductance is an electrical buffer that stabilizes the resting membrane potential against small depolarizing currents. Current ﬂow through the pore of an ion channel is regulated by conformational changes in the protein that open or close access to the pore in a process termed ‘‘gating.’’ The open or closed conformation of the gate is regulated by various stimuli for different channels, such as ligand binding, stretch, intracellular messengers, or voltage itself. The gating properties of ion channels are critically important for orchestrating the precisely timed sequence of events that produce an action potential. The action potential is a wave of membrane depolarization that rapidly propagates in an all-or-none fashion along the length of the muscle ﬁber. At the motor endplate, local depolarization is initiated by ligand activation of nicotinic acetylcholine receptors. This initial depolarization causes voltage-gated Naþ channels to open (‘‘activate’’), producing a sharp rise in sarcolemmal Naþ conductance and the rapid (within 1 msec) upstroke of an action potential toward ENaþ. Repolarization during the latter part of an action potential is dependent on three phenomena: First, Naþ channels inactivate in a slightly delayed, voltage-dependent process, effectively shutting off the Naþ current within milliseconds of its activation. Second, voltagegated Kþ channels activate with delayed kinetics as the membrane depolarizes, increasing the membrane permeability to Kþ and promoting the

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return of the membrane potential toward EKþ. Finally, voltage-gated Cl channels open, further increasing the already large chloride conductance and accelerating the recovery of the resting membrane potential. The availability of Naþ channels, after having recovered from the inactive state to a closed, resting conformation, is a critical determinant of the capability of propagating another action potential. The recovery process is highly voltage dependent (faster and more complete at more negative potentials). Even modest shifts in the resting membrane potential may have a large effect on Naþ channel availability. A depolarization of 10 mV, for example, may inactivate enough Naþ channels to render the sarcolemma inexcitable.

CHLORIDE CHANNEL MYOTONIAS (MYOTONIA CONGENITA) Phenotypes Myotonia congenita is unique among the disorders of sarcolemmal excitability, in that it can be inherited as either a dominant or a recessive trait. The dominant form was ﬁrst described by Asmus Julius Thomsen in 1876, who suffered from the disorder himself. Dominant myotonia congenita or Thomsen’s disease usually becomes clinically apparent during infancy. The more severe recessive myotonia (Becker’s generalized myotonia) typically presents later in the ﬁrst decade, initially affecting the lower extremities then spreading to involve the rest of the skeletal musculature within a few years. Clinical examination readily elicits both grip myotonia (delayed relaxation after a voluntary grasp) and percussion myotonia (sustained contraction of a muscle elicited by percussion with a reﬂex hammer). Myotonic stiffness tends to lessen signiﬁcantly after several repetitions, known as the ‘‘warm-up’’ phenomenon. Cooler ambient temperatures may increase the myotonic activity but does not affect contractile force. Skeletal muscle hypertrophy is often observed, due to chronically high muscular activity. Occasional transient episodes of weakness have been noted in the recessive form, but these last only seconds to a few minutes in contrast to the prolonged episodes in periodic paralysis. In general, symptom penetrance in males is greater than females. Mutation of the C1C-1 Chloride Channel Underlies Myotonia Congenita One of the ﬁrst clues to the pathophysiology of human myotonia congenita arose from electrophysiological studies of goats afﬂicted with a dominant form of myotonia, in which microelectrode recordings revealed a marked reduction in membrane chloride conductance (3). Subsequent to this, a similarly reduced chloride conductance was found in biopsy ﬁbers from

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myotonia congenita patients (4). These electrophysiological insights led to a candidate gene approach to screen for mutations in CLCN1. This gene encodes the C1C-1 channel (5), a 988 amino acid protein that homodimerizes to form the major chloride conductance in skeletal muscle (6). To date, over 60 disease-causing mutations in CLCNl have been reported (for a detailed list, see Ref. 7). Of these, only a few are dominant mutations. The clinical delineation between dominant and recessive phenotypes is slightly blurred at the genotypic level, as a number of individual mutations show incomplete dominance or variable penetrance. Loss of Chloride Conductance in C1C-1 Mutants Causes Myotonia The physiological basis of myotonia in all variants of myotonia congenita is reduced chloride current carried by C1C-1. In addition to the loss of the repolarizing inﬂuence of the Cl current, this defect renders the sarcolemma more susceptible to the use-dependent depolarizing effect of potassium accumulation in the transverse tubules due to the egress of Kþ from the myoplasm with each action potential (8). This Kþ-induced depolarization is thought to elicit self-sustaining trains of action potentials (APs) in the absence of an endplate stimulus. Pharmacological studies indicate that block of up to 70% of the sarcolemmal chloride conductance is well tolerated; only in the setting of more severe decreases in chloride conductance do myotonic discharges appear (9). This phenomenon offers a context in which to differentiate the modes of inheritance of myotonia-inducing mutations. In general, mutations that render the protein incapable of dimer formation through premature truncation of the polypeptide (frameshift insertions or deletions, splice site defects, or nonsense codons), underlie the recessively inherited disease variant (10). In these cases, heterozygotic carriers with one wildtype allele retain 50% of C1C-1 activity, sufﬁcient to prevent myotonic hyperexcitability. Patients with two recessive mutant alleles are unable to generate any C1C-1 channels, probably explaining the greater symptomatic severity in these patients. Given the large number of different recessively linked mutations distributed in the population, patients with the recessive variant are usually compound heterozygotes for two different peptide truncating mutations, rather than homozygotes for the same mutation. Heterozygous carriers are asymptomatic, but latent myotonia may be detected electromyographically in some individuals (11). In contrast, mutations linked to the dominantly inherited variant generally do not prevent dimer formation with subunits derived from the wildtype allele, but rather exert a dominant-negative effect such that the chloride conductance of the heteroallelic dimer pairs is severely impaired (12). Commonly, this dominant negative effect is a positive shift in the voltage dependence of channel activation, such that heteroallelic dimers are signiﬁcantly less likely to be open when the membrane is between 40 mV and 80 mV.

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Treatment Most patients with myotonia congenita manage their symptoms by avoiding maneuvers that provoke myotonic stiffness, and thus do not require pharmacological intervention. In cases of disabling myotonic stiffness, treatment with use-dependent Naþ channel blockers (e.g., mexiletine) is effective in reducing or often completely preventing myotonia.

SODIUM CHANNEL MYOTONIA AND PERIODIC PARALYSIS Phenotypes In contrast to chloride channel myotonia, the sodium channelopathies of skeletal muscle always result in autosomal dominant inheritance with high penetrance. Another unique feature of the Naþ channel disorders is that the phenotypic variability spans the entire spectrum from myotonia only (potassium-aggravated myotonia), to admixtures of myotonia and periodic paralysis (paramyotonia congenita and hyperkalemic periodic paralysis), to periodic paralysis without myotonia (hypokalemic periodic paralysis). The sodium channel disorders associated with myotonia are observed more frequently and share the common feature of underlying gain-of-function defects. Sodium channel mutations are an infrequent cause of hypokalemic periodic paralysis, as described in the next section. Potassium aggravated myotonia (PAM) bears a close phenotypic resemblance to dominantly inherited myotonia congenita. Only after genetic screening became available was it determined that a subset of patients clinically diagnosed with dominantly inherited congenital myotonia (Thomsen’s disease) were instead linked to the Naþ channel gene. In the wake of this revelation, subtle phenotypic differences between the two disorders, ﬁrst noted by Thomsen in his early monographs, became clearer. Like the stiffness characteristic of myotonia congenita, the myotonic stiffness of PAM is the greatest upon initiation of movement, and declines after ‘‘warm-up’’ repetition. As reﬂected in the name, however, elevation of serum potassium tends to increase the frequency and duration of the myotonia in PAM. In addition, myotonic stiffness attributable to PAM is often variable day-today, and is often worsened after very prolonged periods of exercise, in contrast to myotonia congenita. Attacks of weakness usually do not occur and if they do are very infrequent, brief, and mild. Temperature changes do not alter the clinical symptoms. Paramyotonia congenita (PMC) is also a predominantly myotonic phenotype. However, the ‘‘paradoxical’’ myotonia of this disorder allows clinical differentiation from other myotonic disorders. In contrast to the ‘‘warm-up’’ phenomenon of PAM and myotonia congenita, the hallmark of PMC is the exacerbation of myotonia by repeated or continuous effort.

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Myotonia worsens upon exposure to cold, and occasionally episodes of ﬂaccid paralysis may also occur under cooling conditions. The classic presentation of hyperkalemic periodic paralysis (HyperPP) is episodic attacks of ﬂaccid paralysis in association with elevated serum potassium. Myotonic stiffness is usually found between attacks and may be the more prominent complaint in a subset of patients. Paralytic episodes typically start during the ﬁrst decade. Paralysis arises throughout the skeletal musculature within a few minutes, typically sparing bulbar and respiratory muscle function. In many cases, attacks of weakness can be predictably precipitated by exercise or exogenous administration of potassium, although other episodes may occur without obvious provocation. Episodes of paralysis frequently last for several minutes, but durations on the order of hours can occur and full strength may not return for days. The classic ﬁnding of serum hyperkalemia during an attack aids in establishing the diagnosis, but a normal serum potassium level does not rule it out. Cooling neither exacerbates the myotonia nor affects contractile force. A late onset permanent muscle weakness often affects the limb girdle musculature in HyperPP after the fourth decade and is associated with a vacuolar myopathy and accumulation of tubular aggregates. Point Mutations of the Voltage-Gated Na1 Channel Cause PAM PMC and HyperPP Early electrophysiological studies of skeletal muscle ﬁbers derived from biopsies of patients with both PMC and HyperPP identiﬁed an aberrant, non-inactivating Naþ current in both populations (13,14). This discovery implicated SCN4A, the gene encoding the alpha subunit of the voltage-gated sodium channel of skeletal muscle (Nav1.4), as a candidate for linkage screening. The gene product of SCN4A is an 1851 amino acid protein (Navl.4) that forms the main pore-forming subunit of the skeletal muscle Naþ channel. Navl.4 is a member of a large family of voltage-gated Naþ channels distributed in other excitable tissues (15). In all voltage-gated Naþ channels, the a subunit contains four large homologous domains, each of which is composed of six transmembrane segments (S1–S6). The fourth transmembrane segment within each domain contains an evenly spaced series of positively charged residues (either arginine or lysine). Substantial evidence indicates that translocation of these segments in response to changes in the membrane potential confers voltage dependence to channel gating. In skeletal muscle, Nav1.4 is non-covalently co-assembled with an accessory b1 subunit (16). Other Naþ channels in brain and heart are also heteromeric assemblies of a and accessory b subunits. The b subunits appear to be important in the targeting and anchoring of sodium channel complexes and also modulate channel gating. Missense mutations in the b1 subunit

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have been associated with a rare form of generalized epilepsy, but curiously these patients do not have cardiac or skeletal muscle defects (17). More than 30 mutations in SCN4A have been identiﬁed in kindreds with myotonia or periodic paralysis (Fig. 2). In all cases, the mutation results in a missense substitution in the open reading frame. No deletion, nonsense, or splice site defects have been reported. Mutations are clustered in three functional domains of NaVl.4. One group lies in the cytoplasmic loop linking the third and fourth homologous domains. Another cluster is at the cytoplasmic ends of S5 or S6 transmembrane segments, which line the inner mouth of the ion-conducting pore. These regions are thought to form the inactivation gate and its docking site, respectively, and explains why such a widely spread distribution of mutations in the primary sequence disrupt inactivation. The third cluster of mutations is in the S4 segment of the fourth domain, which is a voltage-sensing domain that couples membrane depolarization to inactivation. For the most part, individual missense mutations are consistently linked with PAM, PMC, or HyperPP. However, heterogeneity is often found

Figure 2 Diagrams depict the major structural features of NaV1.4 (A) and CaV1.1 (B) in relation to the plasma membrane. Amino and carboxyl termini for both proteins are oriented intracellularly. S4 membrane-spanning segments carrying positive charges (voltage sensors) are denoted by plus signs. Locations of mutations associated with PAM (gray squares), PMC (gray triangles), HyperPP (gray circles) and HypoPP (black circles) are overlaid.

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in the phenotypic expression of identical mutations between individuals, sometimes within the same kindred. In some cases, incomplete penetrance has been reported, suggesting the existence of modifying factors important for full phenotypic expression (18). The bulk of PAM, HyperPP, and PMC cases are accounted for by a few mutations occurring more frequently in the population, whereas several mutations have been demonstrated in only one kindred or even single individuals. Haplotype analysis has not supported a common founder effect and suggests these more frequently occurring mutant alleles have arisen as independent events (19,20). The Physiological Basis of Myotonia and Paralysis: Na1 Channel Gain of Function Defects The electrical derangements of the sarcolemma in PAM, PMC, and HyperPP all share the common feature that the primary abnormality is a gain-of-function of mutant Naþ channels, usually due to an impairment of inactivation but also caused in some cases by enhanced activation. The mechanism by which both myotonia and periodic paralysis arise from this common defect (and can co-exist in many patients with these disorders) has been established by a combination of experimental and computational models. A toxin-based model has provided direct experimental evidence that even a partial disruption of inactivation is sufﬁcient to produce myotonia. Application of micromolar concentrations of ATXII toxin from sea anemone to rat muscle in vitro prevents inactivation for about 2% of Naþ channels. A single shock elicits a burst of myotonic discharges and prolonged contraction in toxin-exposed ﬁbers. In a computer simulation of the electrical properties of a muscle ﬁber, introduction of a persistent Naþ current due to a small fraction (2%) of inactivation-deﬁcient Naþ channels is sufﬁcient to generate self-sustaining myotonic discharges. A slight increase in the fraction of non-inactivating Naþ channels (3%) induces myotonic runs that dissipate into a persistent membrane depolarization at 40 mV, rendering the sarcolemma incapable of propagating further action potentials. This sustained aberrant depolarization is maintained by the persistent inward Naþ current through the inactivation-deﬁcient fraction of channels. The simulation demonstrates that the critical parameter for determining paralytic and non-paralytic phenotypes is the degree to which inactivation is compromised by a missense mutation. Experimental support for this is found in the observation that those mutants associated with paralytic phenotypes tend to have more signiﬁcant defects of inactivation than those linked to purely myotonic phenotypes (21). Additional features of Naþ channel gating inﬂuence the association between speciﬁc mutations and the clinical phenotype. For example, some mutations slow the rate of inactivation, which otherwise is eventually complete (22). This kinetic defect is sufﬁcient to produce transient bursts of

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myotonic discharges, but cannot cause sustained depolarization and ﬂaccid paralysis. Sodium channels also undergo more than one type of inactivation. In response to prolonged depolarization lasting seconds, or to the more physiologically common scenario of sustained trains of discharges, sodium channels enter a more stable ‘‘slow inactivated’’ state. Slow inactivation is detected by the prolonged duration required for recovery of channel availability at the resting potential, which is in the order of several seconds as compared to the recovery from fast inactivation within a few milliseconds. Slow inactivation is mediated by different structural components in Navl.4 than those responsible for fast inactivation (23). This observation led to the prediction that mutations associated with paralytic phenotypes should also have defective slow inactivation (24). Otherwise, the slow inactivation mechanism would shut off the aberrant persistent Naþ current and allow the ﬁber to hyperpolarize. Indeed, functional testing of NaVl.4 mutants has shown that every mutation that impairs slow inactivation is associated with a paralytic phenotype (HyperPP). The converse is not always true, however, and has led to the notion that defective slow inactivation greatly increases the propensity for attacks of periodic paralysis, but is not necessary. Treatment Most patients with the Naþ channel disorders PAM/PMC/HyperPP minimize the frequency and severity of attacks by changes in lifestyle to avoid environmental triggers. Myotonic stiffness responds well to use-dependent Naþ channel blockers, such as local anesthetic derivatives (mexiletine), antiarrhythmics, and Naþ-selective anticonvulsants. Paralytic attacks can be foreshortened or prevented with the carbonic anhydrase inhibitor acetazolamide, although the mechanism for this effect is not well understood. Potassium wasting diuretics have proven somewhat beneﬁcial at reducing the frequency and severity of paralytic attacks. HYPOKALEMIC PERIODIC PARALYSIS Phenotype Familial hypokalemic periodic paralysis (HypoPP) is transmitted in an autosomal dominant pattern. The classic phenotype is recurrent episodes of severe weakness or even ﬂaccid paralysis associated with serum hypokalemia. The ﬁrst symptomatic attacks usually occur around puberty, which is distinctly later than the onset of HyperPP. The duration of the paralytic episodes is variable, ranging between several minutes to hours or sometimes days. Provocation of the attacks classically occurs hours following heavy carbohydrate ingestion, often late at night or during the early morning hours after a large meal. Similar to HyperPP, prolonged exercise can precipitate

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an attack. Interictal myotonic stiffness is not found in association with HypoPP. The attacks of weakness typically decline in frequency and severity over time, and become rare after the fourth decade. Familial HypoPP can be mimicked by a few acquired disorders. Among these is thyrotoxic periodic paralysis, which is characterized by attacks of hypokalemic ﬂaccid paralysis in the setting of thyrotoxicosis. The increased incidence of thyrotoxic periodic paralysis in the Japanese population suggests a genetic predilection (25), but the majority of cases are sporadic and mapping of susceptibility loci has not been achieved. Laxative abuse or renal Kþ wasting states can induce similar weakness, but life-threatening cardiac arrhythmias usually precede the development of weakness under these circumstances, making the observation of paralysis as an isolated symptom unlikely. Laboratory investigations during an attack often reveal characteristic low serum potassium (usually <2.5 mm), although as with HyperPP, the serum potassium may be normal if the sample is taken during the later stages of an attack. During an attack the ﬁbers are silent, whereas the interictal EMG is normal. The late-onset myopathy is associated with vacuolar changes and T-tubular aggregates that are histologically indistinguishable from the late-onset myopathy in HyperPP (26). Point Mutations of the Voltage-Gated CA21 and Na1 Channels Cause HypoPP HypoPP was linked to chromosome lq.31-32 (27) and mutations were subsequently identiﬁed in the CACNAIS gene (28), which encodes the alpha-lS subunit (CaV1.1) of the skeletal muscle voltage-gated Ca2þ channel, also known as the dihydropyridine-sensitive L-type Ca2þ channel. CaV1.1 shares structural homology to the voltage-gated Naþ channel: it is organized into four domains, each of which is composed of six transmembrane segments (29). HypoPP is caused by three different missense mutations of CaV1.1, all at positively charged residues in the S4 segments of the second or fourth domains (Fig. 2) (28), which, as in the Naþ channel, confer voltage dependence to Ca2þ channel function. In the wake of this initial genotypic characterization, several HypoPP kindreds were found to have no linkage to CACNAIS, indicating genetic heterogeneity of this disorder. More recently, a number of kindreds have been linked to SCN4A (30,31). Four different missense mutations have now been reported, all introducing amino acid substitutions at charged residues in the voltage-sensing S4 segment within the second domain of Navl.4 (Fig. 2). In total, Ca2þ channel mutations are found in 60% and Naþ channel mutations in 20% of kindreds with HypoPP. The genetic basis of the remaining 20% is unknown. The clinical history, frequency and severity of paralytic attacks are indistinguishable in Naþ and Ca2þ-channel linked subtypes, although

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differences in response to treatment have been reported in small patient series (32). Late-onset proximal myopathy is also common for the two subtypes (32). Penetrance in female carriers is variable regardless of genotype, and affected women may develop late-onset proximal myopathy without ever having recognized acute paralytic attacks (33). Physiological Defects Associated with HypoPP Paralytic attacks associated with HypoPP are due to aberrant, persistent depolarization of the resting potential, which inactivates Naþ channels and renders the ﬁber inexcitable. The underlying basis of this pathological depolarization has not been ﬁrmly established. Pharmacological studies of affected muscle ﬁber preparations in vitro have excluded a depolarizing effect of an anomalously high inward current carried by Nav1.4 or CaV1.1 (34,35). Gating behavior of HypoPP mutant Ca2þ and Naþ channels have been studied extensively, but a clear explanation for the depolarization-induced weakness and hypokalemia has not emerged. Caþ channel mutants reduce channel expression level and slow the rate of activation, suggesting a loss-offunction defect (21). The physiological link between Ca2þ channel mutations and membrane depolarization remains unclear. A common consequence of HypoPP-associated Naþ channel mutations is enhancement of slow inactivation (31,36). In some Naþ channel mutations, fast inactivation appears to be mildly enhanced as well (31). The greater propensity of HypoPP mutant Naþ channels to inactivate may contribute to membrane inexcitability, but fails to account for the anomalous depolarization during an attack nor does it explain the paroxysmal nature of the disorder. Other experimental evidence indicates that abnormal regulation of potassium conductances generates the instability of the resting membrane potential in HypoPP. Electrophysiological characterization of intercostal muscle ﬁbers derived from HypoPP patients reveals abnormally low potassium currents near the resting potential (34,35). Reduction in Kþ conductance is predicted to result in depolarization, and may contribute to hypokalemia by trapping Kþ in muscle. Alternatively, reduction of serum Kþ may itself be the precipitating stimulus for a paralytic attack, by triggering an abnormal response in the skeletal muscle. These observations have led to the hypothesis that the pathomechanism in HypoPP is a disruption of the resting Kþ conductance that is indirectly coupled to Ca2þ or Naþ mutations by a means that remains to be established. The reduced Kþ conductance increases the susceptibility to prolonged episodes of depolarization-induced inexcitability and weakness. Treatment Acute attacks are self-limited, and most patients manage their symptoms by avoiding triggers. Break through attacks may respond well to oral Kþ

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administration. Aggressive Kþ administration should be avoided, since total body potassium is not depleted during attacks and overshoot hyperkalemia may result as previously sequestered Kþ shifts back to the extracellular compartments. Daily acetazolamide is effective prophylactically in many patients (37). As with HyperPP, the mechanism by which acetazolamide exerts its prophylactic effect is unknown for HypoPP. Some evidence suggests that Naþ -linked channel mutations may be more likely to be unresponsive or exacerbated by acetazolamide administration (32). ANDERSEN’S SYNDROME Phenotype Andersen’s syndrome (AS) is a triad of periodic paralysis, cardiac arrhythmias, and dysmorphic features (38,39). The morphological changes are modest and include clinodactyly, scoliosis, hypertelorism, micrognathia, and short stature. The cardiac abnormalities are varied and range between asymptomatic long-QT syndrome to recurrent torsade de pointes with cardiac arrest. Episodic attacks of paralysis in AS are often accompanied by hypokalemia, although normokalemic paralysis, and rarely hyperkalemia, has been reported (40). Triggers for paralytic attacks are not well described. As in HyperPP and HypoPP, the electrophysiological basis of paralysis is aberrant depolarization of the sarcolemma. The mode of transmission is autosomal dominant, although the phenotypic expression is highly variable both between and within kindreds. Asymptomatic gene carriers are frequently identiﬁed, as are individual patients exhibiting only one or two of the triad of symptoms, usually with a further marked variability in severity. Andersen’s Syndrome Is Linked to Mutations of the K1 Channel Kir2.1 The genetic basis of AS was recently identiﬁed in several kindreds as mutation of the KCNJ2 gene on the long arm of chromosome 17 (41). KCNJ2 encodes Kir2.1, an inwardly rectifying Kþ channel subunit expressed widely throughout the body, including skeletal muscle, heart, brain, and osteoclasts. The multisystemic manifestations of AS presumably reﬂect this widespread tissue distribution. Most mutations that cause AS are missense mutations resulting in amino acid substitution, distributed throughout the coding sequence (Fig. 3) (41). Two in-frame deletions, one in the ﬁrst transmembrane domain and one near the C-terminus, have been reported Kir2.1 subunits contain two membrane-spanning segments, with an intervening pore-forming loop (42). The channel complex is a tetramer of subunits, which can form as homomultimers of Kir2.1 subunits or heteromultimeric tetramers containing related Kþ channel subunits Kir2.2 and

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Figure 3 The membrane topology of Kir2.1 is diagrammed, and positions of currently reported Andersen syndrome mutations are overlaid. Amino and carboxyl termini are intracellular. Gray circles represent missense mutations, while gray rectangles depict in-frame deletions. Initial reports of the mutations diagrammed are found in (41,44,45,49,50).

Kir2.3 (four further members of this family have been identiﬁed, but their ability to co-assemble with Kir2.1 is not known). The signiﬁcance of potential heteromultimerization in vivo is not well known. Inward rectiﬁer Kþ channels, such as Kir2.1, conduct inward Kþ current at hyperpolarized potentials much more readily than outward Kþ current in depolarized cells. This feature is important for regulating repolarization at the end of an action potential. The Physiological Basis of Andersen’s Syndrome: Loss of Resting K1 Conductance Insights on the pathophysiological basis of AS have been derived from functional expression studies of mutant Kir2.1 channels investigated in heterologous expression systems such as Xenopus oocytes or mammalian ﬁbroblasts. Under these conditions, subunits bearing AS-related mutations co-assemble with wildtype subunits and exert a dominant negative effect, impairing the Kþ conductivity of the heteromultimeric complexes (41,43–46). Some evidence suggests that mutant subunits can impair membrane trafﬁcking of Kir2.1 multimers (47). The severity of this dominant negative effect varies depending on the point mutation; however, there is no clear correlation between the severity of the clinical phenotype associated with a particular mutation and the reduction in Kþ current in these assays. This dominant negative effect is the basis for our understanding of the pathophysiology of AS mutations in vivo. Reduction or loss of an inward rectiﬁer Kþ current is expected to impede the repolarization phase of the cardiac action potential. In skeletal muscle, this same defect may reduce the stability of the resting potential and increase the propensity for sustained depolarization and Kþ trapping in the muscle. The role of the Kir2.1 conductance in skeletal development remains to be delineated, but it is noteworthy that these channels are expressed in osteoclasts. Moreover, the Kir2.1 null mouse has severe craniofacial defects leading to neonatal lethality (48).

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No consistently effective treatment for the episodic weakness is known for AS, although acetazolamide administration has proven modestly effective in a handful of anecdotal cases. CONCLUSIONS The familial non-dystrophic myotonias and periodic paralyses are all disorders of muscle membrane excitability in which the primary defects are mutations in the coding region of ion channel genes. The molecular genetic identiﬁcation of these defects and the experimental assessment of their functional consequences have broadened our understanding of the pathophysiological basis for paroxysmal disorders in excitable tissues and reﬁned our views of the roles that different ionic conductances play in tuning the excitability of muscle. The principles learned from the study of these channelopathies will certainly be of tremendous value in designing therapeutic interventions and will also likely provide insights into other disorders of electrical excitability such as epilepsy, cardiac arrhythmia, and some forms of headache. REFERENCES 1. Barchi RL. Myotonia [Review]. Neurologic Clinics 1988; 6(3):473–484. 2. Bryant SH, Morales-Aguilera A. Chloride conductance in normal and myotonic muscle ﬁbers and the action of monocarboxylic acids. J Physiol 1971; 219:367–483. 3. Lipicky RJ, Bryant SH. Sodium, potassium, and chloride ﬂuxes in intercostal muscle from normal goats and goats with hereditary myotonia congenita. J Gen Physiol 1966; 50:89–111. 4. Lipicky RJ, Bryant SH, Salmon JH. Cable parameters, sodium, potassium, chloride, and water content, and potassium efﬂux in isolated external intercostal muscle of normal volunteers and patients with myotonia congenita. J Clin Invest 1971; 50:2091–2103. 5. Koch MC, Steinmeyer K, et al. The skeletal muscle chloride channel in dominant and recessive human myotonia. Science 1992; 257(5071):797–800. 6. Fahlke C, Knittle T, Gurnett CA, Campbell KP, George AL Jr. Subunit stoichiometry of human muscle chloride channels. J Gen Physiol 1997;109(l): 93–104. 7. Pusch M. Myotonia caused by mutations in the muscle chloride channel gene CLCN1. Hum Mutat 2002; 19(4):423–434. 8. Adrian RH, Bryant SH. On the repetitive discharge in myotonic muscle ﬁbres. J Physiol 1974; 240(2):505–515. 9. Barchi RL. Muscle membrane chloride conductance and the myotonic syndromes. Electroencephalogr Clin Neurophysiol Supplement 1978; 34:559–570. 10. Fahlke C, Zachar E, Riidel R. Chloride channels with reduced single-channel conductance in recessive myotonia congenita. Neuron 1993; 10:225–232. 11. Deymeer F, Lehmann-Horn F, et al. Electrical myotonia in heterozygous carriers of recessive myotonia congenita. Muscle Nerve 1999; 22(1); 123–125.

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12. Steinmeyer K, Lorenz C, Pusch M, Koch MC, Jentsch T. Multimeric structure of C1C-1 chloride channel revealed by mutations in dominant myotonia congenita (Thomsen). EMBO J 1994; 13:737–743. 13. Lehmann-Horn F, Ru¨del R, Dengler R, Lorkovic H, Haass A, Ricker K. Membrane defects in paramyotonia congenita with and without myotonia in a warm environment. Muscle Nerve 1981; 4(5):396–406. 14. Lehmann-Horn F, Ru¨del R, Ricker K, Lorkovic H, Dengler R, Hopf HC. Two cases of adynamia episodica hereditaria: in vitro investigation of muscle cell membrane and contraction parameters. Muscle Nerve 1983; 6(2):113–121. 15. Catterall WA. Molecular properties of voltage-sensitive sodium channels. [Review]. Ann Rev Biochem 1986; 55:953–985. 16. McClatchey AI, Cannon SC, Slaugenhaupt SA, Gusella JF. The cloning and expression of a sodium channel beta 1-subunit cDNA from human brain. Hum Mol Gene 1993; 2(6):745–749. 17. Wallace RH, Wang DW, et al. Febrile seizures and generalized epilepsy associated with a mutation in the Naþ-channel betal subunit gene SCN1B. Nat Genet 1998; 19(4):366–370. 18. McClatchey AI, McKenna-Yasek D, et al. Novel mutations in families with unusual and variable disorders of the skeletal muscle sodium channel. Nature Genetics 1992; 2(2):148–152. 19. Plassart E, Reboul J, et al. Mutations in the muscle sodium channel gene (SCN4A) in 13 French families with hyperkalemic periodic paralysis and paramyotonia congenita: phenotype to genotype correlations and demonstration of the predominance of two mutations. Eur J Hum Genet 1994; 2110–2124. 20. McClatchey Al, Trofatter J, et al. Dinucleotide repeat polymorphisms at the SCN4A locus suggest allelic heterogeneity of hyperkalemic periodic paralysis and paramyotonia congenita [published erratum appears in Am J Hum Genet 1992 Oct; 51(4):942] [see comments]. Am J Hum Gene 1992; 50(5):896–901. 21. Cannon SC. An expanding view for the molecular basis of familial periodic paralysis. Neuromuscul Disord 2002; 12(6):533–543. 22. Chahine M, George AL Jr, et al. Sodium channel mutations in paramyotonia congenita uncouple inactivation from activation. Neuron 1994; 12(2):281-294. 23. Rudy B. Slow inactivation of the sodium conductance in squid giant axons. Pronase resistance. J Physiol 1978; 283:1–21. 24. Ruff RL. Slow Naþ channel inactivation must be disrupted to evoke prolonged depolarization-induced paralysis. Biophys J 1994; 66(2 Pt 1):542–545. 25. Resnick JS, Dorman JD, Engel WK. Thyrotoxic periodic paralysis. Am J Med 1969; 47(5):831–836. 26. Engel AG. Evolution and content of vacuoles in primary hypokalemic periodic paralysis. Mayo Clin Proc 1970; 45(11):774–814. 27. Fontaine B, Vale-Santos J, et al. Mapping of the hypokalaemic periodic paralysis (HypoPP) locus to chromosome lq31–32 in three European families. Nat Genet l994; 6(3):267–272. 28. Ptacek LJ, Tawil R, et al. Dihydropyridine receptor mutations cause hypokalemic periodic paralysis. Cell 1994; 77(6):863–868. 29. Catterall WA, de Jongh K, et al. Molecular properties of calcium channels in skeletal muscle and neurons. Ann NY Acad Sci 1993; 681:342–355.

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30. Bulman DE, Scoggan KA, et al. A novel sodium channel mutation in a family with hypokalemic periodic paralysis. Neurology 1999; 53(9):1932–1936. 31. Jurkat-Rott K, Mitrovic N, et al. Voltage-sensor sodium channel mutations cause hypokalemic periodic paralysis type 2 by enhanced inactivation and reduced current. Proc Natl Acad Sci USA 2000; 97(17):9549–9554. 32. Sternberg D, Maisonobe T, et al. Hypokalaemic periodic paralysis type 2 caused by mutations at codon 672 in the muscle sodium channel gene SCN4A. Brain 2001; 124(Pt 6):1091–1099. 33. Elbaz A, Vale-Santos J, et al. Hypokalemic periodic paralysis and the dihydropyridine receptor (CACNL1A3): genotype/phenotype correlations for two predominant mutations and evidence for the absence of a founder effect in 16 Caucasian families. Am J Hum Genet 1995; 56:374–380. 34. Ru¨del R, Lehmann-Horn F, Ricker K, Kuther G. Hypokalemic periodic paralysis: in vitro investigation of muscle ﬁber membrane parameters. Muscle Nerve 1984; 7(2):110–120. 35. Ruff RL. Insulin acts in hypokalemic periodic paralysis by reducing inward rectiﬁer Kþ current. Neurology 1999; 53(7):1556–1563. 36. Struyk AF, Scoggan KA, Bulman DE, Cannon SC. The human skeletal muscle Na channel mutation R669H associated with hypokalemic periodic paralysis enhances slow inactivation. J Neurosci 2000; 20(23):8610–8617. 37. Griggs RC, Engel WK, Resnick JS. Acetazolamide treatment of hypokalemic periodic paralysis. Prevention of attacks and improvement of persistent weakness. Ann Intern Med 1970; 73(1):39–48. 38. Andersen ED, Krasilnikoff PA, Overvad H. Intermittent muscular weakness, extrasystoles, and multiple developmental anomalies. A new syndrome? Acta Paediatr Scand 1971; 60(5):559–564 39. Tawil R, Ptacek LJ, et al. Andersen’s syndrome: potassium-sensitive periodic paralysis, ventricular ectopy, and dysmorphic features (see comments). Ann Neurol 1994; 35(3):326–330. 40. Sansone V, Griggs RC, et al. Andersen’s syndrome: a distinct periodic paralysis. Ann Neurol 1997; 42(3):305–312. 41. Plaster NM, Tawil R, et al. Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen’s syndrome. Cell 2001; 105(4):511–519. 42. Nichols CG, Lopatin AN. Inward rectiﬁer potassium channels. Annu Rev Physiol 1997; 59:171–191. 43. Preisig-Muller R, Schlichthorl G, et al. Heteromerization of Kir2.x potassium channels contributes to the phenotype of Andersen’s syndrome. Proc Natl Acad Sci USA 2002; 99(11):7774–7779. 44. Andelﬁnger G, Tapper AR, Welch RC, Vanoye CG, George AL Jr, Benson DW. KCNJ2 mutation results in Andersen syndrome with sex-speciﬁc cardiac and skeletal muscle phenotypes. Am J Hum Genet 2002; 71(3):663–668. 45. Tristani-Firouzi M, Jensen JL, et al. Functional and clinical characterization of KCNJ2 mutations associated with LQT7 (Andersen syndrome). J Clin Invest 2002; 110(3):381–388. 46. Lange PS, Er F, Gassanov N, Hoppe UC. Andersen mutations of KCNJ2 suppress the native inward rectiﬁer current IKl in a dominant-negative fashion. Cardiovasc Res 2003; 59(2):321–327.

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47. Bendahhou S, Donaldson MR, Plaster NM, Tristani-Firouzi M, Fu YH, Ptacek LJ. Defective potassium channel Kir2.1 trafﬁcking underlies Andersen-Tawil syndrome. J Biol Chem 2003; 278(51):51,779–51,785. 48. Zaritsky JJ, Eckman DM, Wellman GC, Nelson MT, Schwarz TL. Targeted disruption of Kir2.1 and Kir2.2 genes reveals the essential role of the inwardly rectifying K(þ) current in K(þ)-mediated vasodilation. Circ Res 2000; 87(2):160–166. 49. Donaldson MR, Jensen JL, et al. PIP2 binding residues of Kir2.1 are common targets of mutations causing Andersen syndrome. Neurology 2003; 60(1l):1811–1816. 50. Ai T, Fujiwara Y, et al. Novel KCNJ2 mutation in familial periodic paralysis with ventricular dysrhythmia. Circulation 2002; 105(22):2592–2594.

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10 The Myotonic Dystrophies—Effects of an RNA Mutation John W. Day Department of Neurology, Institute of Human Genetics, University of Minnesota School of Medicine, Minneapolis, Minnesota, U.S.A.

Laura P. W. Ranum Department of Genetics, Cell Biology and Development, Institute of Human Genetics, University of Minnesota School of Medicine, Minneapolis, Minnesota, U.S.A.

INTRODUCTION The ﬁrst pathogenic trinucleotide repeat expansions identiﬁed, in fragile X mental retardation (FMR) (1) and spinobulbar muscular atrophy (SBMA or Kennedy’s disease) (2), revealed two different pathogenic mechanisms—loss of gene product in recessively inherited FMR, and a pathogenic gain of function in SBMA. In FMR, a non-coding CGG repeat expansion at the 50 end of the FMR1 gene results in a loss of function that is consistent with the recessive inheritance of that disease. SBMA, alternatively, is caused by the expansion of a polyglutamine repeat (encoded by a CAG expansion) within the androgen receptor protein, resulting in a pathogenic protein. The repeat expansions in the two forms of myotonic dystrophy do not demonstrate either of these pathogenic mechanisms because they are non-coding mutations that result in a highly penetrant dominant disease. Clinical and molecular characterization of the myotonic dystrophies has demonstrated a novel disease mechanism

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involving pathogenic effects of repeat expansions that are expressed in RNA but do not encode any protein. CLINICAL FEATURES OF THE MYOTONIC DYSTROPHIES History of the Myotonic Dystrophies Steinert (3), and Batten and Gibb (4) independently described the disorder now known as myotonic dystrophy almost 100 years ago. In addition to the observed myotonia, the initial descriptions focused on skeletal muscle weakness and atrophy to distinguish the new disorder from Thomsen’s previously identiﬁed non-dystrophic myotonia (5), which is now known to be caused by point mutations in the gene encoding the muscle chloride channel. The complex multisystemic phenotype of myotonic dystrophy was gradually recognized, with involvement of eyes, heart, endocrine systems, and central nervous system (CNS) that clearly distinguished this disorder from other forms of muscular dystrophy (6). Genetic heterogeneity in myotonic dystrophy was unsuspected until 1992, when testing became available for the ﬁrst identiﬁed form (DM1), which is due to a mutation on chromosome 19 (7,8). In 1994, another multisystemic myotonic disorder was identiﬁed (9,10), and referred to as either proximal myotonic myopathy (PROMM) (9,11), proximal myotonic dystrophy (PDM) (12), or myotonic dystrophy type 2 (DM2) (13,14) in an attempt to emphasize differences or similarities with DMl. The substantial clinical overlap of these diseases led to the adoption of a revised nomenclature (15), with DM1 referring to the chromosome 19 form of the disease, and DM2 referring to the new genetically distinct disorder. The clinical features of the two diseases are given in the Table 1, comparing the ﬁndings in 234 genetically conﬁrmed cases of DM2 to the recognized DM1 phenotype. Onset and Progression of DM DM1 patients are often diagnosed as having congenital, juvenile, or adultonset disease based on age of onset (6). Due to ascertainment biases (e.g., family and patient awareness of deﬁcits, and medical provider awareness of myotonic dystrophy), patients of any age can have a wide range of developmental and degenerative features, which complicates any detailed characterization of the disease by age of onset. Neonatal Onset Patients with congenital DM1, as described by Harper (16), demonstrate an almost pure form of the developmental deﬁcits associated with DM. The pregnancies that produce these infants are complicated by hyperhydramnios, and the children are born with severe neurological, neuromuscular, and

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Table 1 Comparison of Clinical Features in Myotonic Dystrophy Types 1 and 2 Clinical features of DM2 and DM1 Clinical feature

DM2 n ¼ 234 (21–78 yr)

DM1

Skeletal muscle features Myotonia On EMG Weakness Any weakness on exam Neck ﬂexors Thumb or deep ﬁnger ﬂexors Hip ﬂexors Deep knee bend Multisystemic features Cardiac Conduction defect on ECG Cataracts By history or bedside exam

90 82 75 55 64 54

þþþ þþþ þþþ þþþ þ þ

20 60

þþ þþ

Additional laboratory ﬁndings (n) Serology Elevated CK (90) Low IgG (20) Low IgM (20) Low testosterone (22) High FSH (26) Insulin insensitivity (16)

90 65 11 29 65 75

þþ þþ þ/ þþ þþ þþ

Note: The abstracted qualitative frequencies of DM1 features are reported as being expected in all patients (þþþ), common among all patients (þþ), well recognized and common late in the course of the disease (þ), recognized but not common (þ/).

musculoskeletal abnormalities. They have craniofacial abnormalities, including tapered chin, high-arched palate and prominent brow, and may be born with arthrogryposis and talipes in addition to other orthopedic abnormalities (6). Mental retardation and global cerebral atrophy are also common in these patients. Some common degenerative features, including cataracts and central hypersomnia, typically occur during adulthood in these patients rather than occurring in infancy or childhood. No deﬁnite congenital form of DM2 has been reported (17). Juvenile Onset Cases of DM1 that come to medical attention during childhood typically manifest developmental abnormalities that are less severe than seen in congenital onset cases, though this is in part inﬂuenced by ascertainment biases (18). These patients typically have cognitive deﬁcits and learning abnormalities (19) that again distinguish them from DM2, in which a juvenile form has not been described. As in the congenital cases, degenerative features develop as these affected children reach adulthood.

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Adult-Onset DM1 patients typically come to medical attention during adulthood for several reasons: (i) they have congenitally affected children, (ii) other affected family members are identiﬁed, (iii) they develop symptoms of the degenerative disease (e.g., weakness, myotonia, cataracts, or gonadal failure). On examination, although some adult onset patients have no recognizable developmental defects, others manifest clear indicators of developmental abnormalities, including the craniofacial changes and a highly arched palate that indicate the effects of their disease in utero. The degenerative features of the disease occur in all adult onset cases over time, but can progress at different rates, with some middle-aged individuals succumbing to the disease over several years and others having a relatively static course for decades (20,21). DM2, given the absence of developmental effects, typically comes to medical attention in adulthood, though myotonia, myalgias, and cataracts can occur within the ﬁrst two decades of life. Though there may well be disease-speciﬁc differences between the degenerative features of DM1 and DM2, these can only be accurately determined by comparing DM2 patients with DM1 individuals who do not manifest any signiﬁcant developmental components of the disease.

Involvement of Skeletal Muscle in DM The skeletal muscle features in both genetic forms of myotonic dystrophy include progressive weakness, stereotyped changes on biopsy (6,17,22,23), and myotonia. Muscle pain can be a signiﬁcant feature of both disorders (6,17). The initial pattern of muscle weakness in DM1 and DM2 is detailed in Table 1. At onset both forms of the disease affect neck ﬂexors and distal upper extremity muscles [speciﬁcally deep ﬂexors of the thumb and deep ﬂexors of the lateral digits more than medial digits (24)]. As the disease progresses, involvement of the triceps and ankle dorsiﬂexors typically, though not invariably, precedes involvement of shoulder and hip girdle musculature. Although neck and ﬁnger ﬂexor weakness is demonstrable early in the course of DM2, patients with this disease who come to medical attention because of symptomatic weakness typically present with problems associated with hip girdle muscles, which led to the name PROMM (25). In later stages of both DM1 and DM2, diffuse profound weakness can develop, though DM1 involves bulbar, ventilatory, and pelvic ﬂoor musculature more notably than does DM2 (6,17). Neither DM1 nor DM2 is associated with the severe myotonia sometimes seen in chloride channelopathies (6,17), though electrical myotonia is seen in almost all adults with both forms of DM [Table 1 (17)] but is notably absent in infants with congenital onset DM1.

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Other Organ System Involvement in DM The most common cardiac effects of DM1 are atrioventricular and intraventricular conduction abnormalities, and sudden death (26,27); all of these features are also seen in DM2 (17). The cataracts in both diseases are stereotypic and indistinguishable, both being posterior subcapsular opacities that are iridescent on slit lamp examination (6,17). Various other features are common to both diseases, including testicular failure (both hypotestosteronism and oligospermia), hypogammaglobulinemia (serum levels of both IgG and IgM are reduced), and insulin resistance (6,17,28,29). Mental retardation is a recognized feature of congenital and juvenile onset DM1 (6) but has not been causally associated with DM2 [although retarded DM2 individuals have been reported (14)]; both DM1 and DM2 subjects develop CNS white matter abnormalities (30). Central hypersomnia is a recognized feature of DM1 that has not yet been speciﬁcally investigated in DM2, though daytime sleepiness has been reported in some DM2 subjects (14). GENETICS OF THE MYOTONIC DYSTROPHIES Identification of the DM1 Mutation Prior to identiﬁcation of the causative mutation, clinicians had already identiﬁed non-Mendelian features in DM1 inheritance including anticipation and a maternal transmission bias for congenital forms (6,31). The genetic cause of DM1 was identiﬁed in 1992 as a (CTG) n repeat in the 30 -untranslated region of the dystrophia myotonica protein kinase gene DMPK (7,8,32–34), with marked intergenerational instability that was consistent with the observed anticipation. The location of the DM1 mutation in the 30 -untranslated region of a gene meant that DM1 was the ﬁrst dominantly inherited disease found to be caused by an untranslated repeat expansion; the mutation is transcribed into RNA but not translated into protein. In 1995, the DM1 mutation was also found to be in the promoter region of the immediately adjacent homeodomain gene SIX5 (35). The CTG expansion in DM1 can vary from 80 to more than 4000 repeats in affected individuals, with clinically unaffected individuals having repeat sizes between 50 and 100 CTGs. Intergenerational and somatic instability are observed in which repeat size can increase by 50–80 repeats per year. This may be a factor in the variable rate of progression and phenotypic variability (36,37). There is a rough correlation of DM1 repeat size and age of onset for CTGs < 400 repeats (38). Proposed Mechanisms of DM1 Pathogenesis Haploinsufficiency of Genes in the DM1 Locus How a non-coding repeat causes the multisystemic features of DM1 has been controversial, since most dominant disorders are caused by the altered

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function of a mutant protein product. Expression studies suggest that the mutation interferes with production of both DMPK (39–41), and Six5, possibly through regional effects produced by repeat-induced alterations in chromatin structure (42,43). However, neither DMPK knock-out nor Six5 knock-out mice show the multisystemic features of the disease; DMPK knock-outs have isolated myopathy and cardiac abnormalities (44–46), and Six5 knockouts demonstrate only nuclear cataracts without the iridescent opacities characteristic of DM patients (47,48). The fact that no DMPK or Six5 point mutations have been associated with a DM phenotype further suggests that the multisystemic features of DM1 are not simply caused by DMPK or Six5 haploinsufﬁciency. RNA Mediated Pathogenesis Another hypothesized mechanism is that the enlarged CUG-containing transcripts accumulate as intranuclear foci and disrupt cellular function (28,49–54). Support for this model comes from a transgenic mouse model (55), in which the CTG expansion is inserted into the 30 end of the gene encoding human skeletal muscle actin, a gene not directly involved in DM1 but with expression limited to skeletal muscle. This mouse model expresses an mRNA with a CUG repeat tract of 250 repeats, and causes the myotonia and myopathic features characteristic of DM1, but because the CUG-containing transgene expression is limited to skeletal muscle the role of the CUG expansion in the multisystemic features of DM was not addressed. Additive Model of DM1 Pathogenesis An additive model has been proposed (56) in which DM1 pathogenesis results from multiple mechanisms: CTG expression in RNA results in myotonia and myopathy; DMPK haploinsufﬁciency causes the myopathy and cardiac abnormalities; Six5 haploinsufﬁciency is responsible for the cataracts; and altered expression of additional regional genes is responsible for other aspects of the complex phenotype, including DMWD (57), the DM gene with WD repeats that has been proposed to cause gonadal and CNS abnormalities (58), and FCGRT, the gene encoding the IgG Fc Fragment Receptor and Transporter, located 4 Mb from the CTG expansion and suggested to underlie the hypogammaglobulinemia (59). However, the discovery of the mutation in DM2 makes this model less tenable. Identification of the DM2 Mutation We began studying DM2 in 1992 as an independent approach to deﬁning the underlying pathogenesis of the myotonic dystrophies. In 1998, we linked the DM2 mutation to a 3 cM region at 3q21 (13,14), and subsequently, in 2001, demonstrated that DM2 is caused by a transcribed but untranslated

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CCTG repeat expansion located in intron 1 of the zinc ﬁnger protein 9 (ZNF9) gene (60). The DM2 repeat tract contains the complex motif (TG)n (TCTG)n (CCTG)n; the TG, TCTG, and CCTG tracts are all polymorphic in the general population, but only the CCTG portion expands in affected individuals. The CCTG portion of the repeat tract is usually interrupted on normal alleles, but as in other expansion disorders these interruptions are lost on affected alleles [and in an allele of an unaffected individual who possibly carries a premutation (61)]. Although DM2 is generally a milder disease than DM1, the DM2 CCTG expansions can be much larger than the expansions in DM1; CCTG expansions range in size from 75 to 11,000 CCTG repeats (mean 5000 CCTGs) in affected alleles. The smallest pathogenic size is not clear because uncommon shorter expansions are found in individuals with multiple allele sizes in lymphocyte DNA (17,60). The lack of correlation between repeat size and disease severity, and the recent observation that individuals homozygous for large DM2 repeats do not have a more severe disease (62), indicate that larger repeats do not result in increasingly severe pathogenic effects. The DM2 CCTG expansion mutations show both somatic and intergenerational instability to a greater extent than is seen in DM1. In peripheral blood samples, the mutation size heterogeneity caused by somatic instability is so extreme that 20% of DM2 expansions are not detectable by Southern analysis because the DNA at the DM2 locus forms a broad smear without any deﬁnable bands. Consequently, DM2 genetic diagnosis depends on the use of a PCR-based assay of the repeat that is not necessary for diagnosis of DM1 or other repeat expansion disorders (17). Although intergenerational decreases in the age of onset have been reported in DM2 families based on clinical criteria (17,63), this result may have been affected by ascertainment biases. Furthermore, the classically expected trend of longer repeat expansions in patients with earlier onset disease has not been observed in DM2, though somatic instability of the CCTG repeat expansion clearly complicates this analysis (17,36). Common Pathogenesis of DM1 and DM2 Both the DM2 CCTG expansion within intron 1 of ZNF9 and the DM1 CTG expansion in DMPK are transcribed into RNA but do not alter the proteincoding portion of either gene. The normal function of ZNF9 as a nucleic acid binding protein appears unrelated to any of the proteins encoded in the DMl region of chromosome 19 (64,65). Similarly, genes in the DM2 region (KIAA1160, Rab 11B, glycoprotein IX, FLJ11632, and FLJ12057) bear no obvious similarity or relationship to the genes at the DMl locus (DMPK, SIX5, DMWD, and FCGRT). Even if the DM2 expansion alters the regulation of ZNF9 or other genes in the DM2 region, dysregulation of these different sets of proteins at the DMl and DM2 loci would not be expected to result in diseases with such strikingly similar multisystemic features. Although the

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additive model of DMl proposes that CUG repeats in RNA cause myotonia and muscular dystrophy, the other DM features, including effects on the heart, eye, endocrine system, and immunoglobulin levels have been ascribed to haploinsufﬁciency of genes in the DM1 region. The molecular and clinical parallels between DM1 and DM2 strongly indicate that the clinical features common to both diseases including myotonia, muscular dystrophy, cataracts, cardiac arrhythmias, insulin insensitivity and diabetes, hypogammaglobulinemia, and testicular failure are caused by the pathogenic effects of RNA containing the CUG and CCUG expansions (14,17). MOLECULAR PATHOGENESIS OF THE MYOTONIC DYSTROPHIES Evidence of a Toxic RNA Effect Efforts to understand how the DMl CUG expansion expressed at the RNA level could mediate a dominant effect on other genes not localized to the DM1 locus (the so-called ‘‘trans’’ effect) have focused on the identiﬁcation of RNA binding proteins that might be dysregulated by the CUG repeat motifs. Recent suggestions that ribonuclear inclusions in DM1 and DM2 sequester transcription factors have not yet been conﬁrmed in tissues from affected patients (66). Direct evidence that RNA containing the repeat expansion is responsible for DM pathogenesis includes: (i) a CTG expansion in the 30 UTR of DMPK mRNA inhibits myoblast differentiation (67); (ii) transgenic models with CTG expansions expressed at the RNA level cause myotonia and muscular dystrophy (55,68); (iii) CUG and CCUG containing transcripts accumulate as RNA foci (49,60,69,70); (iv) CUG containing transcripts alter the regulation or localization of RNA binding proteins, including CUG-BP (51) and three different forms of muscleblind (MBNL1, MBLL, and MBXL) (54,69–71); (v) altered RNA binding protein activity caused by the CUG and CCUG RNA expansions result in altered splicing and the abnormal function of several genes, detailed below, that are related to DM pathophysiology including cardiac troponin T, the insulin receptor, chloride channels, tau protein, and myotubularin (28,52,72,73). Gene Splicing Abnormalities in the Myotonic Dystrophies Cardiac Troponin T (cTNT) A landmark discovery showed increased CUG-BP activity in DM1 muscle, leading to altered splicing of cardiac troponin T (cTNT) by binding intronic CUG repeat splicing signals in cTNT pre-mRNA (52). In adult DM1 cardiac and skeletal muscle, cTNT transcripts contain exon 5, resulting in a splice form normally seen in fetal tissue. This was the ﬁrst demonstrated splicing target of CUG-BP and the ﬁrst demonstration that the presence

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of elongated CUG repeat expansions in RNA leads to trans alterations in gene splicing. Insulin Receptor A classic clinical feature in both DM1 and DM2 patients is insulin resistance, predisposing patients to diabetes (74). Alternative splicing of the insulin receptor (IR) pre-mRNA is aberrantly regulated in DM1 skeletal muscle in that exon 11 is preferentially excluded, which results in predominant expression of the insulin insensitive splice form, IR-A (28,75). These results have now also been duplicated in DM2 (29), further demonstrating the common pathogenic mechanism responsible for both diseases, and supporting a model in which increased activity of CUG-BP leads to insulin resistance and diabetes in DM1 and DM2 by alternative splicing of the IR. Chloride Channel A classic feature of both DM1 and DM2 is clinical and electrical myotonia, in which voluntary muscle contraction is followed by involuntary repetitive action potentials that prevent relaxation. Skeletal muscle from a transgenic mouse model of DM1 has diminished muscle transmembrane chloride conductance, aberrant splicing of the muscle chloride channel (C1C-1), and loss of C1C-1 protein from the surface membrane. Skeletal muscle from DM1 and DM2 subjects (76) also have aberrant C1C-1 splicing and reduced chloride channel protein, demonstrating the pathophysiological basis of this part of the complex DM phenotype. CUG-BP, which is elevated in DM1 skeletal muscle, binds to the C1C-1 pre-mRNA, resulting in the aberrant pattern of C1C-1 splicing (72). Thus, the C1C-1 splicing abnormalities cause the chloride channelopathy that underlies myotonia in both DM1 and DM2, substantiating the trans-dominant pathogenic role of CUG and CCUG expansions in disease pathogenesis. Tau and Myotubularin Splicing alterations of the microtubule-associated tau mRNA have been observed in CNS tissue from DM1 patients (73) and in a murine model (68), which may underlie various CNS alterations in DM1 and DM2. In addition, altered splicing of the myotubularin-related 1 (MTMR1) gene has been reported in congenital DM1 muscle cells in culture and in skeletal muscle samples from congenital DM1 patients, suggesting a role for MTMR1 in myotonic dystrophy, possibly in the profound muscle atrophy of congenital DM1 (77). Role of Muscleblind and CUG-BP in DM Pathogenesis The role of different RNA binding proteins (CUG-BP and MBNL) in DM pathogenesis has been unclear. Although CUG-BP is upregulated in DM1

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muscle through an yet unknown mechanism, and increased CUG-BP results in a trans-dominant effect on gene splicing, CUG-BP does not co-localize with the ribonuclear inclusions; in contrast, the RNA binding proteins in the muscleblind family do co-localize with the ribonuclear inclusions, but were not initially associated with any speciﬁc molecular pathogenic effects. Direct evidence supporting the role of muscleblind in disease pathogenesis comes from the recently developed MBNL1 knockout mouse model of myotonic dystrophy, which has myotonia, myopathy, cataracts, and RNA splicing abnormalities characteristic of DM1 and DM2 (78). There appears to be a relationship between effects of CUG-BP and MBNL1, in that overexpression of CUG-BP causes the same speciﬁc alternative splicing changes that occur with depletion of MBNL1. In general, CUG-BP appears to promote splice forms normally involved in fetal development while MBNL1 preferentially leads to adult splice forms. These data predict that overexpression of CUG or CCUG repeat expansions, overexpression of CUG-BP, or depletion of MBNL1 would all result in a similar set of splicing alterations, the downstream effects of which would lead to characteristic molecular and physiological features of the myotonic dystrophies. Pathophysiological Implications of Differences Between DM1 and DM2 Although DM1 and DM2 phenotypes are strikingly similar, they are not identical. DM2 does not show a congenital form, with the attendant craniofacial and musculoskeletal abnormalities, and does not manifest the severe CNS involvement sometimes encountered in DM1 (17). The clinical distinctions between these diseases could result from differences in temporal or spatial expression patterns of the genes containing the expanded repeats (DMPK and ZNF9) and the genes for the various RNA binding proteins, or could be caused by differences in the downstream effects of CUG as opposed to CCUG expansions. Alternatively, the differences between DM1 and DM2 could involve locus-speciﬁc genes such as DMPK, SIX5 or DMWD for DM1, and ZNF9 for DM2. A possible mechanism for congenital DM1 is the demonstrated methylation of a genetic insulator at the DM1 locus in congenital cases, which alters DMPK expression so that higher levels of CUG containing transcripts are associated with the more severe congenital phenotype (79). Further clinical and molecular comparisons of DM1 and DM2 are needed to clarify the differences between the two disorders, which will help determine whether there are distinct pathogenic mechanisms responsible for the phenotypic differences, or, alternatively, to what extent the clinical distinctions simply reﬂect disease-speciﬁc differences in pathogenic severity of the CUG and CCUG repeat expansions in RNA.

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REPORTS OF A THIRD FORM OF MYOTONIC DYSTROPHY Our 2001 discovery of the DM2, CCTG expansion allowed for further analysis of the genetic heterogeneity of DM. It now appears that previously reported families with dominant multisystemic myotonic disorders, whether reported as having PROMM, PDM, or DM2, result from either DM1 or DM2 expansions (17). Linkage disequilibrium and haplotype analysis indicate that single founder mutations led to both the CTG expansion in DM1 (80–82) and the CCTG expansion in DM2 (61,83). A family recently suggested to have DM3 was reported and has several clinical features that have not been described in DM1 or DM2 (spongioform encephalopathy, motor neuron degeneration, and dementia), in addition to having features typical of the myotonic dystrophies including cataracts and electrical myotonia (though it remains unclear whether the abnormal EMG spontaneous activity is an intrinsic feature of muscle as is true of the myotonia seen in DM1 and DM2, or is secondary to the observed motor neuron loss in this family) (84). No investigations of ribonuclear inclusions or splicing abnormalities have been reported. Additional genetic and clinical testing will be needed to determine whether the pathogenic pathway involved in this family is related to DM1 and DM2, or is more consistent with the molecular pathogenesis of the tauopathies. Additional families recently reported as possibly having DM3, have characteristic features of myotonic dystrophy, though complete clinical and genetic characterization is pending (85).

SUMMARY The recent clinical and genetic characterization of DM1 and DM2 has now substantiated a third pathogenic mechanism that results from elongated repeat tracts. In addition to the previous recognition that repeat expansions can cause pathogenic loss of gene expression in recessive disorders (e.g., Friedreich ataxia), or cause a novel pathogenic protein in dominantly inherited disorders (e.g., Huntington’s disease), we now realize that in the dominantly inherited myotonic dystrophies, expression of RNA containing CUG and CCUG repeat expansions alters processing of multiple transcripts, leading to abnormal splicing of the chloride channel, insulin receptor, and other genes that underlie the complex multisystemic phenotype. Other molecular mechanisms may be involved in aspects of the myotonic dystrophy phenotype, such as the features that are seen in DM1 but not DM2, but we now have a primary pathophysiological target against which to direct possible therapeutic regimens. Pharmacological approaches can be developed to correct some of the speciﬁc features of the disease (e.g., myotonia, and insulin resistance), while stem cell and other genetic therapies are being developed to correct the underlying genetic abnormalities (86).

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71. Fardaei M, Larkin K, Brook JD, Hamshere MG. In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts. Nucleic Acids Res 2001; 29(13):2766–2771. 72. Charlet BN, Savkur RS, Singh G, Philips AV, Grice EA, Cooper TA. Loss of the muscle-speciﬁc chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Mol Cell 2002; 10(1):45–53. 73. Sergeant N, Sablonniere B, et al. Dysregulation of human brain microtubuleassociated tau mRNA maturation in myotonic dystrophy type 1. Hum Mol Genet 2001; 10(19):2143–2155. 74. Moxley RT, Griggs RC, Goldblatt D. Muscle insulin resistance in myotonic dystrophy: effect of supraphysiologic insulinization. Neurol 1980; 30(10):1077–1083. 75. Seino S, Bell GI. Alternative splicing of human insulin receptor messenger RNA. Biochem Biophys Res Commn 1989; 159(1):312–316. 76. Mankodi A, Takahashi MP, et al. Expanded CUG Repeats Trigger Aberrant Splicing of C1C-1 Chloride Channel Pre-mRNA and Hyperexcitability of Skeletal Muscle in Myotonic Dystrophy. Mol Cell 2002; 10(1):35–44. 77. Buj-Bello A, Furling D, et al. Muscle-speciﬁc alternative splicing of myotubularin-related 1 gene is impaired in DM1 muscle cells. Hum Mol Genet 2002; 11(19):2297–2307. 78. Kanadia RN, Johnstone KA, et al. A muscleblind knockout model for myotonic dystrophy. Sci 2003; 302(5652):1978–1980. 79. Filippova GN, Thienes CP, et al. CTCF-binding sites ﬂank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus. Nat Genet 2001; 28(4):335–343. 80. Imbert G, Kretz C, Johnson K, Mandel JL. Origin of the expansion mutation in myotonic dystrophy. Nat Genet 1993; 4(1):72–76. 81. Neville CE, Mahadevan MS, Barcelo JM, Korneluk RG. High resolution genetic analysis suggests one ancestral predisposing haplotype for the origin of the myotonic dystrophy mutation. Hum Mol Genet 1994; 3(1):45–51. 82. Deka R, Majumder PP, et al. Distribution and evolution of CTG repeats at the myotonin protein kinase gene in human populations. Genome Res 1996; 6(2):142–154. 83. Bachinski LL, Udd B, et al. Conﬁrmation of the type 2 myotonic dystrophy (CCTG)n expansion mutation in patients with proximal myotonic myopathy/ proximal myotonic dystrophy of different European origins: a single shared haplotype indicates an ancestral founder effect. Am J Hum Genet 2003; 73(4): 835–848. 84. Le Ber I, Martinez M, et al. A non-DM1, non-DM2 multisystem myotonic disorder with frontotemporal dementia: phenotype and suggestive mapping of the DM3 locus to chromosome 15q21-24. Brain 2004; 127(9):1979–1992. 85. Meola G, Sansone V, Milanese SD, Udd B, Vihola A, Mancinelli E, Pazzi A, Bachinski L, Moxley R, Krahe R. Lack of DM1-(CTG)n and DM2-(CCTG)n mutations in two families with autosomal dominant muscle weakness, myotonia, and cataracts: DM3? Neurology 2004; 62(suppl 5):A354. 86. Langlois MA, Lee NS, Rossi JJ, Puymirat J. Hammerhead ribozyme-mediated destruction of nuclear foci in myotonic dystrophy myoblasts. Mol Ther 2003; 7(5 Pt 1):670–680.

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11 Spinal Muscular Atrophy Stephen J. Kolb and J. Paul Taylor Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A.

INTRODUCTION Spinal muscular atrophy (SMA) was ﬁrst described in 1891 independently by Guido Werdnig working at the Institute of Pathological Anatomy in Graz, Austria, and Johann Hoffman of Heidelberg University (1,2). Their reports presented the clinical and pathological aspects of infantile SMA, including early onset, occurrence among siblings of unaffected parents, progressive weakness, hand tremor, and death from respiratory failure in early childhood. Since the ﬁrst description of this disease, a broader range of severity and age of onset has been appreciated, and a few separate disease entities have been deﬁned. In 1991, the International Consortium on Childhood SMA deﬁned three clinical subgroups to reﬂect the broad range of clinical severity (3). All types of SMA linked to chromosome 5q11 are associated with a relative deﬁciency in the product of the survival motor neuron (SMN ) gene (described below). SMA is one of the most common autosomal recessive diseases, affecting approximately 1 in 6000–10,000 live births, and is the leading hereditary cause of infant mortality (4–7). De novo mutations in the gene responsible for SMA are relatively frequent, and the carrier frequency for disease-causing mutations is estimated at approximately 1 in 50 (8). For unknown reasons, there is a slight male predominance for SMA, particularly with respect to the later onset forms of the disease.

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CLINICAL FEATURES OF AUTOSOMAL RECESSIVE SMA The classical form of severe spinal muscular atrophy (SMA type I; Werdnig– Hoffmann disease) has a consistent clinical phenotype that is well recognized by pediatricians and pediatric neurologists (9,10). Children typically present with general hypotonia plus axial and limb weakness within the ﬁrst few months of life. The legs are affected more than the arms and proximal muscles more than distal muscles leaving residual spontaneous activity in the feet and in the forearms and hands. Facial muscles are typically spared so that the infant usually has a bright normal expression. The intercostal muscles are always affected, whereas the diaphragm is spared, allowing adequate spontaneous respiratory activity until the infants are precipitated into respiratory failure by an incidental respiratory infection or aspiration. With rare exception, children with SMA type I die by two years of age with a median life span of seven months. Nearly 80% of children with SMA type I die by the time they are one year old (10). SMA type II is a less severe form of the disease with onset typically between 6 and 18 months of age. Children affected by SMA type II achieve the ability to sit although they cannot stand or walk unaided. SMA type III, also known as Kugelberg–Welander disease, is a later onset and more slowly progressive form of the disease with signs and symptoms becoming apparent after they attain the ability to walk. Zerres and Rudnik-Schoneborn have proposed that SMA type III be subdivided into types IIIa (onset before three years of age) and IIIb (onset after three years of age) to reﬂect the different clinical course experienced by these groups (11). The probabilities of being ambulatory 10, 20, and 40 years after onset are 73, 44, and 34%, respectively, in patients with SMA type IIIa, and 97, 89, and 67%, respectively, in patients with SMA type IIIb (11). Very late onset SMA (after the age of 35 years of age) has been referred to as ‘‘SMA type IV’’ and represents the mildest form within the spectrum of the SMA phenotype (12). ‘‘SMA type 0’’ has been used by some to describe severe SMA that presents prenatally (9,13). In this condition fetal movements are absent or markedly reduced and profound weakness and hypotonia are found at birth (13). All patients with SMA share clinical features that result from skeletal muscle denervation (14). Thus, physical examination is universally associated with symmetrical muscle weakness and muscle atrophy as well as decreased or absent deep tendon reﬂexes. Often, tongue fasciculations and hand tremors are seen. Sensory abnormalities are not detected and sphincter function is normal. Nerve conduction studies reveal normal sensory and motor nerve conduction velocities and electromyogram shows ﬁbrillation potentials, sharp waves, and large motor units consistent with a pattern of acute and chronic denervation. Muscle biopsy shows ﬁber type grouping and/or atrophic and hypertrophic ﬁbers also characteristic of acute and chronic denervation.

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MOLECULAR GENETICS OF AUTOSOMAL RECESSIVE SMA SMA types I, II, and III were all mapped to chromosome 5q11.2-13.3 in 1990, providing support for the contention that they are allelic disorders (15–18). Two years later, the ﬁrst prenatal predictions were performed using polymorphic markers linked to the SMA locus (19,20). The physical map of the SMA locus was found to be highly complex containing multicopy repetitive sequences, pseudogenes, and retrotransposon-like sequences suggesting that this region might be unstable (21–23). Indeed, at least 2% of SMA patients have de novo rearrangements (23,24). In 1995, the SMA locus was reﬁned further to reveal an inverted duplication containing four genes: the survival of motor neurons (SMN) gene (25), the gene coding for neuronal apoptosis inhibitory protein (NAIP) (26), the gene coding for p44, a subunit of the basal transcription factor TFIIH (27), and the H4F5 gene (28). Many patients have deletions that span more than one of these genes, however the smallest deletions characterized in SMA patients involved the telomeric copy of the SMN gene (SMN1) (29). The centromeric counterpart, SMN2, differs from SMN1 by a single nucleotide change (C/T transition) at position þ6 in exon 7 (30). This alteration does not change the primary amino acid sequence but alters splicing such that most transcripts produced by SMN2 lack exon 7 (31–33). Thus, approximately 90% of the protein produced by SMN2 is a truncated form that fails to oligomerize and appears to be unstable since it is rapidly degraded. In individuals with normal SMN1 alleles, SMN2 is dispensable since 5–10% of normal individuals lack both copies (34–36). Approximately 94% of individuals with clinically typical SMA lack both copies of SMN1 exon 7 (29). Loss of SMN1 can occur by deletion (these are typically large and include the whole gene) or by gene conversion of SMN1 to SMN2. In the remaining 6% of SMA patients that do not lack both copies of SMN1, there are a substantial number of intragenic (nondeletion) mutations, including missense, nonsense, or splice site mutations (37). These intragenic mutations provide solid evidence that SMN1 is indeed the SMA gene since, unlike many of the large deletions, they do not affect the neighboring genes within the original candidate region. Intragenic mutations are more frequently associated with milder forms of SMA. The severity of the SMA phenotypes appears to be inversely correlated to the level of full-length SMN protein produced (38,39). In the case of SMA, it is the remaining SMN2 genes that are left to provide that full-length protein. Indeed, SMN2 gene copy number is a strong modiﬁer of disease phenotype (40–44). The most commonly used method to genetically conﬁrm the diagnosis of SMA is a qualitative polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) assay (also known as the ‘‘SMN1 deletion assay’’) to detect the homozygous absence of SMN1 exon 7 (37). The PCR-RFLP assay takes advantage of the base differences in exons

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7 and 8 to distinguish SMN1 from SMN2. After PCR ampliﬁcation of this region of the SMN1 gene, a restriction endonuclease is used that digests only the SMN2 exon 7 PCR products because of a restriction site generated by a mismatched reverse primer (37). Approximately 6% of individuals with SMN1-related SMA do not have homozygous SMN1 deletions and will not be identiﬁed by the PCRRFLP assay. Most of these affected individuals are compound heterozygotes, having an SMN1 deletion on one chromosome and a point mutation or other nondeletion mutation on the other chromosome (29). In these cases, quantitative SMN1 gene dosage analysis is required to determine whether the patient carries only one copy of SMN1 exon 7. When the index of suspicion is high, and an individual carries only one copy of SMN1 exon 7, sequence analysis may be performed to evaluate for known, or potentially novel, loss-of-function mutations (8). As many as 2% of individuals with SMA have a de novo mutation, i.e., they have a new SMN1 mutation that is not detectable in samples from the parents (24). Since the PCR-RFLP assay cannot distinguish between carriers with one copy of SMN1 and normal individuals with two copies of SMN1, quantitative SMN1 gene dosage analysis is also required to determine SMA carrier status (8). However, approximately 4% of individuals have two or more copies of SMN1 on the same chromosome (4). Thus, there is the potential for a falsely negative test in an individual carrying an SMN1 deletion on one chromosome, but two or more SMN1 copies on the other (8). Thus, risk assessment is an important component of SMA genetic testing to reduce the possibility of a false result. Because of the complexity of SMA genetics, SMA genetic testing should be performed at experienced centers. SMA UNRELATED TO MUTATIONS IN SMN1 There are a number of inherited motor neuron diseases that share overlapping features with SMA, but are genetically distinct and have been referred to as the ‘‘SMA plus’’ syndromes (45). These include SMA with pontocerebellar hypoplasia, SMA with myoclonic epilepsy, and the Brown–Vialetto–van Laere syndrome—a progressive bulbar palsy with progressive neurosensory deafness. SMA with respiratory distress (SMARD), also known as ‘‘diaphragmatic SMA,’’ is a genetically heterogeneous disorder that features progressive loss of anterior horn motor neurons, and is not linked to 5q11.2-13 (46). Unlike SMN1-related SMA, SMARD is characterized by distal-predominant weakness and diaphragmatic paralysis that leads to life-threatening respiratory distress. Arthrogryposis is a heterogeneous syndrome characterized by joint contractures present at birth (47). The major clinical form, arthrogryposis multiplex congenita (AMC), is caused by decreased fetal movements in utero attributable to a large number of conditions, including neuropathies,

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myopathies, connective tissue diseases, and the limited space within the uterus (47). SMA with congenital bone fractures is a rare syndrome characterized by ﬂoppiness in infancy and by multiple fractures of the long bones for which linkage to chromosome ﬁve has been excluded (48). A particularly severe form of SMN1-unrelated SMA is X-linked and associated with arthrogryposis and congenital fractures (49). Arthrogryposis also has been associated with SMN1-related SMA. In two small series, a total of 6 of 16 patients with ‘‘neurogenic AMC’’ had homozygous deletions for SMN1 (50,51). Another related disease is spinal and bulbar muscular atrophy (SBMA), also known as Kennedy’s disease. SBMA is an X-linked disorder characterized by slowly progressive weakness resulting from degeneration of motor neurons of the brainstem and spinal cord (52). Patients with SBMA often exhibit mild signs of feminization such as gynecomastia and, less frequently, infertility despite normal serum androgen levels. The androgen insensitivity observed in this disorder provided clues to underlying cause, and in 1991 the molecular basis of SBMA was found to be trinucleotide (CAG) repeat expansion in the ﬁrst exon of the androgen receptor (AR) gene (53). More recently, an autosomal form of SBMA, with more prominent distal weakness, was found to be caused by mutations affecting the dynactin component of the dynein motor complex (54). Of course, the largest group of diseases with phenotypic similarities to SMA is amyotrophic lateral sclerosis (ALS). BASIC RESEARCH ON AUTOSOMAL RECESSIVE SMA: THE SMN COMPLEX SMN is an essential protein. Complete knock-out of SMN results in embryonic lethality in divergent organisms including human, mouse, chicken, C. elegans and S. Pombe (55). In experimental systems, SMN function appears necessary for cell survival independent of cell type. Therefore, it is intriguing and enigmatic that homozygous deletions of SMN1 should lead to selective motor neuron loss in patients with SMA. The basis of cell type speciﬁc neuron loss with relative depletion of SMN is a central issue that remains to be resolved. The SMN protein is a 294 amino acid polypeptide that is expressed in all eukaryotes tested so far (except Saccharomyces cerevisiae) and in all cell types of vertebrate organisms. SMN is found in the cytoplasm and the nucleoplasm where it is highly concentrated within discrete bodies called Gems (55,56). The SMN complex is the native functional entity of the SMN polypeptide and is comprised of SMN and six distinct proteins known as Gemins: Gemin2 (formerly SIP1), Gemin3/DP103 (a DEAD-box RNA helicase), Gemin4, Gemin5/p175 (a WD-repeat protein), Gemin6, and Gemin7 (57–64). The Gemin proteins colocalize with SMN in Gems and are present throughout

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the cytoplasm. The Gemins are readily isolated by co-immunoprecipitation experiments using anti-SMN antibodies or antibodies to the Gemins, even under stringent high salt conditions and can thus be considered integral components of the SMN complex (65). The most prominent known structural motifs within the Gemin proteins are a DEAD-box RNA helicase motif in Gemin3 (58,60) and an array of 13 WD repeats in Gemin5 (61). It is presumed that the RNA helicase activity of Gemin3 is critical for the function of the formed SMN complex as a whole. The presence of multiple WD repeats in Gemin5 suggests that this protein forms multiple protein– protein interactions and may serve as a platform for the assembly of protein complexes. Taken together, the presence of an RNA helicase and multiple WD repeats in the SMN complex suggests a role in the assembly of ribonucleoproteins. The pre-mRNA splicing reaction is carried out by the spliceosome in the nucleus. The spliceosome contains ﬁve small nuclear ribonucleoproteins (snRNPs) that are responsible for recognizing 50 and 30 splice sites located at exon–intron junctions. The assembly of the ﬁve snRNPs onto the intron is a highly dynamic process which culminates in the juxtaposition of 50 and 30 splice sites leading to ligation of the adjacent exons and release of the intervening intron. The formation of snRNPs occurs in the cytoplasm and is a stepwise process that requires the ATP-dependent assembly of a seven-membered ring (the Sm core) of common spliceosomal substrate proteins (Sm proteins) around a highly conserved sequence motif of less than 10 nucleotides, called the ‘‘Sm site,’’ present in most snRNAs. The SMN complex interacts with several substrate proteins. These include the Sm proteins and Sm-like (Lsm) proteins of snRNPs, which are essential components of the splicing machinery. Additional substrates are the snoRNP protein ﬁbrillarin and GAR1, hnRNP U, Q and R, RNA helicaseA, coilin, nucleolin, and Epstein–Barr virus nuclear antigen 2 (66–72). It is intriguing that most of the substrates identiﬁed currently are constituents of various RNP complexes that are involved in diverse aspects of RNA processing. Remarkably, the SMN complex also binds with high afﬁnity to the snRNA component of snRNPs directly and in a sequence-speciﬁc manner (73,74). These observations led to the demonstration of an essential role of the SMN complex in the assembly of the spliceosomal snRNPs (75). The SMN complex not only mediates snRNP assembly, but provides stringent control of the process by ensuring that Sm cores are only formed on the correct snRNA molecules (Fig. 1). In addition to its roles in spliceosomal snRNP assembly, the SMN complex may function in the formation of other RNPs. The SMN complex may play a role in the biogenesis of snoRNP which are involved in the posttranslational processing and modiﬁcation of ribosomal RNAs (67,68). The SMN complex may also be involved in the biogenesis of RNA polymerase II transcription and processing machinery in the nucleus (76,77). It is clear

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Figure 1 The role of the SMN complex in the assembly of spliceosomal snRNAs. The Sm site-containing snRNAs, U1, U2, U4, U5 as well as the minor spliceosome snRNAs, are transcribed in the nucleus by RNA polyermase II. The primary transcripts contain a monomethylated m7GpppG (m7G) cap structure at the 50 -end and are rapidly exported to the cytoplasm with the nuclear CBC proteins 20 and 80 and the export adaptor phax. The formation of the Sm core is required for the subsequent hypermethylation of the 7-methyl guanosine (m7G) cap of these snRNAs to convert it into a 2,2,7-trimethyl guanosine (m3G or TMG) and for 30 -end maturation (113,114). A properly assembled Sm core and the TMG-cap structure are prerequisite for snRNP import into the nucleus. In the nucleus, the newly imported snRNPs are initially concentrated in Cajal bodies from which they transit to pre-mRNAs for splicing. The SMN complexes in the nucleus are found throughout the nucleoplasm but are particularly concentrated in Gems, the twins of the snRNP-rich CBs. Abbreviations: SMN, survival motor neuron; CBC, cap binding complex; TMG, 2,2,7-trimethyl guanosine; CBs, Cajal bodies. Source: From Ref. 72 with permission.

that the SMN protein, as part of the SMN complex, is involved in the assembly of RNP particles that play critical roles at multiple steps in the processing of mRNAs in cells. It is currently unclear how these functions relate to the loss of motor neurons seen in SMA patients. Much attention is currently being focused on possible unique roles for SMN in the motor neuron. Primary motor neurons isolated from a transgenic SMA mouse model exhibit reduced axon growth in culture compared to motor neurons isolated from wildtype mice (78). Also, overexpression of

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SMN in differentiated PC12 cells promotes neurite outgrowth and modulates the localization of b-actin mRNA suggesting that SMN is involved in the transport of speciﬁc mRNA molecules in neuronal processes (78). It is clear that SMN has functions, as exempliﬁed by its requirement for snRNP assembly, that are common to all cells, and that SMN is found in neuronal processes. The resolution of whether the clinical symptoms of SMA are caused by deﬁciencies in functions of SMN that are speciﬁc to motor neurons or that are common to all cells but at higher demand in motor neurons will be a critical advance in our understanding of SMA. MODEL SYSTEMS In mice, there is one SMN gene that is equivalent to SMN1. Complete loss of this gene results in an embryonic lethal phenotype as would be expected given the housekeeping functions of SMN (79). Introduction of one or two copies of SMN2 rescues the embryonic lethal phenotype resulting in mice with severe SMA whereas 8–16 copies of SMN2 result in rescue (80,81). This indicated that SMN2 can produce the SMN required to correct the SMA phenotype although it is not currently clear as to when during development or in which speciﬁc tissue(s) high levels of SMN are needed. Expression of a known mild SMA missense mutation (A2G) on the severe SMA mouse background results in a mild phenotype (82). Overall, these mouse models can mimic the human disease. However, it is not clear from these murine models whether the global reduction of SMN activity primarily affects neurons, muscle, glia, or all of these cell types to result in the selective loss of motor neurons. Investigators have also disrupted SMN expression in speciﬁc cell types including neurons, myocytes, and hepatocytes using the Cre-Lox recombination system (83–85). In each case, this has led to failure of the targeted cell type to develop normally (83–85). C. elegans and Drosophila melanogaster models of SMA have also been developed (86–88). Because of their short generation time, well-annotated genomes, vast array of genetic tools, and the ability to perform unbiased genetic screens, both model systems offer a number of advantages that are a strong complement to mouse model systems. The availability of these models will permit unbiased forward genetic screens that may illuminate the normal function of SMN and its role in disease. THERAPEUTICS Initial treatment strategies for SMA paralleled those for other neuromuscular diseases, in particular ALS. Candidate therapeutic compounds were chosen as potential SMA therapies either because they were known to directly increase muscle strength clinically or they have been shown to improve outcomes in animal models of ALS. Thyrotropin-releasing hormone (TRH)

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produced a modest, transient improvement in muscle tone with no change in strength in one pilot clinical trial involving SMA type II and SMA type III patients (86). Albuterol, which had been shown to increase muscle strength in healthy volunteers, was similarly ineffective (90). Gabapentin, a drug approved for use in patients with postherpetic neuralgia and used by neurologists for neuropathic pain syndromes and refractory generalized epilepsy, prolongs survival in a mouse model of ALS (88). Two large clinical trials using gabapentin in SMA showed no improvement in strength, forced vital capacity (FVC) or functional rating scale (89,90). Riluzole, the only compound thus far proven to slow the progression of ALS, albeit mildly (94,95), has some promise as an SMA therapy. It is well tolerated in SMA patients (96) and improved median survival in an SMA mouse model (97), and a large clinical trial with Riluzole is currently underway. However, while therapies developed for ALS are a logical starting point for SMA therapy development, there is currently no mechanistic link between the two motor neuronopathies. And as opposed to the majority of ALS cases, there is a clear therapeutic goal in SMA: to increase the expression of full-length SMN protein. Thus, the current focus of SMA treatment development is based upon the potential to increase the expression of fulllength SMN protein, either by increasing exon 7 inclusion in SMN2 transcripts or by increasing the overall transcription from the SMN2 gene. All patients with SMA carry functional SMN2 alleles, irrespective of their disease-producing SMN1 mutation, providing a clear therapeutic target. High-throughput screens for small molecule modulators of the SMN2 gene have yielded many compounds that increase full-length SMN levels in cell culture. These include sodium butyrate (98), phenylbutyrate (99), sodium vanadate (100), aclarubicin (101), and indoprofen (102). Of these, the most promising class of compound is the histone deacetylase (HDAC) inhibitor. Indeed, valproic acid, which belongs to this class and is already approved for use in epilepsy has been shown to increase full-length SMN levels in cell culture models of SMA (103,104). HDAC inhibitor activity results in an increase in histone acetylation that can relax tertiary structure of chromatin and facilitate access of the transcriptional machinery to target genes. These compounds appear to increase overall transcription of the SMN2 gene resulting in increases in the functional, full-length SMN protein. Clinical trials using two HDAC inhibitors, valproic acid and sodium butyrate, are currently underway. Genetic strategies to increase exon 7 inclusion are also being pursued. In transient cotransfection experiments, the RNA splicing regulators Tra2a and Tra2b have been shown to favor SMN2 exon 7 inclusion (104,105). Inhibition of hnRNP A1 protein, which functions to inhibit splicing, restores SMN2 exon 7 inclusion in vivo suggesting that the C/T transition in SMN2 creates an exonic splicing silencer at that region (106). This approach has not been limited to the augmentation of RNA splicing modulators.

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Antisense oligonucleotides directed toward the 30 splice site of exon 8 have also resulted in SMN2 exon 7 inclusion (107). Finally, chimeric molecules have been designed to mimic the functions of splicing factors that can promote exon 7 inclusion in vitro (108). Targeted expression of exogenous SMN protein to motor neurons remains an additional genetic approach. For this approach, a major hurdle is to deliver the transgene to the relevant, disease-associated cell types. A recent report described the successful expression of human SMN in mouse motor neurons and muscle in vivo using a lentiviral-based gene transfer system (109). This expression improved motor neuron survival and resulted in a small but signiﬁcant increase in overall survival when applied to a mouse model of SMA. Adenovirus-mediated delivery of SMN to patient cells in culture has also been reported (110). Another potential strategy for SMA therapy is to replace the function of the SMN protein. As mentioned above, SMN is essential for the assembly of snRNPs, and it may play a role in the assembly of other RNA–protein complexes. Cell-based and in vitro assays that measure snRNP assembly will provide interesting tools with which to investigate these fundamental RNA processing mechanisms and to identify small molecules that can enhance these biological processes. There are signiﬁcant challenges ahead. The design of therapeutic clinical trials for SMA patients is complicated by its distinctive clinical course (111). SMA is unusual among neurodegenerative diseases in that the rate of functional decline appears to slow with the passage of time. These clinical impressions are supported by neurophysiological studies demonstrating precipitous loss of motor units early in the course of the disease (112). Transition from a phase of more obvious decline to a slower chronic phase occurs at widely varying ages. At one end of the spectrum are infants with severe SMA (SMA type I) who appear to enter the slow chronic phase as early as a few months of life if at all; at the other end are individuals with milder forms of SMA (SMA type III) who may not manifest clinical disability until late teen or even early adult years. For this reason, it may be useful to group SMA patients by severity of disease and to design outcome measures and therapies for each group. Current efforts to develop reliable molecular surrogate markers, such as a quantitative blood test of SMN protein expression, hold the promise of providing additional, objective indicators that can be used for prognosis, for determining the natural history of the disease, and for the detection of a therapeutic response in clinical trials. It is further not clear to what degree the weakness seen in SMA patients will be attenuated once therapies are shown to increase SMN levels in patients. Despite these challenges, there is also much reason to be hopeful for SMA patients and their families. The SMN gene was discovered 10 years ago and since then there has emerged an entire, dedicated, multidisciplinary ﬁeld of study committed to understanding and treating SMA. Remarkable

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coordination between laboratories in the private and public sectors is accelerating the rate of discovery of candidate therapeutic compounds and is creating an infrastructure to facilitate the development and execution of clinical trials for those candidates. Further basic science research to understand where, when, and how much SMN expression is required to prevent motor neuron loss is joined by neuroscientists, geneticists, and cell biologists. This is an exciting and fast paced research area that, over the next 10 years, will yield important lessons not only about how to effectively treat SMA, but about the nature of other neurodegenerative diseases, the motor neuron, RNA processing and more.

REFERENCES 1. Werdnig G. Zwei fru¨hinfantile heredita¨re Fa¨lle von progressiver Muskelatrophie unter dem Bilde der Dystrophie, aber auf neurotischer Grundlage. Arch Psychiatri Nervenkrankheiten, Berlin, 1891; 22:437–481. 2. Hoffmann J. Weitere Beitra¨ge zur Lehre von der progressiven neurotischen Muskeldystrophie. Deutsche Zeitschrift fu¨r Nervenheilkunde, Berlin, 1891; 1: 95–120. 3. Munsat TL, Davies KE. International SMA consortium meeting (26–28 June 1992, Bonn, Germany). Neuromuscul Disord 1992; 2(5–6):423–428. 4. Czeizel A, Hamula J. A Hungarian study on Werdnig-Hoffmann disease. J Med Genet 1989; 26:761–763. 5. Emery AE. Population frequencies of inherited neuromuscular diseases – a world survey. Neuromuscul Disord 1991; 1:19–29. 6. Pearn J. Incidence, prevalence, and gene frequency studies of chronic childhood spinal muscular atrophy. J Med Genet 1978; 15:409–413. 7. Tangsrud SE, Halvorsen S. Child neuromuscular disease in southern Norway. Prevalence, age and distribution of diagnosis with special reference to nonDuchenne muscular dystrophy. Clin Genet 1988; 34:145–152. 8. Ogino S, Wilson RB. SMN dosage analysis and risk assessment for spinal muscular atrophy. Am J Hum Genet 2002; 70:1596–1598. 9. Dubowitz V. Very severe spinal muscular atrophy (SMA type 0): an expanding clinical phenotype. Eur J Paediatr 1999; 3(2):49–51. 10. Thomas NH, Dubowitz V. The natural history of type I (severe) spinal muscular atrophy. Neuromuscul Disord 1994; 4:497–502. 11. Zerres K, Rudnik-Scho¨neborn S. Natural history in proximal spinal muscular atrophy. Clinical analysis of 445 patients and suggestions for a modiﬁcation of existing classiﬁcations. Arch Neurol 1995; 52:518–523. 12. Brahe C, Servidei S, Zappata S, Ricci E, Tonali P, Neri G. Genetic homogeneity between childhood-onset and adult-onset autosomal recessive spinal muscular atrophy. Lancet 1995; 346:741–742. 13. MacLeod MJ, Taylor JE, Lunt PW, Mathew CG, Robb SA. Prenatal onset spinal muscular atrophy. Eur J Paediatr Neurol 1999; 3(2):65–72. 14. Nicole S, Diaz CC, Frugier T, Melki J. Spinal muscular atrophy: recent advances and future prospects. Muscle Nerve 2002; 26(1):4–13.

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74. Yong J, Pellizzoni L, Dreyfuss G. Sequence-speciﬁc interaction of U1 snRNA with the SMN complex. Embo J 2002; 21(5):1188–1196. 75. Yong J, Wan L, Dreyfuss G. Why do cells need an assembly machine for RNA-protein complexes? Trends Cell Biol 2004; 14(5):226–232. 76. Pellizzoni L, Charroux B, Rappsilber J, Mann M, Dreyfuss G. A functional interaction between the survival motor neuron complex and RNA polymerase II. J Cell Biol 2001; 152(1):75–86. 77. Pellizzoni L, Kataoka N, Charroux B, Dreyfuss G. A novel function for SMN, the spinal muscular atrophy disease gene product, in pre-mRNA splicing. Cell 1998; 95(5):615–624. 78. Rossoll W, Jablonka S, Andreassi C, Kroning AK, Karle K, Monani UR, Sendtner M. SMN, the spinal muscular atrophy-determining gene product, modulates axon growth and localization of beta-actin mRNA in growth cones of motoneurons. J Cell Biol 2003; 163(4):801–812. 79. Schrank B, Gotz R, Gunnersen JM, Ure JM, Toyka KV, Smith AG, Sendtner M. Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos. Proc Natl Acad Sci USA, 1997; 94(18):9920–9925. 80. Hsieh-Li HM, Chang JG, Jong YJ, Wu MH, Wang NM, Tsai CH, Li H. A mouse model for spinal muscular atrophy. Nat Genet 2000; 24(1):66–70. 81. Monani UR, Sendtner M, et al. The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(-/-) mice and results in a mouse with spinal muscular atrophy. Hum Mol Genet 2000; 9(3): 333–339. 82. Monani UR, Pastore MT, et al. A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy. J Cell Biol 2003; 160(1):41–52. 83. Frugier T, Tiziano FD, Cifuentes-Diaz C, Miniou P, Roblot N, Dierich A, Le Meur M, Melki J. Nuclear targeting defect of SMN lacking the C-terminus in a mouse model of spinal muscular atrophy. Hum Mol Genet 2000; 9(5): 849–858. 84. Cifuentes-Diaz C, Frugier T, Tiziano FD, Lacene E, Roblot N, Joshi V, Moreau MH, Melki J. Deletion of murine SMN exon 7 directed to skeletal muscle leads to severe muscular dystrophy. J Cell Biol 2001; 152(5):1107–1114. 85. Vitte JM, Davoult B, et al. Deletion of murine SMM exon 7 directed to liver leads to severe defect of liver development associated with iron overload. Am J Pathol 2004; 165(5):1731–1741. 86. Miguel-Aliaga I, Culetto E, Walker DS, Baylis HA, Sattelle DB, Davies KE. The Caenorhabditis elegans orthologue of the human gene responsible for spinal muscular atrophy is a maternal product critical for germline maturation and embryonic viability. Hum Mol Genet 1999; 8(12):2133–2143. 87. Miguel-Aliaga I, Chan YB, Davies KE, van den Heuvel M. Disruption of SMN function by ectopic expression of the human SMN gene in Drosophila. FEBS Lett 2000; 486(2):99–102. 88. Chan YB, Miguel-Aliaga I, Franks C, Thomas N, Trulzsch B, Sattelle DB, Davies KE, van den Heuvel M. Neuromuscular defects in a Drosophila survival motor neuron gene mutant. Hum Mol Genet 2003; 12(12):1367–1376.

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12 Hereditary Spastic Paraplegia Kleopas A. Kleopa The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

INTRODUCTION Hereditary spastic paraplegia (HSP) encompasses a heterogeneous group of disorders characterized by progressive lower limb spasticity that were ﬁrst described by Stru¨mpell, Lorrain, and Seeligmu¨ller in the late nineteenth century. Autosomal dominant (AD), autosomal recessive (AR), and X-linked inheritance of HSP have been characterized, as have early and late onset forms of HSP, as well as uncomplicated and complicated HSP phenotypes (1,2), with considerable intrafamilial variation in the age of onset and severity (3–7). The discovery of the molecular genetic mechanisms underlying many forms of HSP in the last decade has provided the basis for a new classiﬁcation scheme according to genes and loci for these disorders. At least 10 loci for dominant forms, 11 loci for recessive forms, and 3 loci for X-linked HSP have been identiﬁed along with an increasing number of causative genes (Table 1). These discoveries provide an insight into the neurobiology of these disorders. CLINICAL AND PATHOLOGICAL FEATURES OF HSP The reported prevalence of HSP varies in different studies from 2 to 10 per 100,000, reﬂecting differences in epidemiological methodology, the diagnostic criteria used, or geographical factors (2,8). The ﬁrst symptom of HSP is difﬁculty walking or delayed walking when onset is in infancy. Leg stiffness, urinary disturbance, and premature wear on footwear are also early

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Table 1 Molecular Genetic Classiﬁcation of Inherited Spastic Paraparesis Disease (OMIM number)

Locus

Affected gene/ protein

Phenotype

Autosomal dominant HSP (spastic paraplegia-SPG) SPG3 (182600) 14q11-21 SPG3A/ Uncomplicated Atlastin SPG4 (182601) 2p22-21 SPAST/ Uncomplicated Spastin SPG6 (600363) 15q11.1 NIPA1 Uncomplicated SPG8 (603563) 8q23-24 Uncomplicated SPG9 (601162) 10q23.3-24.2 With cataracts, gastroesophageal reﬂux and motor neuronopathy SPG10 (604187) 12q13 KIF5A Uncomplicated SPG12 (604805) 19q13 Uncomplicated SPG13 (605280) 2q24-34 HSPD1/Hsp60 Uncomplicated SPG17 (27068511q12-14 BSCL2/Seipin With distal Silver syndrome) amyotrophy SPG19 (607152) 9q33-34 Uncomplicated Autosomal recessive HSP SPG5 (270800) 8p12-13 Uncomplicated SPG7 (602783) 16q24.3 SPG7/ Pure or Paraplegin complicated phenotypes SPG11 (604360) 15q13-15 Pure and complicated phenotypes SPG14 (605229) 3q27-28 With mental retardation and peripheral neuropathy SPG15 (270700) 14q22-24 With distal amyotrophy, mental retardation, retinopathy SPG20 (27590013q12.3 SPG20/Spartin With distal Troyer syndrome) amyotrophy (hands) SPG21 (24890015q21-22 ACP33/ With dementia Mast syndrome) Maspardin and white matter abnormalities

References 55,58,61 36,40 69,70 72 92

76,77 80 83,84 89,90 94 121 95,96

102

124

109

112

27

(Continued)

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Table 1 Molecular Genetic Classification of Inherited Spastic Paraparesis (Continued ) Disease (OMIM number) SPG23

Locus

Affected gene/ protein

1q24-32

SPG24 (607584) 13q14 Infantile onset HSP 2q33 (IAHSP) (607225allelic to ALS2, JPLS) X-linked HSP Xq28 SPG1 (303350allelic to MASA and X-linked hydrocephalus)

SPG2 (312920allelic to PMD)

Xq22

SPG16 (300266)

Xq11.2

ALS2/Alsin

L1CAM

PLP

Phenotype

References

With pigmentary abnormalities of skin and hair, premature aging Uncomplicated With tetraplegia, dysarthria, dysphagia, slow eye movements

118

Mental retardation, hydrocephalus, aphasia, adducted thumbs Pure and complicated phenotypes Pure and complicated phenotypes

125 119

131

138

147–149

Note: Updated information on HSP genes can be found at OMIM (http://www.ncbi.nlm.nih. gov.80/entrez/) and GeneClinics (www.genetests.org) (for abbreviations see text).

manifestations. The age of onset can be from infancy to the eighth decade of life, showing one peak in incidence before six years of age and another one between the second and fourth decades (1). Some patients may have only subclinical involvement (8). The rate of progression and disease severity vary between families and, to a lesser extent, within the same family, while life expectancy is usually normal (1,9). Neurological examination of patients with pure HSP shows symmetric, often severe leg spasticity, predominantly affecting the hamstrings, quadriceps, and ankles, causing the characteristic circumduction and toe walking. Pes cavus and other foot deformities may be found in up to 30% of patients. Mild distal weakness and muscle atrophy, affecting mostly the intrinsic foot muscles and the tibialis anterior, are less common but well-recognized features in patients with long disease duration. However, muscle weakness in HSP is much milder than the spasticity and the reverse should raise doubt

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about the diagnosis (1–3). Hyperreﬂexia is almost always found in the legs with extensor plantar responses in the majority of the affected individuals, but ankle reﬂexes may be absent in some patients. The upper limbs are typically spared, except for mild hyperreﬂexia in some cases and more severe involvement in rare syndromes discussed further (1,3,10). Mild sensory abnormalities occur in 10–65% of typical HSP cases, especially reduced vibration sense and paresthesias in the lower limbs (1,3). In most patients nerve conduction studies are normal (9,11), suggesting that the clinical ﬁndings are due to a central axonopathy rather than peripheral nerve involvement. There is also involvement of the bladder in up to 50% of HSP patients with urgency, hesitancy, and frequency, sometimes progressing to incontinence (1,12). Anal sphincter involvement is unusual and has only been described in combination with urinary symptoms (13). Sexual dysfunction has been reported in only one family, occurring about 10–15 years after the onset of disease (14). The autonomic nervous system has not been systematically studied, but autonomic function tests have been normal in HSP patients with sphincter disturbances (12). HSP is classiﬁed as uncomplicated (also referred to as ‘‘pure’’) and complicated. In uncomplicated HSP, spastic paraparesis is the dominating clinical feature, with few other features except for the mild neuropathic, sensory and autonomic features mentioned above. In complicated HSP, spastic paraparesis remains the major manifestation, but additional features are present, including dementia, peripheral neuropathy, epilepsy, optic atrophy, retinopathy, mental retardation, extrapyramidal dysfunction, ataxia, deafness, and icthyosis. Some types of complicated HSP are extremely rare and may have only been described in one or two families. Epilepsy may occur in both AD and recessive forms of complicated HSP syndromes (15–18), and can manifest with many seizure types including myoclonic, simple complex partial, and generalized seizures. The seizures may predate or follow the onset of the spastic paraparesis and are often accompanied by cognitive impairment (16,17). Peripheral neuropathy is a well-recognized feature of complicated HSP, but electrophysiological ﬁndings of peripheral neuropathy can also be found in patients with ‘‘pure’’ HSP (19). The clinical severity ranges from asymptomatic sensory involvement detected on clinical examination (20), to severe sensory loss with chronic painless cutaneous ulcers (21). Nerve biopsy shows features of axonal neuropathy (20,22). Coexisting manifestations include optic atrophy (22) or mental retardation (23,24). Dementia is a frequent manifestation of complicated HSP, either as the only additional feature (2,25), or in combination with ataxia, dysarthria, athetosis, or epilepsy (15,16,26,27). Some patients also have a thin corpus callosum and thalamic hypometabolism (28). The dementia in HSP patients is mainly subcortical with impairment of attention, recent memory, visuomotor coordination, and perceptual speed. Features of cortical involvement such as

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dysphasia, agnosia, or dyscalculia are typically not present (25). Cognitive decline, typically after the age of 40 years, has also been increasingly recognized in some forms of ‘‘pure’’ HSP (25,29–32), especially when patients are carefully evaluated by neuropsychological tests (33). There are only a few reports on the neuropathological changes in HSP (see Ref. 2 for review). The major feature is axonal degeneration that is maximal in the terminal portions of the longest descending and ascending tracts within the spinal cord, including the crossed and uncrossed corticospinal tracts to the lower limbs, and from the dorsal column pathways the fasciculus gracilis ﬁbers from the lower limbs. Demyelination and gliosis may accompany the axonal loss. The spinocerebellar tracts can be affected in about 50% of cases (34). In rare cases, neuronal loss in the motor cortex, Clarke’s column, cerebellum, basal ganglia, and anterior horns have also been found (13). Thus, the distal part of the longest central nervous system (CNS) axons is preferentially affected in HSP, indicating a ‘‘dying back’’ process with degeneration beginning distally and then proceeding towards the cell body. GENETICS AND NEUROBIOLOGY OF HSP The extensive clinical heterogeneity of HSP is matched by the emerging genetic heterogeneity. Increasing numbers of different loci and causative genes have been identiﬁed in the last decade (Table 1 and Fig. 1), providing important insight into the pathogenic mechanisms of the disease. Autosomal Dominant HSP Autosomal dominant is the most frequent mode of inheritance in pure HSP and accounts for approximately 70% of all cases (35) with linkage to 10 different loci thus far. In most of these loci, genes harboring mutations responsible for HSP have been identiﬁed, but clinical genetic testing is not always available or informative. This reﬂects the large number of genes and the frequent occurrence of speciﬁc mutations limited to single families (‘‘private’’ mutations). Chromosome 2p Locus (SPG4) Linkage to the SPG4 locus on chromosome 2p was demonstrated in Autosomal dominant HSP (ADHSP) families from different countries (36,37), distinguishing this disorder from ADHSP mapped earlier to chromosome 14. Subsequent studies have shown that the HSP associated with a chromosome 2p locus is by far the most common ADHSP, accounting for up to 40% of pedigrees (5,38,39). SPG4-linked families are characterized by a mean age of onset ranging from 12 to 43 years (4–6). Marked interfamilial and intrafamilial variation may result from allelic heterogeneity, modifying

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Figure 1 Localization and putative function of gene products involved in the pathogenesis of HSP. Diagram of a motor neuron with a long myelinated axon. Abbreviations: HSP60, hereditary spastic paraplegia 60; NIPAI, nonimprinted in Prader-Willi/Angelman loci 1; PLP, proteolipid protein; N, nucleus; mitoch, mitochondrion; TGN, trans-Golgi network; ER, endoplasmic reticulum.

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genes, environmental factors, or even difﬁculty in recognizing the precise onset of symptoms that typically begin insidiously. There is also marked variation in the disease severity, from subclinical involvement in asymptomatic patients with pyramidal signs in the lower limbs and normal or only slightly abnormal gait to patients who are chair bound or bedridden. The majority of SPG4 patients retain the ability to walk independently or with minimal support (4,5). SPG4-linked families frequently manifest late onset dementia or subclinical cognitive dysfunction (16,25,29,31,32). In addition to the typical pathological features of pure HSP, in one SPG4 family with dementia unique features including neuronal loss in the substantia nigra associated with Lewy bodies, and tau positive neuroﬁbrillary tangles in the hippocampus have been described (32). In contrast, prolongation of central motor conduction times or signs of peripheral neuropathy are less frequent in SPG4 linked families compared to other HSP forms (19). SPAST, the ﬁrst ADHSP gene to be identiﬁed by using a positional cloning strategy, is composed of 17 exons spanning a region of 90 kb and encodes for a 616-amino-acid protein of approximately 67.2 kD called spastin (40). Spastin belongs to the group of AAA proteins (ATPases associated with diverse cellular activities) and is expressed in neurons where it is predicted to have a nuclear localization (40,41). AAA proteins play an important role as chaperones in various cellular functions, including cell cycle regulation, protein degradation, organelle biogenesis, and vesicle mediated protein function (42). They share a common 230-amino-acid domain mediating the ATPase function, including the ATP binding and hydrolysis sites, located between amino acids 342 and 599 of spastin (40). Outside this domain spastin shows homology only to a particular subclass of AAA proteins, including the yeast 26S proteasome subunits involved in protein degradation (40). Over 100 different spastin mutations have been identiﬁed to date, including missense, nonsense, and splice site mutations in almost all exons, as well as small deletions and insertions leading to major changes in the amino acid sequence of the AAA cassette (39,40,43–49). A speciﬁc mutation appears to be unique to most individual kindreds and is rarely shared by unrelated individuals (50), making it difﬁcult to establish clear genotype– phenotype correlations (39,43). However, clinical and electrophysiological differences among patients depending on the type of spastin mutation have been reported (51). Splice site and frameshift mutations lead to unstable mRNA and cause a loss of spastin function through haploinsufﬁciency (41,45,49). Loss of spastin function leads to disrupted intracellular transport (52), as the threshold level of spastin expression may be critical for axonal preservation in the corticospinal tract (40). Spastin missense mutations do not appear to cause more severe phenotypes as opposed to truncating ones (45). However, the possibility of dominant-negative effects has

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been raised by recent studies showing that spastin mutants may lead to redistribution of the microtubule cytoskeleton (53). Spastin mutations also cause abnormal perinuclear clustering of mitochondria and peroxisomes, indicating an impairment of kinesin-mediated organelle transport (54). Such alterations may cause energy deﬁcits leading to length-dependent axonal degeneration. Chromosome 14q Locus (SPG3) The ﬁrst ADHSP locus on 14q was initially discovered in a family with early onset HSP (55). Several families have been subsequently linked to 14q and the SPG3 interval was eventually narrowed to a 2.7 cM region (56–58). SPG3 typically presents in the ﬁrst or second decade, but mostly before ﬁve years of age (7,55–59), and can be mistaken for cerebral palsy (60). Despite earlier onset, eventual disease severity is generally similar to other subtypes of ADHSP (7,56,57). Screening of candidate genes in affected kindreds led to the identiﬁcation of speciﬁc missense mutations in a novel gene (61). SPG3A gene has 14 exons spanning approximately 69 kb and encodes a protein called atlastin (61). Atlastin shows homology with several members of the dynamin family of large GTPases, particularly guanylate-binding protein-1. The dynamins play an important role in endocytosis, secretion and maintenance of mitochondrial form, and in the formation of transport vesicles from the trans-Golgi network through their GTPase activity (62,63). Expression of dominant dynamin mutants in mammalian cells leads to disruption of Golgi structure and function (64). Atlastin is enriched in pyramidal neurons of the cerebral cortex and hippocampus and colocalizes with markers of the Golgi apparatus in cultured neurons (65). Since an intact Golgi apparatus is required for axonal growth (66), the role of atlastin in Golgi membrane dynamics suggests that impaired long axon formation may be a relevant mechanism in SPG3 in addition to distal degeneration of axons. This would be the basis for the very early onset, which led some authors to describe it as a neurodevelopmental, rather than a neurodegenerative disorder. Most reported atlastin mutations affect conserved amino acids in the N-terminal region that contains the conserved GTPase domain (61), or in the RD motif affecting the active side of the GTPase (59). In contrast to spastin, for which most reported mutations are private/unique for each family, half of the SPG3 families reported carry the R239C atlastin mutation. This may be a commonly occurring new mutation or may represent a founder effect from a common ancestral mutation (67). Atlastin mutations are second in frequency to spastin mutations and have been found in 9–38% of ADHSP families (7,61,67,68). Atlastin mutations may be found in families with early or late onset HSP (68). A mutation located close to the carboxy terminus was identiﬁed in a family with infancy onset severe HSP

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(60), whereas a truncating mutation was found in another family with milder phenotype (7). Whether atlastin missense mutations could cause a more severe phenotype than the truncating ones suggesting dominant-negative effects remains to be determined. Chromosome 15q Locus (SPG6) Linkage to the SPG6 locus on 15q was reported in a large North American family (69). The age at onset was between 12 and 35 years with a phenotype typical of pure HSP, although relatively severe compared to other loci. Almost one third of affected members required a wheelchair, some as early as 40 years of age. Pes cavus was almost always present in affected members. Using the candidate gene approach, a disease-causing mutation in the NIPA1 (nonimprinted in Prader-Willi/Angelman loci 1) gene was identiﬁed in the original and in one further SPG6-linked family (70). The T45R amino acid substitution in the NIPA1 protein affects a highly conserved amino acid in the predicted ﬁrst of nine transmembrane domains (70). Hydrophobicity analysis indicates that NIPA1 could be a membrane receptor or transporter, but the exact function remains to be determined (71). Although individuals with Prader–Willi syndrome or Angelman syndrome have 15q deletions that include NIPA1 (71), they do not exhibit progressive spastic paraplegia indicating that NIPA1 haploinsufﬁciency does not cause a phenotype. Therefore, the NIPA1 T45R missense mutation is likely to be pathogenic through a dominant gain-of-function mechanism (70). Chromosome 8q Locus (SPG8) Three ADHSP families from different countries were linked to 8q24 allowing reﬁnement of the SPG8 locus to a 3.4 cM region, but the SPG8 gene has not yet been identiﬁed (72–74). The mean age at onset was comparable to SPG4 families, but the otherwise typical ADHSP phenotype was more severe, particularly in the American family, in which two-thirds of affected members over 40 years required the use of a wheelchair (75). One patient from the American family had marked spinal cord atrophy seen in MRI (75). Chromosome 12q Locus (SPG10) The SPG10 locus on 12q13 was identiﬁed in a large ADHSP family in which affected family members presented with the typical phenotype of pure HSP and onset ranging from 8 to 40 years of age. Disease severity was variable with some patients requiring a wheelchair whereas others remained asymptomatic at the age of 40 years (76). In the same family, a missense mutation in the motor domain of the neuronal kinesin heavy chain gene KIF5A was subsequently identiﬁed (77).

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Kinesin proteins form a large family of ATPases that function as molecular motors using microtubules as tracks (78). Each kinesin transports speciﬁc organelles allowing the intracellular movement of macromolecular cargo from the neuronal cell body to the distal tip of the axon. As most proteins must be transported down the axon after being synthesized in the perikaryon owing to limited protein synthesis within axons themselves, kinesins play an essential role in axonal survival. The SPG10 mutation affects a conserved asparagine residue and likely impairs the stimulation of the motor ATPase by microtubule binding (77). Targeted disruption of the neuronal KIF5A in mice causes axonal degeneration by impairing microtubuledependent axonal transport and accumulation of at least one cargo, neuroﬁlament subunits, in cell bodies (79). The molecular mechanism of SPG10 adds to the growing evidence that both CNS and PNS axonopathies are length-dependent because the longest axons are the most vulnerable to defects in axonal transport. Chromosome 19q Locus (SPG12) The SPG12 locus on 19q13 was ﬁrst identiﬁed in a large Welsh family with ADHSP (80). Subsequent linkage studies narrowed the minimum candidate gene region to a 3.3-cM interval (81,82). In the original SPG12 family mean age at onset was seven years (ranging from 5 to 22) with typical ADHSP phenotype (5,80), whereas in a large SPG12-linked Italian family onset was later, around 14 years, and the phenotype was more severe. Most affected members developed rapidly progressive spastic paraplegia and required use of a wheelchair within four years after onset. As with other HSP subtypes, anticipation was suspected but not yet proven (82). Chromosome 2q Locus (SPG13) The SPG13 locus on 2q24-q34 was ﬁrst reported in a large French family with ADHSP (83). Compared to SPG4, in the SPG13 family there were signiﬁcantly more patients with increased reﬂexes in the upper limbs and with severe functional handicaps. A mutation in the gene encoding mitochondrial chaperonin Hsp60 (heat shock protein 60) was subsequently identiﬁed in affected members of the same family (84). Like other chaperonins, Hsp60 is involved in folding and assembly of proteins and is located in the mitochondrial matrix (85). The affected members of the SPG13 family carry the V72I Hsp60 mutation. When co-expressed with the co-chaperonin Hsp10, wild-type human Hsp60, but not the V72I mutant, can support growth of E. coli cells in which the homologous chaperonin genes have been deleted (84). The dominant inheritance in SPG13 may result from haploinsufﬁciency of Hsp60, especially under stress conditions, or from dominant-negative effects of the mutant on

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the wild-type protein lowering the overall chaperonin activity. Thus, SPG13 can be deﬁned as ‘‘chaperonopathy,’’ emphasizing the role of these proteins in the maintenance of long axons in the CNS. Chromosome 11q Locus (SPG17, Silver Syndrome) Silver ﬁrst described two families with ADHSP and amyotrophy of the hands (86). Families with similar phenotypes were subsequently reported from different countries (10,87,88). Onset of symptoms varied widely from 8 to 63 years of age and some family members had very mild disease indicating a broad phenotypic variation (88). The typical HSP phenotype is characteristically combined with amyotrophy and weakness of intrinsic hand muscles, most marked in the thenar eminence, so that the disease in some families had been mistaken for amyotrophic lateral sclerosis (10). Nerve conduction velocities were normal or mildly reduced. The disease was linked to a new locus on 11q12-q14 in one of the original families reported by Silver (89). Another family with similar phenotype did not show linkage to 11q, indicating genetic heterogeneity (89). Subsequent linkage studies reduced the candidate gene interval (88) allowing the identiﬁcation of heterozygous mutations in the BSCL2 gene (90). Eight related Austrian families with distal spinal muscular atrophy (DSMA-V) had the same mutation found in the Silver syndrome, establishing that these are allelic disorders. The BSCL2 gene contains 11 exons spanning at least 14 kb of DNA. It encodes a 398-amino-acid protein, seipin, with at least two hydrophobic amino acid stretches, which is highly expressed in the CNS and in testis (91). Seipin is a glycosylated integral membrane protein of the endoplasmic reticulum and the discovered mutations alter the N-glycosylation site causing aggregate formation (90). Homozygous null mutations of the same gene are associated with congenital lipodystrophy with severe insulin resistance (91), indicating an important role for this protein in diverse cellular functions. Other ADHSP Loci A complicated form of ADHSP in a large Italian pedigree has been linked to the SPG9 locus on chromosome 10q23.3-24.2. Age at onset varied from the ﬁrst to third decade of life with evidence for anticipation. Spastic paraparesis was associated with gastroesophageal reﬂux causing persistent vomiting, distal muscle atrophy due to axonal motor neuropathy, and bilateral cataracts (92). Whether the almost identical phenotype of congenital cataracts and spastic paraparesis described previously in another pedigree proves to be associated with the SPG9 locus remains to be determined (93). Another ADHSP locus (SPG19) on 9q was found in an Italian family with typical HSP and onset from 36 to 55 years (94). Motor and sensory nerve conduction velocities obtained in some of the patients were slow in

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the lower limbs. The disorder progressed slowly and was relatively benign, with only one patient conﬁned to a wheelchair. Autosomal Recessive HSP Autosomal recessive HSP (ARHSP) is less common than the dominant form and includes both uncomplicated and complicated phenotypes. Multiple loci have been identiﬁed through linkage studies, often in single, consanguineous families, and some of the responsible genes have been discovered. Chromosome 16q Locus (SPG7) The SPG7 locus on chromosome 16q24.3 was ﬁrst identiﬁed in a large consanguineous Italian family with ARHSP and onset between 25 and 42 years of age. Apart from the typical ﬁndings of pure HSP, dysarthria, dysphagia, and optic atrophy were present in some of the affected family members. Nerve conduction studies in one of the patients showed mild axonal neuropathy. All affected individuals were able to walk independently except for the oldest patient (95). Further studies showed that the SPG7 phenotype is heterogeneous with both pure and complicated forms (96,97). In affected individuals of the original SPG7 family a homozygous 9.5 kb deletion was identiﬁed in the gene that encodes paraplegin (96). The SPG7 gene is organized into 17 exons spanning approximately 52 kb (98). Screening of further unrelated ARHSP families for paraplegin mutations revealed additional deletion and insertion mutations leading to frameshift (96). However, paraplegin mutations appear to be an overall uncommon cause of HSP, found only in one of 30 English HSP families (99). Paraplegin is a member of the mitochondrial metalloproteases, a subgroup of the AAA protein family, which have both proteolytic and chaperonelike activities at the inner mitochondrial membrane (96). Analysis of muscle biopsies from patients with paraplegin mutations showed typical signs of mitochondrial oxidative phosphorylation defects, including COX-negative and ragged red ﬁbres (96,99). Furthermore, the degree of mitochondrial abnormality correlated with the severity of the disease. Functional analysis at the cellular level showed that paraplegin co-assembles in the mitochondrial inner membrane with the homologous protein, AFG3L2, to form a high molecular mass complex. Loss of paraplegin in ﬁbroblasts from SPG7 patients leads to reduced complex I activity in mitochondria and increased sensitivity to oxidative stress, which can be rescued by exogenous expression of wild-type paraplegin (100). Moreover, paraplegindeﬁcient mice have abnormal mitochondria and impairment of axonal transport leading to distal axonal degeneration (101). Mitochondrial morphologic abnormalities in distal axonal regions occur prior to the ﬁrst signs of axonal damage and correlate with onset of motor impairment. Axonal swellings result from massive accumulation of organelles and neuroﬁlaments (101).

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Thus, it appears that loss of paraplegin function causes mitochondrial dysfunction, resulting in failure of axonal transport and axonal degeneration (96,101). Chromosome 15q Locus (SPG11) A high percentage of families with recessive HSP have been linked to 15q13–15, the most common ARHSP locus (102). In SPG11-linked families age at onset ranges from the ﬁrst to the ﬁfth decade of life and the phenotype is variable with both uncomplicated and complicated forms. Some of the additional features reported include atrophy or agenesis of the corpus callosum, periventricular white matter changes demonstrated on MRI, mental retardation in one case, sensorimotor neuropathy in one family, and mild cerebellar dysfunction. The association of HSP with agenesis of the corpus callosum and mental retardation was initially reported in Japanese families that linked to 15q13–15, within the SPG11 locus (103,104) and subsequently in other countries (24,105,106). However, the SPG11 locus is distinct from the locus responsible for agenesis of the corpus callosum and peripheral neuropathy (ACCPN) syndrome. No mutations in the SLC12A6 gene that cause ACCPN could be found in an SPG11 family with thin corpus callosum (106). Some families with the SPG11 phenotype did not link to 15q134–15, suggesting further genetic heterogeneity (104,105). Chromosome 14q22 Locus (SPG15, Kjellin Syndrome) Following rare reports associating macular degeneration and spastic paraplegia, Kjellin reported in 1959 two pairs of brothers from two kindreds who had spastic paraplegia, distal amyotrophy of the hands, mental retardation, and central retinal degeneration (107). While mental retardation was present since birth in all patients, the onset of spastic paraplegia was in the third decade. Similar families were subsequently described with an earlier age of onset, mostly in the second decade, and additional features included sphincter disturbances, dysarthria, mild cerebellar ataxia, and further intellectual deterioration in the second decade (26,108). Two of the families linked to a new locus on 14q22-q24 (109), which is distinct from the SPG3 locus. Chromosome 13q Locus (SPG20, Troyer Syndrome) The combination of spastic paraplegia with distal muscle wasting, especially of the intrinsic hand muscles, was ﬁrst described in an Ohio Amish group and was called the Troyer syndrome for the surname of many of the affected persons (110). The disorder was also described in a consanguineous Kuwaiti family (111). Onset was in early childhood with dysarthria, distal muscle wasting, difﬁculty in learning to walk, and in some patients drooling and mild cerebellar signs. Patients usually lost the ability to ambulate by the third or

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fourth decade (110). Troyer syndrome was linked to 13q12.3 in the original Amish kindred. All affected individuals were homozygous for a frameshift mutation caused by a deletion of A at nucleotide 1110 in the SPG20 gene (112). SPG20 is ubiquitously expressed and encodes for spartin, a 666-amino acid protein of 72 kD that shares similarity with molecules involved in endosomal membrane trafﬁcking and microtubule dynamics, including spastin (112,113). Chromosome 15q21 Locus (SPG21, Mast Syndrome) Mast syndrome is a complicated form of HSP associated with presenile dementia, also described in an Ohio Amish isolate (114). Onset is in the late teens or twenties with slow progression that may lead to akinetic mutism in the most severely affected patients. Developmental delay, motor difﬁculties, mild incoordination and awkward running, were sometimes noted in childhood (27). In contrast, other patients were perceived as relatively normal until early adulthood, before decline in walking and mental function was evident in the third to ﬁfth decade of life. Thin corpus callosum and white matter abnormalities were found in the MRI (27). The Mast syndrome locus was mapped to 15q22.31 and a homozygous 1-bp insertion in the ACP33 gene was found in all affected patients. The encoded protein, maspardin, localizes to the endosomal trans-Golgi transportation vesicles and may play a role in protein transport and sorting (27). Chromosome 1q Locus (SPG23) A distinctive syndrome consisting of disordered skin and hair pigmentation, progressive spastic paraparesis beginning in childhood, and peripheral neuropathy was described in a Jordanian family. Sural nerve biopsy revealed axonal degeneration. Skin biopsy revealed abnormal epidermal pigmentation but skin ﬁbroblast repair studies were normal and no biochemical defect was found (115,116). A similar phenotype was reported in a few sporadic cases and in a second consanguineous family with spastic paraparesis, muscle wasting, microcephaly, mental retardation, skeletal deformities, and cutaneous manifestations (117). Follow up of that family years later revealed prematurely aged facial appearance with greying of eyebrows and eyelashes. The disorder was linked to a new locus on 1q24-q32, designated SPG23 (118). Infantile Onset HSP Linked to Chromosome 2q33 Eymard-Pierre et al. (119) studied 15 patients from 10 families of different ethnic origins who presented with severe spastic paraparesis starting in the ﬁrst two years of life with ascending progression to the upper limbs within the next few years. Despite progression to tetraplegia, anarthria, dysphagia, and slow eye movements in the ﬁrst decade of life, the disease was compatible with long survival. Motor evoked potentials and magnetic resonance imaging

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demonstrated dysfunction of the upper motor neurons, while lower motor neurons were not involved. Linkage analysis demonstrated that Infantile onset HSP (IAHSP) is allelic to juvenile amyotrophic lateral sclerosis at the ALS2 locus on 2q33-q35 and truncation mutations in the ALS2 gene encoding alsin were identiﬁed in 4 of the 10 IAHSP families (119). A 1-bp deletion in the ALS2 gene has also been identiﬁed in a large consanguineous Pakistani family with a similar phenotype (120). Mutations in regulatory ALS2 regions or genetic heterogeneity may account for the failure to ﬁnd ALS2 mutation in other IAHSP families. Thus, ALS2 mutations may cause a spectrum of disorders ranging from early onset retrograde (IAHSP) or juvenile onset degeneration of the upper motor neurons with ALS2, or without (juvenile-onset primary lateral sclerosis, JPLS) lower motor neuron involvement. Other ARHSP Loci A locus for pure HSP on chromosome 8p12-q13 (SPG5) was discovered in four Tunisian families (121) and subsequently in families from other countries (122,123). Age at onset varied widely from 1 to 40 years. Further linkage studies narrowed the SPG5A interval, but mutations in several candidate genes were excluded (123). Three further ARHSP loci associated with complicated phenotypes were discovered in single families. The SPG14 locus on 3q27-28 was discovered in a consanguineous Italian family with HSP accompanied by bilateral pes cavus and mild mental retardation. Average age of onset was 30 years. There was evidence of distal motor neuropathy, while sural nerve biopsy in one patient was normal. All affected individuals were able to walk unaided (124). In a consanguineous Saudi Arabian family, ﬁve siblings presented at about one year of age when they started to stand with the tendency to walk on tiptoes, spasticity, scissoring gait, clonus, and hyperreﬂexia. This very early onset HSP was linked to 13q14 (SPG24) (125). Three of the affected and two unaffected individuals also had sensorineural deafness, which segregated as an independent trait. In another consanguineous Italian family four of eight siblings suffered multiple disc herniations presenting with spastic paraplegia, which improved partially after surgery (126). The susceptibility locus for disc herniation and spastic paraplegia was mapped to 6q22.31-24.1 (SPG25).

X-linked HSP Although X-linked HSP (XHSP) is rare, its molecular genetic basis was the ﬁrst to be discovered in HSP and is relatively well understood. Three different loci have been identiﬁed including SPG1, SPG2, and SPG16. Complicated HSP occurs in all types, whereas the pure phenotype has only been linked to SPG2 and SPG16.

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Xq28 Locus (SPG1) The syndrome of mental retardation, aphasia, shufﬂing gait, and adducted thumbs (MASA) affecting mostly boys was ﬁrst described in a MexicanAmerican family (127). In addition, the patients had small body size, microcephaly, exaggerated lumbar lordosis, and hyperreﬂexia in the lower limbs. The absence of hydrocephalus distinguished the MASA syndrome from X-linked aqueductal stenosis. In similar patients, spastic paraparesis and MASA syndrome were reported as X-linked ‘‘complicated’’ spastic paraplegia type 1 and were linked to Xq28 (128). After the discovery that mutations in the L1 cell adhesion molecule (L1CAM) gene cause X-linked hydrocephalus (129), two different groups showed that mutations in the same gene are also responsible for XHSP (SPG1) and the MASA syndrome (130,131). These allelic disorders appear to form a clinical spectrum with both inter- and intrafamilial phenotypic variability. Affected members with various phenotypes may be found within one family. The L1CAM-related diseases are now referred to as being part of the clinical syndrome corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraplegia, and hydrocephalus (CRASH). L1CAM is an integral membrane glycoprotein member of the immunoglobulin superfamily of cell adhesion molecules mediating cell-to-cell adhesion. It is expressed by neurons and Schwann cells (SC) and plays an essential role in axon outgrowth, myelination and path ﬁnding (132). In a mouse model of the CRASH syndrome mutant mice are smaller and less sensitive to touch and pain than wild-type mice. Their hind legs appear weak and uncoordinated, the size of the corticospinal tract is reduced, and the lateral ventricles are often enlarged. Ensheathment of axons is impaired (133). Xq22 Locus (SPG2) X-linked recessive HSP was described in a family, in which the disorder began as ‘‘pure’’ spastic paraparesis, but the patients later developed nystagmus, dysarthria, sensory disturbance, mental retardation, optic atrophy in half of the cases, muscle wasting, and joint contractures, resulting in wheelchair dependency by early adult life (134). Subsequent reports of XHSP included both uncomplicated (135) and ‘‘complicated’’ phenotypes (136), which may even coexist within the same family (137). Linkage studies demonstrated mapping of the SPG2 locus on Xq21 (135–137). Disease causing mutations were found in the proteolipid protein (PLP) gene, conﬁrming that SPG2 and Pelizaeus–Merzbacher disease (PMD), a severe dysmyelinating disease of the CNS, are allelic disorders (138,139). Several PLP mutations have been identiﬁed in both ‘‘complicated’’ and pure HSP pedigrees (138–141). Clinical severity correlates with the nature of the mutation and there seems to be a spectrum ranging from pure HSP to severe connatal PMD, with all variations in between.

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The PLP gene encodes two major myelin proteins, DM-20 and PLP, which differ by alternative splicing of exon 3, whose distal end is retained in the mature PLP but not in DM-20 (142). DM-20 is expressed before PLP during development and may play a role in oligodendrocyte maturation (143), while PLP is expressed later and is crucial to myelin sheet compaction (138). The PLP mutation found in SPG2 patients (139) is identical to the mutation identiﬁed in the ‘‘rumpshaker’’ mice (144), which, although myelin-deﬁcient, have normal longevity and morphologically normal oligodendrocytes. They show a generalized tremor at about 12 days of age, which then becomes conﬁned to the rear end. In contrast, ‘‘jimpy’’ mice resemble the PMD phenotype and show failure of development and differentiation of oligodendrocytes leading to early death. The jimpy mutation as well as subsequently identiﬁed lethal PLP mutations result in truncation of the fourth transmembrane domain in both the PLP and DM-20 (142). They cause increased oligodendrocyte cell death, perhaps due to toxic gain of function effect of misfolded PLP products retained in the endoplasmic reticulum (145). As in the mutant mice, it appears that human mutations altering the structure of both PLP and DM-20 cause different forms of PMD, whereby toxic gain of function mutants are associated with the most severe, connatal PMD phenotypes (146). In contrast, PLP-speciﬁc mutations, which leave DM-20 intact, cause SPG2 (138,141). Other XHSP Loci A family with severe XHSP has been reported, in which the patients presented with quadriplegia, motor aphasia, reduced vision, mild mental retardation, and dysfunction of the bowel and bladder. Onset was in the ﬁrst three months of life with nystagmus and dorsal ﬂexion of the great toes. Motor development was delayed, and spasticity progressed from the lower to the upper extremities. The patients never learned to walk (147). In this and in another pedigree with pure HSP, the disease was linked to Xq11.2 (148). However, mutation analysis on the PLP gene was negative, suggesting the existence of a third XHSP locus (SPG16). An insertion of nucleolus organizer region (NOR) into Xq11.2 was found in a Japanese family with pure HSP, perhaps disrupting the putative HSP gene situated at Xq11.2 (149). DIAGNOSTIC APPROACH AND TREATMENT IN HSP Before making the diagnosis of HSP, either pure or complicated, other inherited disorders should be considered, in which lower extremity spastic weakness is a prominent feature (2). These include inherited leukodystrophies, Friedreich ataxia, Machado–Joseph disease, familial Alzheimer’s disease, familial motor neuron disease, and dopa-responsive dystonia, which are discussed in other chapters of this book. Furthermore, several forms of inherited spastic ataxia (SAX) including SAX1 (150), spastic paraplegia, ataxia, mental retardation

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(SPAR) (151), AR spastic ataxia of Charlevoix-Saguenay (ARSACS) (152), as well as the Sjogren–Larsson and Fitzsimmons syndromes may be considered, the discussion of which is beyond the scope of this review. Acquired disorders that are potentially treatable may also mimic HSP. Among others, one should exclude multiple sclerosis, tropical spastic paraparesis, neurosyphilis, AIDS, vitamin B12 or E deﬁciency, and structural spinal cord disorders such as Arnold-Chiari malformation, neoplasms, compression, and arteriovenous malformation. Although useful in excluding alternative diagnoses, laboratory investigations add little to the clinical diagnosis of HSP. Central motor responses are usually delayed or absent from the lower limbs, with normal values from the upper limbs (2,4,19). Somatosensory evoked potentials from the lower limbs may also be small or absent, while nerve conduction studies and EMG are normal in most cases (1,9,19). MRI may exclude focal or diffuse white matter disease, and in some patients with HSP it may show atrophy of the spinal cord and occasionally of the corpus callosum (75,153). Thus, the diagnosis of HSP is based on clinical features (including inheritance pattern and the ‘‘complicated’’ vs. uncomplicated phenotype) in conjunction with exclusion of other disorders. With the discovery of multiple causative genes, molecular genetic testing is now available for HSP. A limited number of clinical laboratories (listed in www.genetests.org) may currently test for mutations in L1CAM, PLP, SPG3A, SPAST (SPG4), NIPA1, SPG7, and SPG20. Prenatal diagnosis is possible for SPG1, SPG2 and SPG7. Thus, molecular genetic diagnosis is possible in over 50% of families with ADHSP but in a fewer number of patients with ARHSP (5,38,39). Research laboratories may sequence additional HSP genes. However, with the number of potential genes to be assessed, it is important to obtain a detailed family history to stratify the molecular diagnostic approach. Treatment for HSP patients is currently symptomatic. Regular physiotherapy should be offered to prevent contractures and improve mobility. Orthopaedic interventions and/or assistive devices may maximize function. Medications that treat spasticity include oral baclofen, dantrolene, tizanidine, as well as intrathecal baclofen in severe cases. Bladder spasticity may improve with oxybutynin. The Spastic Paraplegia Foundation offers resources for patient information and support (http://sp-foundation.org/). CONCLUSION The emerging neurobiological complexity underlying HSP provides the basis for the clinical and genetic heterogeneity. A growing number of different disorders classiﬁed as pure or ‘‘complicated’’ HSP share ﬁnal common pathways of impaired cellular trafﬁcking dynamics and axonal transport. HSP genes encode for proteins that play an essential role in CNS myelination and

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development (PLP/DM20, L1CAM), in mitochondrial function (paraplegin, HSP60), in chaperon and protein trafﬁcking related activities (spastin, atlastin, paraplegin, HSP60, BSCL2, spartin, maspardin, and alsin), and in axonal transport (KIF5A). Dysfunction of these genes leads to the characteristic clinical and neuropathological ﬁndings of length-dependent degeneration that is predominant in, but not limited to the motor axons in the CNS. The increasing knowledge of the molecular mechanisms underlying HSP is expected to open the way for the development of effective treatments in the future.

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149. Tamagaki A, Shima M, Tomita R, Okumura M, Shibata M, Morichika S, Kurahashi H, Giddings JC, Yoshioka A, Yokobayashi Y. Segregation of a pure form of spastic paraplegia and NOR insertion into Xq11.2. Am J Med Genet 2000; 94:5–8. 150. Meijer IA, Hand CK, Grewal KK, Stefanelli MG, Ives EJ, Rouleau GA. A locus for autosomal dominant hereditary spastic ataxia, SAX1, maps to chromosome 12p13. Am J Hum Genet 2002; 70:763–769. 151. Hedera P, Rainier S, Zhao XP, Schalling M, Lindblad K, Yuan Q-P, Ikeuchi T, Trobe J, Wald JJ, Eldevik OP, Kluin K, Fink JK. Spastic paraplegia, ataxia, mental retardation (SPAR): a novel genetic disorder. Neurology 2002; 58: 411–416. 152. Engert JC, Berube P, et al. ARSACS, a spastic ataxia common in northeastern Quebec, is caused by mutations in a new gene encoding an 11.5-kb ORF. Nat Genet 2000; 24:120–125. 153. Krabbe K, Nielsen JE, Fallentin E, Fenger K, Herning M. MRI of autosomal dominant pure spastic paraplegia. Neuroradiology 1997; 39:724–727.

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13 Mitochondrial Disorders Clotilde Lagier-Tourenne and Michio Hirano Department of Neurology, Columbia University College of Physicians and Surgeons, New York, New York, U.S.A.

INTRODUCTION Mitochondria are essential cellular organelles that convert carbohydrates, lipids, and proteins into usable energy in the form of adenosine triphosphate (ATP) through aerobic metabolism. They originated from aerobic bacteria that formed symbiotic relationships with eukaryotic cells, which beneﬁted from their newly acquired capacity to utilize oxygen. This relationship is unique due, in part, to the fact that mitochondria are the only mammalian subcellular organelles—aside from the nucleus—that contain genetic material. Although mitochondria contain their own DNA (mtDNA), over evolutionary time most of the genes necessary for mitochondrial functions have been transferred to the nuclear DNA (nDNA). Therefore, mitochondrial diseases, that is, genetic diseases resulting in mitochondrial dysfunction, can be due to mutations in either genome. The ubiquitous nature of mtDNA and the peculiar rules of mitochondrial genetics contribute to the extraordinary clinical heterogeneity of mitochondrial disorders. Impairment of oxidative ATP production affects mainly tissues with high-energy requirements, such as skeletal muscle and brain, and results in mitochondrial myopathies or mitochondrial encephalomyopathies. Nevertheless, these disorders affect virtually every organ system in varying combinations resulting in protean clinical manifestations.

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To better understand mitochondrial disorders, the functions and properties of mitochondria are reviewed brieﬂy, followed by a description of the molecular mechanisms leading to mitochondria dysfunction. Next, the clinical, histological, and biochemical features shared by mitochondrial diseases will be described followed by a review of the most common mitochondrial syndromes. PRINCIPLES OF MITOCHONDRIAL GENETICS Biochemical Properties Mitochondria are essential to eukaryotic cells because they contain the enzymatic machinery necessary for aerobic metabolism, by which fatty acids, carbohydrates, and amino acids are broken down to form CO2, H2O, and, most importantly, to convert adenosine diphosphate (ADP) to ATP, the energy currency of the cell. The biochemical processes of aerobic metabolism can be classiﬁed into ﬁve major steps: (1) transport, (2) substrate utilization, (3) Krebs, citric acid, or tricarboxylic acid (TCA) cycle, (4) electron transport chain, and (5) oxidation–phosphorylation coupling (Fig. 1) (1). The electron transport chain, comprised of four enzymes (complexes I–IV), has been the subject of intense clinical and basic investigations over the last two decades. Reducing equivalents (electrons) are carried along these enzymes to create an efﬂux of protons from the mitochondrial matrix across the inner mitochondrial membrane to generate a transmembrane proton gradient. Oxidative– phosphorylation is the process by which the proton gradient across the mitochondrial inner membrane is converted into ATP. This chapter focuses primarily on diseases caused by respiratory chain enzyme defects which can result either from primary mutations in mtDNA or nuclear DNA. Mitochondrial Genetic Concepts Human mtDNA is a small circular, double-stranded molecule composed of 16,569 base pairs and encodes 37 genes: 13 polypeptides, 22 transfer ribonucleic acids (tRNA) molecules, and 2 ribosomal RNAs (rRNAs) (2). All 13 mtDNAencoded polypeptides are subunits of the respiratory chain (seven in complex I, one in complex III, three in complex IV, and two in complex V). The mitochondrial genome differs from nDNA in several aspects. First, there are no introns within the mtDNA and therefore, the genome is tightly packed with information. Second, each cell contains hundreds to thousands of copies of mtDNA. Third, mtDNA undergoes spontaneous mutations more rapidly than nDNA. Fourth, mtDNA is maternally inherited. These unique properties of mtDNA are responsible for the characteristics that distinguish mitochondrial genetics from mendelian genetics and help to explain many of the clinical peculiarities of mtDNA-related disorders. An important principle of mtDNA genetics is heteroplasmy. Each mitochondrion contains 2–10 copies of mtDNA and, in turn, each cell

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Figure 1 Mitochondrial metabolic pathways.

contains multiple mitochondria; therefore, there are numerous copies of mtDNA in each cell (polyplasmy). Alterations of mtDNA may be present in some (heteroplasmy) or in all (homoplasmy) of the mtDNA molecules. Usually, neutral mutations (or polymorphisms) are homoplasmic. In contrast, most (but not all) pathogenic mtDNA mutations are heteroplasmic. As a consequence of heteroplasmy, the proportion of a deleterious mtDNA mutation can vary widely and a minimum critical number of mutant genomes must be present before tissue dysfunction becomes evident (and related clinical signs become manifest), a concept aptly termed the threshold effect. However, this is a relative concept: tissues with high metabolic demands, such as brain, heart, and muscle, tend to have lower tolerance for mtDNA mutations than metabolically less active tissues (3). Moreover, an individual who harbors a large proportion of mutant mtDNA will be more severely afﬂicted by the mitochondrial dysfunction than a person with a low percentage of the same mutation; therefore, there is a spectrum of clinical severity among patients with a given mitochondrial mutation.

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Another factor that can inﬂuence the expression of the same mtDNA mutation in different individuals is the tissue distribution of that mutation. This tissue distribution is dependent on the concept of mitotic segregation. Both organellar division and mtDNA replication are apparently stochastic events unrelated to cell division; thus, the number of mitochondria (and mtDNA) can vary not just in space (i.e., among cells and tissues) but also in time (i.e., during development or aging). Moreover, at cell division, the proportion of mutant mtDNAs in daughter cells may drift, allowing relatively rapid changes in genotype that can translate into changes in phenotype, including the clinical picture, if and when the threshold is crossed. A ﬁfth unusual characteristic of mtDNA is maternal inheritance (4). During the formation of the zygote, mtDNA derives exclusively from the oocyte. Therefore, a mother carrying an mtDNA mutation will pass it on to all of her children, males and females, but only her daughters will transmit it to their progeny in a ‘‘vertical,’’ matrilinear line. This inheritance pattern is important to recognize in determining whether a family is likely to harbor an mtDNA mutation. However, the other features of mitochondrial genetics (e.g., heteroplasmy and the threshold effect) often mask maternal inheritance by causing striking intrafamilial clinical heterogeneity. Maternal relatives harboring lower percentages of an mtDNA mutation may have fewer symptoms (oligosymptomatic) than the proband or may even be asymptomatic. Therefore, in taking the family history, it is important to ask about the presence of subtle symptoms (e.g., short stature, hearing loss, migrainous headache, and isolated exercise intolerance) in maternally related family members. PRIMARY MITOCHONDRIAL DNA MUTATIONS In 1988, the ﬁrst mtDNA point mutation and large-scale deletions were described (5–8). Since then, there has been an outburst of information relating mtDNA mutations to human mitochondrial disorders including: duplications, single or multiple deletions, and pathogenic point mutations (9). mtDNA Deletions Holt et al. ﬁrst identiﬁed large-scale mtDNA deletions in mitochondrial myopathy patients and soon thereafter, Zeviani et al. pointed out the speciﬁc association with progressive external ophthalmoplegia (PEO) or Kearns–Sayre syndrome (KSS) (5,8). The mtDNA deletions range from about 2.0–10.4 kilobases (kb) in length and are mainly conﬁned to an 11-kb region that does not include the origins of mtDNA replication or mtDNA promoter regions (10). About one-third of the mtDNA deletions involve an identical 4977 base pair (bp) segment often referred to as the ‘‘common deletion’’. The majority of mtDNA deletions are ﬂanked by direct DNA sequence repeats, which suggests that they may arise from homologous

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recombination events or slip mispairing (11–13). Most patients with single deletions of mtDNA are sporadic cases suggesting that the recombination events occur in the oocytes or early in embryogenesis. Large-scale mtDNA deletions are often undetectable in mitotically active cells, such as ﬁbroblast and lymphocytes; therefore, molecular diagnosis requires muscle biopsy. Because multiple mtDNA deletions can be identiﬁed by polymerase chain reaction (PCR) in muscle from normal aged individuals, clinicians must be careful in their interpretation of PCR-detectable mtDNA deletions. In contrast, Southern blot analysis generally does not reveal mtDNA deletions in aged individuals and is the most appropriate technique to identify single mtDNA duplications, and single or multiple mtDNA deletions. mtDNA Duplications Duplications of mtDNA were initially reported in two patients with KSS, who also harbored single mtDNA deletions (14). Later, maternally inherited duplications associated with deletions were also described (15). Duplicated mtDNA molecules consist of a wild-type mtDNA molecule plus an additional segment corresponding to the deleted mtDNA molecule. Duplications may be the underlying cause of maternally transmitted ‘‘deletions’’ of mtDNA. Studies of one mitochondrial myopathy patient, who harbored both a deletion and a corresponding duplication of the mtDNA, revealed that the deletions, not the duplications, were responsible for the histological abnormalities in skeletal muscle, implying that duplications per se may not be pathogenic (16). mtDNA Point Mutations The ﬁrst point mutation in human mtDNA was identiﬁed by Wallace et al. in patients with Leber hereditary optic neuropathy (LHON) (7). Since then, numerous mtDNA mutations have been related to maternally inherited or sporadic mitochondrial diseases. These mutations can either affect mitochondrial protein synthesis as mutations in rRNA and tRNA genes, or can occur in protein-coding genes causing defects of a speciﬁc respiratory chain complex (17). In contrast to deletions and duplications, mtDNA point mutations are generally detectable in DNA from leukocytes, urine sediment, and buccal mucosa (18). NUCLEAR DNA MUTATIONS The nuclear genome encodes hundreds of mitochondrial proteins that are vital for all functions of the organelles. Consequently, mutations of nDNA transmitted by mendelian inheritance can affect the respiratory chain directly or indirectly, potentially impairing a wide variety of mitochondrial

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functions. These include mitochondrial motility, protein or metabolite importation, and maintenance of the membrane milieu (Table 1). Finally, a last group of genetic mitochondrial disorders includes defects of intergenomic communication, due to mutations of nDNA genes controlling replication and expression of the mitochondrial genome that in turn alter mtDNA integrity. Mutations in Structural Subunits or Assembly Genes of the Respiratory Chain Complexes Pathogenic mutations in nDNA-encoded structural subunits of complexes I and II have been identiﬁed leading to severe infantile or childhood neurological disorders such as Leigh syndrome or leukoencephalopathy (19,20). Other clinical phenotypes associated with complex I deﬁciency include fatal infantile lactic acidosis, hepatopathy and tubulopathy, cardiomyopathy with or without cataracts, macrocephaly with progressive leukodystrophy, and unspeciﬁed encephalomyopathies (21,22). Mutations in complex II subunits have also been associated with familial paragangliomas and pheochromocytomas (23,24). In contrast, autosomal deﬁciencies of complexes III, IV, or V have not been linked to mutations in nDNA-encoded subunits, but rather to defects of nDNA genes encoding ancillary proteins required for enzyme assembly or insertion of cofactors. For example, mutations in six genes involved in assembly of cytochrome c oxidase (SURF1, SCO1, SCO2, COX10, COX15, and LRPPRC ) have been identiﬁed as causes of autosomal recessive cytochrome c oxidase (COX) deﬁciencies (25–31). In addition, mutations in the assembly factor for complex III, BCS1L, are associated with neonatal proximal tubulopathy, hepatopathy, and encephalopathy (32). Mutations in Genes in Mitochondrial Importation and Motility Identiﬁcation of mutations in other autosomal genes is leading to new concepts of mitochondrial diseases. For example, the identiﬁcation of DDP1 gene mutations in X-linked deafness-dystonia (Mohr-Tranebjaerg syndrome) as well as in opticoacoustic nerve atrophy with dementia (Jensen syndrome), comprise the ﬁrst defect in a mitochondrial importation factor (33). Moreover, mutations in the OPA1 gene in autosomal dominant optic atrophy 1 (Kjer type optic atrophy) indicate that defects of mitochondrial division or biogenesis can cause diseases (34,35). Defects of Intergenomic Communication In 1989, Zeviani et al. described multiple mtDNA deletions in a family with autosomal dominant progressive external ophthalmoplegia (adPEO) (13). The mendelian inheritance pointed to a nDNA mutation and an abnormal interaction of a mutated nDNA-encoded factor with the mitochondrial

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Table 1 Nosological Classiﬁcation of Mitochondrial Respiratory Chain Disorders Due to Nuclear DNA Mutations Biochemical defect

Clinical phenotype

Defective gene product (if known)

Mutations in genes encoding structural subunits of respiratory chain enzymes Complex I deﬁciency Encephalomyopathy or NDUFS2, NDUFS4, Leigh syndrome NDUFS8, NDUFV1, NDUFS1, NDUFS7 Complex II deﬁciency Leigh syndrome Flavoprotein subunit of SDH Hereditary paraganglioma SDHB, SDHC, SDHD or pheochromocytoma Mutations in genes encoding assembly factors for respiratory chain enzymes BCSIL Complex III deﬁciency Encephalopathy, tubulopathy and hepatopathy Complex IV deﬁciency Leigh syndrome SURF1 Infantile SCO2 cardioencephalopathy Infantile encephalopathy SCO1, COX 10 Defects of intergenomic communication Autosomal recessive PEO MNGIE Thymidine with multiple D- mtDNA phosphorylase arPEO POLG ARCO Unknown Autosomal dominant PEO adPEO ANT1, Twinkle, POLG with multiple D-mtDNA MtDNA depletion Infantile myopathy TK2 Infantile encephalopathy dGK and hepatopathy Alpers disease POLG Infantile myopathy or Unknown hepatopathy Navaho neurohepatopathy Unknown Defects of the mitochondrial membrane Unknown Coenzyme Q10 deﬁciency Myoglobinuria, encephalopathy, ragged-red ﬁbers Cerebellar ataxia Unknown Impaired processing of Barth syndrome Tafazzin mitochondrial phospholipids Defects of mitochondrial importation Impaired importation of X-Iinked deafness-dystonia Tim 8/9 proteins (Continued)

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Table 1 Nosological Classification of Mitochondrial Respiratory Chain Disorders Due to Nuclear DNA Mutations (Continued ) Biochemical defect

Clinical phenotype

Defects of mitochondrial division and biogenesis Impaired mitochondrial Autosomal dominant optic atrophy 1 Defects of mitochondrial metal metabolism Iron-sulfur metabolism Friedreich ataxia Iron export defect X-linked sideroblastic anemia Other Hereditary spastic paraplegia

Defective gene product (if known) OPA1

Frataxin ABC7

Paraplegin

genome was postulated. Subsequently, mutations in different nuclear genes were identiﬁed as causes of secondary mtDNA defects such as multiple deletions, depletion, or both. Multiple mtDNA deletions of different sizes described in adPEO (13) have been associated with mutations in three nuclear genes: adenine nucleotide translocator 1 (ANT1) (36), Twinkle (37), and polymerase gamma (POLG) (38). Mutations in POLG are also responsible for autosomal recessive PEO (arPEO) (38); however, the molecular defect leading to the multiple deletions present in autosomal recessive cardiomyopathy and ophthalmoplegia (ARCO) is not yet known (39). Depletion of mtDNA is the other major defect of intergenomic communication, together with multiple mtDNA deletions described earlier. As the name implies, this is a quantitative rather than qualitative mtDNA abnormality, consisting of markedly decreased levels of mtDNA in one or more tissues (40). The clinical spectrum of mtDNA depletion has been incompletely characterized and is probably more heterogeneous than was initially thought. Four syndromes stand out, all inherited as autosomalrecessive traits: congenital myopathy, infantile myopathy, hepatopathy, and hepatocerebral mtDNA depletion. Loss-of-function mutations in the gene encoding the thymidine phosphorylase (TP) led to the novel concept that nucleotide pool imbalances can affect mtDNA quantity and integrity (41). Indeed, mutations in the TP gene were identiﬁed in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), which is associated with multiple deletions, depletion and site-speciﬁc point mutations of mitochondrial DNA (42–44). Deﬁciency of TP leads to dramatically elevated levels of circulating thymidine and deoxyuridine (45). Alterations of pyrimidine nucleoside metabolism

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are hypothesized to cause imbalances of mitochondrial nucleotide pools that, in turn, may cause somatic alterations of mtDNA. This concept was reinforced when defects of ANT1 were associated with multiple deletions in adPEO (36), and mutations in genes encoding mitochondrial deoxyguanosine kinase (dGK) and thymidine kinase (TK2) were found to cause mtDNA depletion. In these forms of mtDNA depletion syndrome (MDS), defects of mitochondrial nucleoside kinases are thought to cause deﬁciency of mitochondrial dNTPs, which impedes mtDNA replication. Moreover, an iatrogenic form of mtDNA depletion has also been recognized in patients with acquired immunodeﬁciency syndrome in whom a mitochondrial myopathy developed during treatment with a nucleoside analogue, zidovudine (Retrovir) (46,47). Thus, inborn and acquired defects of nucleoside and nucleotide metabolism comprise a new group of mitochondrial disorders. GENERAL FEATURES OF MITOCHONDRIAL DISEASES Defects in the electron transport chain and in oxidation–phosphorylation coupling will impair ATP synthesis. In turn, deﬁciencies in ATP will affect virtually all tissues, causing multisystem diseases collectively described as mitochondrial myopathies or mitochondrial encephalomyopathies. While the clinical heterogeneity of these disorders can be confusing, there are general features that can be helpful in making a preliminary diagnosis. Clinical Features of Mitochondrial Diseases Some patients with mitochondrial diseases have isolated myopathies, encephalopathies, or other single-organ disorders; however, most develop multisystem disorders. Despite the plethora of manifestations, some are frequently present together and should alert the clinician to a mitochondrial etiology (Table 2). Ptosis, PEO, or both are hallmarks of KSS, sporadic PEO, and autosomal dominant or recessive forms of PEO. The onset of ophthalmoparesis is usually insidious and complaints of diplopia and blurred vision due to dysconjugate gaze are uncommon. Patients may note mild worsening of symptoms with fatigue, but the ptosis and ophthalmoparesis do not ﬂuctuate as widely as in myasthenia gravis. Other, ophthalmological signs such as pigmentary retinopathy, optic atrophy, cataracts, and visual ﬁeld defects are also often present. Limb myopathy is frequent in patients with mitochondrial disorders. Neck ﬂexors may be affected earlier and more severely than neck extensors. As with most myopathies, proximal limb muscles are disproportionately weaker than distal muscles. Patients with mitochondrial myopathies are generally thin with decreased muscle bulk. Many patients with deﬁciencies

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Table 2 Clinical Features of Mitochondrial Diseases Due to Nuclear DNA Mutations Leigh syndrome Onset typically in infancy or childhood, but can occur in adulthood Infantile-onset Typically before age six months in a previously normal infant Developmental arrest or regression, hypotonia, feeding difﬁculty, respiratory abnormalities, vision loss, oculomotor palsies, and nystagmus. Brain MRI reveals symmetric lesions of the basal ganglia and midline brainstem. Childhood-onset Similar to the infantile-onset patients, but PEO, dystonia, or ataxia may be prominent. Infantile myoclonic epilepsy and leukodystrophy due to complex I deﬁciency (NDUFVl mutations) Myoclonic epilepsy Hypotonia Developmental delay Leukodystrophy Infantile cardioencephalomyopathy due to complex I deﬁciency (NDUFS2 mutations) Hypertrophic cardiomyopathy Encephalopathy (ataxia, pyramidal signs, basal ganglia and white matter lesions) Myopathy Optic atrophy Infantile cardioencephalopathy due to SCO2 mutations Progressive hypertrophic cardiomyopathy Encephalopathy with seizures and variable atrophy, migrational defects, neuronal defects, and gliosis Respiratory insufﬁciency Hypotonia Fatal infantile myopathy (FIM) with cytochrome c oxidase deﬁciency Diffuse weakness, hypotonia, and respiratory insufﬁciency due to myopathy Sometimes de Toni-Debre´-Fanconi syndrome Benign infantile myopathy (BIM) with cytochrome c oxidase deﬁciency Diffuse weakness, hypotonia, and respiratory insufﬁciency due to myopathy Spontaneous improvement by age 2–3 years Autosomal dominant progressive external ophthalmoplegia (adPEO) Ptosis and PEO generally beginning in young adulthood Ragged-red and cytochrome c oxidase (COX) deﬁcient ﬁbers in skeletal muscle Multiple D-mtDNA Other features include: proximal limb weakness, respiratory insufﬁciency, depression, peripheral neuropathy, sensorineural hearing loss, cataracts, endocrinopathies. (Continued)

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Table 2 Clinical Features of Mitochondrial Diseases Due to Nuclear DNA Mutations (Continued ) Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) Ptosis and PEO Gastrointestinal dysmotility Demyelinating peripheral neuropathy Cachexia Leukoencephalopathy on MRI scan Autosomal recessive cardiomyopathy ophthalmoplegia (ARCO) Ptosis and PEO Proximal muscle weakness Hypertrophic cardiomyopathy Infantile mtDNA depletion syndromes Myopathic form Feeding difﬁculty, failure to thrive, hypotonia, weakness, and occasionally PEO Elevated serum creatine kinase (2–30 times the upper limit of normal) Myopathy with COX-deﬁcient ﬁbers and sometimes RRF Hepatopathy Liver failure manifesting as persistent vomiting, failure to thrive, hypotonia, and hypoglycemia. Alpers syndrome Normal early development followed by rapid episodic psychomotor regression, intractable seizures, liver disease Navaho neurohepatopathy (NHH) 4 of the following 6 criteria or 3 plus a positive family history of NHH Sensory neuropathy Motor neuropathy Corneal anesthesia, ulcers, or scars Liver disease Documented metabolic or immunologic derangement Central nervous system demyelination Coenzyme Q10 deﬁciency Myopathic form Myoglobinuria Encephalopathy (seizures or mental retardation) RRF in muscle biopsy Cerebellar form Cerebellar ataxia with prominent cerebellar atrophy in brain MRI scans Barth syndrome X-Iinked Dilated cardiomyopathy Neutropenia (due to impaired leukocyte maturation) Myopathy with abnormal mitochondria (Continued)

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Table 2 Clinical Features of Mitochondrial Diseases Due to Nuclear DNA Mutations (Continued ) Mohr-Tranebjaerg Syndrome (X-linked deafness-dystonia syndrome) X-Iinked Sensorineural hearing loss Dystonia Other features: spasticity, dysphagia, intellectual decline, paranoia, cortical blindness Autosomal dominant optic atrophy 1 (Kjer type optic atrophy) Childhood-onset of insidious optic atrophy manifesting as loss of visual acuity, color vision loss, temporal optic disc pallor, centrocecal scotoma) X-linked sideroblastic anemia with ataxia syndrome (XLSA/A) Hypochromic microcytic anemia Ring sideroblasts in bone marrow Ataxia Corticospinal tract signs

of respiratory chain enzymes may exhibit normal muscle strength with manual testing; yet, they complain of functional impairment due to premature fatigue. While this type of exercise intolerance is intuitively compatible with a defect in aerobic metabolism, the subjective nature of the complaint makes formal exercise testing with measurements of oxygen consumption, maximum work rate, and serum lactate very helpful. Exercise intolerance may be the sole manifestation of an mtDNA mutation (48). Sensorineural hearing loss is also frequently associated with mitochondrial encephalomyopathies. The hearing loss may be asymmetric and ﬂuctuating in severity. A particular mtDNA point mutation has been associated with cases of aminoglycoside-induced deafness (AID) (49). Maternally inherited deafness and diabetes (DAD) is another frequent phenotypic combination observed in patients with mtDNA mutations (50). Finally, peripheral neuropathy, dementia, migraine-like headaches, hypertrophic cardiomyopathy, renal tubular acidosis, short stature, multiple lipomas, and hypoparathyroidism are also common clinical manifestations of respiratory chain enzyme defects. Laboratory Tests in Mitochondrial Diseases Clinical Laboratory Tests Laboratory evaluation of patients with mitochondrial diseases should begin with routine blood tests including complete blood count, serum electrolytes (including calcium and phosphate), liver function tests, blood urea nitrogen, creatinine, lactate, and pyruvate. These tests may reveal dysfunction of the

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parathyroid, kidney, or liver. Serum lactate at rest is frequently elevated and out of proportion to pyruvate in patients with defects of the respiratory chain (e.g., lactate to pyruvate ratio 20:l); however, many patients with well-documented mitochondrial diseases have normal baseline lactate levels that may increase dramatically after moderate exercise. Formal exercise testing with careful measurements of oxygen consumption and near-infrared spectroscopy, although not widely available, are very useful in such patients. Lumbar puncture may show elevated CSF protein, especially in KSS patients where it is often greater than 100 mg/dl. It may also reveal elevated lactate and pyruvate levels, sometimes in patients with normal serum levels. Electrocardiograms may reveal preexcitation or heart block. Electromyography and nerve conduction studies are typically consistent with a myogenic process although neurogenic changes may also be detected. Neuropathologic and neuroradiologic changes fall into ﬁve main patterns: 1. Microcephaly and ventricular dilatation, sometimes associated with agenesis of the corpus callosum, are seen in infants with severe congenital lactic acidosis. 2. Bilateral, symmetric lesions of basal ganglia, thalamus, brainstem, and cerebellar roof nuclei are the signatures of Leigh syndrome. 3. Multifocal encephalomalacia, usually involving the cortex of the posterior cerebral hemispheres, corresponds to the ‘‘strokes’’ of the Mitochondrial Encephalomyopathy, Lactic Acidosis, with Stroke-like episodes (MELAS) syndrome. 4. Spongy encephalopathy, predominantly in white matter, is characteristic of KSS. 5. Calciﬁcations of the basal ganglia can be seen in all of these disorders, but in a minority of patients with any syndrome. Special Laboratory Tests Clinical research has contributed greatly to our understanding of mitochondrial disorders and to our diagnostic capabilities. Specialized evaluation for oxidative–phosphorylation defects has evolved from laboratory research to include muscle histochemistry and immunohistochemistry, measurement of oxidative–phosphorylation enzyme activities, and molecular genetic analyses. In patients whose clinical manifestations conform to one of the welldeﬁned mitochondrial diseases associated with speciﬁc point mutations of mtDNA, direct genetic testing can be performed using DNA from leukocytes, urinary sediment, or buccal mucosa (18). Muscle biopsy: In the mid-1960s, Shy, Gonatas, and Perez described the typical ultrastructural alterations seen in mitochondrial myopathies (51). These changes include: (1) an overabundance of ultrastructually normal

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mitochondria, ‘‘pleoconial myopathy,’’ (2) enlarged mitochondria with disoriented cristae, ‘‘megaconial myopathy,’’ and (3) inclusions within mitochondria, ‘‘paracrystalline’’ and ‘‘osmiophilic’’ inclusions (51). Engel and Cunningham developed a modiﬁed Gomori trichrome stain which has been commonly used to identify ﬁbers with subsarcolemmal accumulations of mitochondria, which are referred to as ‘‘ragged-red ﬁbers’’ (RRF) (52). RRFs are generally associated with primary mtDNA mutations that affect mitochondrial protein synthesis, such as point mutations in tRNA genes or large-scale mtDNA deletion that eliminate at least one tRNA gene. Both types of mutations lead to a paucity of normal mitochondrial tRNAs, hence impaired protein synthesis. While none of the known primary nDNA mutations directly affects mitochondrial protein synthesis, defects of intergenomic communication lead to secondary depletion and multiple deletions of mtDNA, thereby producing RRF. Histochemical and immunohistochemical stains for mitochondrial enzymes are also used to identify excessive mitochondrial proliferation and to demonstrate defects of speciﬁc enzymes. Indeed, the histochemical stain for succinate dehydrogenase (SDH), which functions as complex II (succinate-CoQ reductase) of the mitochondrial respiratory chain, provides a more sensitive indicator of mitochondrial proliferation than Gomori trichrome stain. The histochemical stain for cytochrome c oxidase (COX; complex IV) often provides vital morphological information. In patients with a pathogenic nDNA mutation in a COX subunit or in a COX assembly factor, histochemistry will show a generalized reduction or absence of COX staining in skeletal muscle. By contrast, in patients with primary or secondary mtDNA mutations, COX histochemistry typically shows a mosaic pattern of ﬁbers with normal and decreased staining. In KSS, MELAS, and MERRF, mitochondrial proliferation is almost always identiﬁed in skeletal muscle by the Gomori trichrome stain and SDH histochemistry. In MELAS patients, there is often excessive SDH staining within blood vessel walls, so-called strongly SDH-reactive vessels or SSVs (53,54). Another peculiar characteristic of skeletal muscle from MELAS patients is the relative preservation of COX staining in RRF, in contrast to muscle from KSS and MERRF patients, in which there are abundant COX-negative RRF on serial or double-stained (SDH and COX) sections. Unfortunately for clinicians, the histological abnormalities described above are neither speciﬁc nor sensitive enough to deﬁne all mitochondrial diseases. Morphologically abnormal muscle mitochondria are sometimes detected in conditions that are not due to primary oxidative–phosphorylation defects, such as inﬂammatory myopathies (55) and myotonic dystrophy (56). Conversely, some primary mitochondrial disorders have subtle or no morphological abnormalities of mitochondria, including Neuropathy Ataxia, and Retinitis Pigmentosa (NARP), Leber’s hereditary optic neuropathy (LHON), and Leigh syndrome (LS).

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Biochemistry (Respiratory chain enzyme assays): Activities of mitochondrial respiratory chain enzymes can be measured in crude muscle extracts or in isolated mitochondria. Biochemical studies in patients with mtDNArelated disorders yield two main types of results. The ﬁrst pattern consists of partial defects in the activities of multiple complexes containing mtDNAencoded subunits (I, III, and IV), contrasting with normal or increased activities of SDH (complex II), because all subunits of this enzyme complex are encoded by nDNA. The second pattern is severe deﬁciency in the activity of one respiratory complex. As a rule, the ﬁrst pattern is found in patients with mutations impairing mitochondrial protein synthesis such as tRNA genes mutations and mtDNA deletions or depletion. The second is typical of a mutation in a mtDNA or nDNA gene encoding a polypetide subunit of a respiratory chain enzyme complex. Identiﬁcation of an isolated enzyme deﬁciency pinpoints the biochemical problem and focuses molecular genetic analyses to identify the causative mutation. For example, isolated monoenzymopathies can be diagnosed for some of the clinical syndromes described above, such as the fatal and benign COX-deﬁcient myopathies. However, some mitochondrial disorders, such as LHON and NARP, may not have any detectable abnormalities of respiratory chain enzymes with routine assays.

SPECIFIC MITOCHONDRIAL DISEASES Progressive External Ophthalmoplegia PEO is deﬁned clinically as a slowly progressive limitation of eye movements until there is complete immobility (ophthalmoplegia). It is usually accompanied by eyelid droop (ptosis). There may or may not be weakness of muscles of the face, oropharynx, neck, or limbs. This condition is clearly heterogeneous, but several distinct syndromes can be recognized, some related to primary abnormalities of mtDNA (with sporadic single deletions or maternally inherited point mutations), and others related to autosomal genes directly affecting mtDNA (defects of intergenomic signaling with multiple deletions of mtDNA). PEO with Single mtDNA Deletions PEO with or without limb weakness: This is a relatively benign condition, often compatible with a normal lifespan. Symptoms usually begin in childhood but may be delayed to adolescence or adult years. Ptosis is often the ﬁrst symptom, followed by ophthalmoparesis. The disorder is bilateral and symmetric, so diplopia is exceptional. Some patients also have pharyngeal and limb weakness. Although typically sporadic, women with PEO or KSS due to a single-mtDNA deletion have a 4% chance of transmitting the mutation to each child (57).

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Kearns–Sayre syndrome: In 1958, Kearns and Sayre deﬁned a syndrome as the combination of: retinitis pigmentosa, external ophthalmoplegia, and complete heart block (58). In 1983, Rowland et al. redeﬁned the KSS by the invariant triad of ophthalmoplegia, pigmentary retinopathy, and onset before age 20 years plus at least one of the following three additional features: cardiac conduction block, cerebellar syndrome, or cerebrospinal ﬂuid (CSF) protein greater than 100 mg/dL (59). In addition to the diagnostic clinical features, KSS patients often have hearing loss and limb weakness. Associated endocrinopathies include diabetes mellitus, hypoparathyroidism, irregular menses, and growth hormone deﬁciency. Seizures are infrequent and are usually associated with electrolyte disturbances, as may occur in hypoparathyroidism, one of several endocrine disorders sometimes associated with KSS (60). The evolution is characterized by gradual progression of the multisystemic disorders. Placement of a cardiac pacemaker in the setting of impending complete heart block can extend a KSS patient’s life by years. The ataxia and myopathy can be severely debilitating to the extent that some patients are wheelchair-bound in their twenties. Virtually all KSS patients have been sporadic. Laboratory abnormalities: In both conditions, muscle biopsy shows RRFs that are devoid of histochemically demonstrable COX activity. Raised levels of lactate and pyruvate are usually found in blood in both sporadic PEO and KSS. Computed tomography or magnetic resonance imaging (MRI) sometimes shows evidence of leukoencephalopathy, and there may be calciﬁcation of basal ganglia. Correspondingly, at postmortem examination, typical cases of KSS show spongy degeneration of the brain. Maternally Inherited PEO with Point Mutations of mtDNA A substantial number of patients with mitochondrial PEO (i.e., PEO and RRF in the muscle biopsy) show maternal inheritance of their syndrome. PEO predominates in this syndrome but is often associated with various combinations of other symptoms, including hearing loss, endocrinopathy, heart block, cerebellar ataxia, or pigmentary retinopathy. There is usually lactic acidosis, and muscle biopsies show RRF. The most common mutation in these patients is the typical A3243G MELAS mutation, but other mutations have also been described, including the A8344G mutation typically seen in MERRF syndrome. The reason for the appearance of PEO in patients with the MELAS mutation is not known, but it may be related to a selective accumulation of mutant mtDNA in extraocular muscle. Autosomal PEO with Multiple Deletions of mtDNA PEO has been described in numerous families with adPEO or autosomalrecessive (arPEO) inheritance. Patients with adPEO gradually develop ptosis

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and ophthalmoparesis during early adulthood and may manifest other symptoms including: proximal limb weakness, respiratory insufﬁciency, severe depression, ataxia, peripheral neuropathy, sensorineural hearing loss, cataracts, endocrine dysfunction, and parkinsonism (61–63). By contrast, arPEO disorders begin in childhood or adolescence and manifest as multisystem disorders such as mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or autosomal recessive cardiomyopathy ophthalmoplegia (ARCO) (64). MNGIE is due to mutations in the gene encoding TP and is characterized by ptosis, PEO, peripheral neuropathy, and severe gastrointestinal dysmotility leading to cachexia (65). MRI shows diffuse leukodystrophy, although patients rarely show cognitive defects. Death usually occurs in the fourth or ﬁfth decade. ARCO was identiﬁed in two large families from the eastern Arabian peninsula with childhood-onset of PEO, proximal muscle weakness and severe hypertrophic cardiomyopathy (39). The cause of ARCO is not known. Laboratory Abnormalities In all these conditions, muscle biopsy usually shows RRF and COX-negative ﬁbers. Lactic acidosis is usually present but may not be very marked. In MNGIE, nerve conduction studies and nerve biopsies have shown features of both axonal and demyelinating neuropathy. Moreover, biochemical detection of elevated plasma thymidine and deoxyuridine or decreased TP activities in MNGIE patients provides more rapid and cost-effective screening tests than sequencing of the TP gene (66). Molecular Genetics Southern blot analyses of muscle mtDNA in these conditions show multiple bands representing species of mtDNA molecules harboring deletions of different sizes. As previously described, mutations in nuclear genes are responsible for defects of intergenomic communication leading to these secondary mtDNA deletions. Myoclonus Epilepsy with Ragged-Red Fibers Identiﬁed as a clinical syndrome in 1980 by Fukuhara et al. myoclonus epilepsy with ragged-red ﬁbers (MERRF) is deﬁned by the clinical features: myoclonus, generalized seizures, ataxia, and COX-negative RRF in the muscle biopsy (67). Symptoms usually begin in childhood, but adult-onset has also been described. Although progression is slow, patients can be severely impaired by the myoclonus, ataxia, or both. The ataxia has been attributed to degeneration of both the posterior column and the cerebellum. Other common clinical manifestations include: hearing loss, family history of affected individuals in the maternal lineage, dementia, exercise intolerance, and lactic

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acidosis (60). Large lipomas have been seen in MERRF patients, as well as in oligosymptomatic relatives (68). MERRF was the ﬁrst multisystem disorder to be associated with an mtDNA point mutation, speciﬁcally, A8344G in the transfer RNA lysine (tRNALys) gene (69). Approximately 80% of patients with MERRF have the A8344G mtDNA mutation; however, at least six other mtDNA point mutations and multiple deletions of mtDNA have been pathogenically linked to MERRF (70). MELAS Identiﬁed as a distinct clinical syndrome in 1984 by Pavlakis et al. MELAS is characterized by: (1) strokes at a young age and typically before age 40 years, (2) encephalopathy evident as seizures, dementia, or both, and (3) lactic acidosis, RRF or both as manifestations of the respiratory chain defects (71,72). In addition to these cardinal manifestations, other frequent clinical features include: normal early development, myogenic limb weakness, ataxia, myoclonus, migraine-like headaches, recurrent nausea and vomiting, and hearing loss (72). Although the clinical features of ataxia, myoclonus, seizures, and ragged-red ﬁbers are common to both MERRF and MELAS patients, the clinical course of MELAS is punctuated by strokes which are abrupt and often devastating. Typically, the strokes affect the occipital cortex, but may affect other regions of the brain. Both clinical and radiological defects can improve over weeks to months, but virtually always recur (72). The cerebral lesions often do not conform to territories of large vessels, a ﬁnding that has led some individuals to use the term stroke-like episodes to distinguish the cerebral insults in MELAS from the more typical ischemic strokes (72). In a few cases, the lesions have affected the temporal lobe mimicking herpes simplex encephalitis (73). Another intriguing feature of MELAS is the infrequent occurrence of the full-syndrome in more than one member within a pedigree, in contradistinction to MERRF families where multiple members typically have the complete syndrome. Usually, maternal relatives of MELAS patients are oligosymptomtic or asymptomatic. Laboratory abnormalities include elevated blood and CSF lactate. Muscle biopsy shows RRF, which are uncharacteristically COX-positive, rather than COX-negative as in most other mtDNA-related diseases. Electrocardiograms may reveal preexcitation in MELAS or MERRF, and heart block in MELAS. Electromyography and nerve conduction studies are typically consistent with a myogenic process although neurogenic changes may be detected in MERRF or MELAS. Fundoscopy may reveal pigmentary retinopathy in MELAS or MERRF, and optic atrophy is sometimes detected in MERRF patients.

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In about 80% of patients, the molecular defect is a point mutation (A3243G) in the tRNALeu(UUR) gene of mtDNA. In the remaining patients, 15 other mutations have been described, some of which affect proteinencoding genes rather than tRNA genes (74). A3243G is the most frequent pathogenic mtDNA mutation with prevalence estimates ranging from 1.4– 16.3 per 100,000 (75,76). In addition to MELAS and maternally inherited PEO, the A3243G mutation has been associated with diabetes mellitus alone or in combination with deafness. The heterogeneity in the clinical expression of the A3243G mutation may be due to varying degrees of heteroplasmy in different tissues; however, other inﬂuences, such as nuclear DNA factors, may also play a role. Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) The combination of NARP is a maternally transmitted multisystem disorder of young adult life, comprising, in various combinations, sensory neuropathy, ataxia, seizures, dementia, and retinitis pigmentosa. Lactic acid in blood may be normal or slightly elevated, and muscle biopsy does not show RRF. The most common molecular defect is a point mutation (T8993G) in the gene that encodes ATPase 6 (77). When this mutation approaches homoplasmic levels, onset is in infancy, and the clinical and neuropathologic features are those of Leigh syndrome [maternally inherited Leigh syndrome (MILS)] (78). A different mutation at the very same nucleotide (T8993C) causes a phenotype similar to MILS but generally milder. Other mutations in the ATPase 6 gene have been associated with Leigh-like syndromes or with familial bilateral striatal necrosis. Leigh Syndrome Leigh syndrome is the most common mitochondrial disorder in infants and children, but is rare in adults (79). Infants may present symptoms at birth or may develop normally for months; however, onset is typically before age six months. The initial manifestations include hypotonia, psychomotor delay, and respiratory insufﬁciency followed by impaired vision, hearing loss, seizures, ataxia, limb weakness, pyramidal tract signs, and intellectual deterioration. Nystagmus is also common. In patients with later onset, PEO, dystonia, or ataxia may be present. CSF protein content is mildly elevated in 25% of patients. Lactate and pyruvate levels are almost always increased in CSF and, to a lesser degree, in blood and urine. These ﬁndings and the histopathology lead to a search for an abnormality of oxidative metabolism. Electroencephalogram changes and abnormal evoked responses are nonspeciﬁc. MRI of the brain reveals characteristic symmetric lesions of the periaqueductal region of the midbrain and pons and in the medulla adjacent to the fourth ventricle. Other parts of the central nervous system and peripheral nerves may also be

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affected. The lesions are a combination of cell necrosis, demyelination, and vascular proliferation. The condition may be inherited in an autosomal-recessive, X-linked, or maternal pattern, and mutations have been identiﬁed in many mitochondrial or nuclear genes involved in energy metabolism. Leber Hereditary Optic Neuropathy (LHON) LHON is a maternally inherited disorder characterized by loss of central vision (80,81). Onset usually occurs in adolescence or early-adult years but may occur from ages 5 to 80 years. Cloudiness of central vision progresses painlessly over weeks, usually ﬁrst in one eye, to a larger, denser centrocecal scotoma. Both eyes are usually affected within weeks or months, sometimes simultaneously. Color vision is affected, and acuity drops to 20/200 or ﬁnger counting. Sometimes, there is later improvement, infrequently striking and occasionally sudden, with severity and possibility of improvement varying with the particular mutation. The fundus may appear normal until optic atrophy supervenes, but at onset, the disc often appears blurred and suggestive of edema. Circumpapillary telangiectatic microangiopathy, without evidence of abnormal vascular permeability, is sometimes observed. The vascular abnormalities may be seen in presymptomatic and asymptomatic relatives and do not invariably predict imminent visual loss, which may never occur. In most patients with LHON, visual disturbance is the only symptom, but cardiac preexcitation is frequently associated. Neurologic examination, however, may reveal subtle neurologic abnormalities, including postural tremor, dystonia, motor tics, parkinsonism with dystonia, or peripheral neuropathy. Several patients (mostly women) with LHON have had multiple sclerosis-like manifestations. The molecular genetic features of LHON are complex and unique. The ﬁrst and most common primary mtDNA mutation associated with LHON was found in the gene for subunit 4 of complex 1 (7). Two additional common mtDNA mutations in complex I (ND) subunits have been identiﬁed: G3460A in ND1 and T14484C in ND6. While these three mtDNA mutations account for >90% of LHON patients, additional mtDNA mutations have been identiﬁed in families with LHON. Penetrance rates of the LHON mutations are uncertain; however, some reports estimate that symptoms appear in 20% to 83% of men and 4% to 32% of women at risk (80,81). Sixty percent to 90% of LHON patients are men. The molecular basis for male predominance is not known. No treatment is of proven value. Antioxidants (e.g., idebenone and coenzyme Q10) have also been used to reduce possible damage from reactive oxygen species generated by the impaired oxidative metabolism. According to consensus, tobacco and alcohol should be avoided in family members at risk.

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Infantile Myopathies with Cytochrome c Oxidase (COX) Deficiency Infantile myopathies with COX deﬁciency are important for clinicians to recognize because the patients with the benign infantile myopathy (BIM) can recover fully if proper medical care is administered (82). Both BIM and fatal infantile myopathy (FIM) with COX deﬁciency present with congenital generalized muscle weakness, with respiratory muscle insufﬁciency and lactic acidosis; however, infants with FIM die within the ﬁrst year of life despite aggressive medical interventions (82). Patients with BIM also present with severe myopathy and lactic acidosis soon after birth, but improve dramatically and are virtually normal by age two and three years (83). Lactic acidosis and muscle histological abnormalities also resolve. Because of the remarkable spontaneous improvement, the syndrome has been called reversible infantile myopathy or BIM. The latter term is misleading because the disorder can be fatal if the patients are not properly managed in the critical ﬁrst few months of life. The common biochemical and histochemical feature of these patients is COX deﬁciency in skeletal muscle, sparing heart, brain, and liver. Recurrence of these syndromes in siblings indicates that these disorders are hereditary and probably autosomal recessive. mtDNA Depletion The clinical presentation of this mitochondrial syndrome is variable, even within a single family (40,84). Onset may be congenital or soon after birth, generally leading to death within the ﬁrst year of life. However, onset can also be in infancy or in childhood with survival through the ﬁrst decade. A variety of speciﬁc clinical phenotypes have been reported: (1) Congenital myopathy, with neonatal weakness and hypotonia requiring assisted ventilation, and death before age one year; renal dysfunction (DeToni–Fanconi syndrome) may also be present, (2) myopathy usually developing around age one year with rapid worsening causing respiratory insufﬁciency and death within a few years, (3) hepatopathy with hepatomegaly and intractable liver failure causing death within the ﬁrst year, (4) hepatocerebral mtDNA depletion with onset within the ﬁrst six months and death usually in the ﬁrst year, (5) a lower motor neuron disorder resembling spinal muscular atrophy (SMA), and (6) Alpers’ syndrome (infantile poliodystrophy), a progressive encephalohepatopathy that causes death in early childhood (40,84–89). Peripheral neuropathy has also been noted in some patients with later onset. Lactic acidosis is severe in infants with congenital myopathy or hepatopathy, but lactate can be normal or only mildly elevated in children with infantile myopathy. Serum creatine kinase is markedly elevated in children with myopathy. COX-negative ragged-red ﬁbers are prominent in muscle biopsies from patients with congenital myopathy, but may not be seen in early biopsies

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from children with infantile myopathy. Southern blot analysis shows a profound defect of mtDNA in muscle (less than 10% of normal) in the congenital form. Patients with the infantile myopathy present only partial mtDNA depletion in muscle (about 30% of normal), with some ﬁbers virtually devoid of mtDNA while others look normal. In the hepatic form, liver biopsy shows mitochondrial proliferation in hepatocytes, and biochemical analysis shows very low activities of all respiratory chain complexes containing mtDNA-encoded subunits. Severe mtDNA depletion in liver is found by Southern blot. Pathogenic mutations in TK2 have been identiﬁed in patients with the myopathy or SMA-Uke phenotypes, while mutations in dGK have been associated with hepatocerebral mtDNA depletion (87,90). Some patients with Alpers’ syndrome have had mutations in POLG (89). Thus, abnormalities of nucleoside/nucleotide metabolism are emerging as common causes of intergenomic communication. REFERENCES 1. DiMauro S, Bonilla E. Mitochondrial Encephalomyopathies. In: Rosenberg R, Prusiner S, DiMauro S, Barchi R, eds. The Molecular and Genetic Basis of Neurological Disease. 2nd ed. Boston: Butterworth-Heinemann, 1997:201–235. 2. Anderson S, Bankier AT, Barrel BG, DeBruijin M, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith A, Staden R, Young IG. Sequence and organization of the human mitochondrial genome. Nature 198l; 290:457–465. 3. Wallace DC. Diseases of the mitochondrial DNA. Annu Rev Biochem 1992; 61: 1175–1212. 4. Giles RE, Blanc H, Cann RM, Wallace DC. Maternal inheritance of human mitochondrial DNA. Proc Natl Acad Sci USA 1980; 77:6715–6719. 5. Holt IJ, Harding AE, Morgan Hughes JA. Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 1988; 331:717–719. 6. Lestienne P, Ponsot G. Keams–Sayre syndrome with muscle mitochondrial DNA deletion. Lancet 1988; 1:885. 7. Wallace DC, Singh G, Lott MT, Hodge JA, Schurr TG, Lezza A, Elsas LJ, Nikoskelainen EK. Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 1988; 242:1427–1430. 8. Zeviani M, Moraes CT, DiMauro S, Nakase H, Bonilla E, Schon EA, Rowland LP. Deletions of mitochondrial DNA in Kearns-Sayre syndrome. Neurology 1988; 38:1339–1346. 9. Servidei S. Mitochondrial encephalomyopathies: gene mutations. Neuromusc Disord 2004; 14:107–116. 10. Schon EA, Rizzuto R, Moraes CT, Nakase H, Zeviani M, DiMauro S. A direct repeat is a hotspot for large-scale deletions of human mitochondrial DNA. Science 1989; 244:346–349. 11. Mita S, Rizzuto R, Moraes CT, Shanske S, Arnaudo E, Fabrizi GM, Koga Y, DiMauro S, Schon EA. Recombination via ﬂanking direct repeats is a major

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49. Prezant TR, Agapian JV, Bohlman MC, Bu X, Oztas S, Qiu W-Q, Arnos KS, Cortopassi GA, Jaber L, Rotter JI, Shohat M, Fischel-Ghodsian N. Mitochondrial ribosomal RNA mutation associated with both antibiotic-induced and non-syndromic deafness. Nat Genet 1993; 4:289–294. 50. van den Ouweland JMW, Lemkes HHPJ, Ruitenbeek W, Sandkuij LA, de Vijlder MF, Strnyvenberg PAA, van dKJJP, Maassen JA. Mutation in mitochondrial tRNALeu(UUR) gene in a large pedigree with maternally transmitted type II diabetes mellitus and deafness. Nat Genet 1992; 1:368–371. 51. Shy GM, Gonatas NK, Perez M. Childhood myopathies with abnormal mitochondria. I. Megaconial myopathy-pleoconial myopathy. Brain 1966; 89:133–158. 52. Engel WK, Cunningham CG. Rapid examination of muscle tissue: An improved trichrome stain method for fresh-frozen biopsy sections. Neurology 1963; 13:919–923. 53. Hasegawa H, Matsuoka T, Goto I, Nonaka I. Strongly succinate dehydrogenase-reactive blood vessels in muscles from patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes. Ann Neurol 1991; 29:601–605. 54. Sakuta R, Nonaka I. Vascular involvement in mitochondrial myopathy. Ann Neurol 1989; 25:594–601. 55. Carpenter S, Karpati G, Eisen AA. A morphologic study of muscle in polymyositis: clues to pathogenesis of different types. Amsterdam: Excerpta Medica, 1975:374–379. 56. Fardeau M. Ultrastructural lesions in progressive muscular dystrophies: a critical study of their speciﬁcity. Amsterdam: Excerpta Medica, 1970:98–108. 57. Chinnery PF, DiMauro S, Shanske S, Schon EA, Zeviani M, Mariotti C, Carrara F, Lombes A, Laforet P, Ogier H, Jaksch M, Lochmuller H, Horvath R, Deschauer M, Thorburn DR, Bindoff LA, Poulton J, Taylor RW, Matthews JN, Turnbull DM. Risk of developing a mitochondrial DNA deletion disorder. Lancet 2004; 364:592–596. 58. Kearns TP, Sayre GP. Retinitis pigmentosa, external ophthalmoplegia, and complete heart block. Arch Ophthal 1958; 60:280–289. 59. Rowland LP, Hays AP, DiMauro S, De Vivo DC, Behrens M. Diverse clinical disorders associated with morphological abnormalities of mitochondria. In: Cerri C, Scarlato G, eds. Mitochondrial Pathology in Muscle Diseases. Padua: Piccin Editore, 1983:141–158. 60. Hirano M, DiMauro S. Clinical features of mitochondrial myopathies and encephalomyopathies. In: Lane RJM, ed. Handbook of Muscle Disease. New York: Marcel Dekker Inc. USA, 1996:479–504. 61. Servidei S, Zeviani M, Manfredi G, Ricci E, Silvestri G, Bertini E, Gellera C, Di Mauro S, Di Donato S, Tonali P. Dominantly inherited mitochondrial myopathy with multiple deletions of mitochondrial DNA: clinical, morphologic, and biochemical studies. Neurology 1991; 41:1053–1059. 62. Suomalainen A, Kaukonen J. Diseases caused by nuclear genes affecting mtDNA stability. Am J Med Genet 2001; 106:53–61. 63. Luoma P, Melberg A, Rinne JO, Kaukonen JA, Nupponen NN, Chalmers RM, Oldfors A, Rautakorpi I, Peltonen L, Majamaa K, Somer H, Suomalainen A. Parkinsonism, premature menopause, and mitochondrial DNA polymerase

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14 Friedreich Ataxia Martin B. Delatycki, Michael C. Fahey, and Louise Corben Bruce Lefroy Centre for Genetic Health Research, Murdoch Childrens Research Institute, Royal Children’s Hospital, Victoria, Australia

Andrew Churchyard Monash Institute for Neurological Disease, Southern Health, Monash Medical Centre, Victoria, Australia

INTRODUCTION The disease now known as Friedreich ataxia (FRDA) was originally described in ﬁve publications by Nikolaus Friedreich over the period 1863–1877 (1–5). Since then a great deal has been learned about its pathogenesis, which has led to signiﬁcant conceptual therapeutic advances although the translation to treatment that alters the natural history is yet to be made. FRDA is the most common inherited ataxia (6). Prior to the availability of molecular diagnosis, FRDA was estimated to affect about 1:50,000 Caucasians with an estimated carrier prevalence of 1:110 (7). More recent studies based on molecular data suggest a higher incidence with a carrier rate of 1:60–1:90 (8,9). FRDA is seen in India, North Africa, and the Middle East but is exceedingly rare among Asians and in those of African descent (10).

CLINICAL FEATURES Diagnostic criteria for FRDA were proposed by Geoffrey (11) and revised by Harding (12). Since the molecular basis of FRDA has been deﬁned, these

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Table 1 Comparison of Symptom Frequency in Four Series of FRDA Patients. The ﬁgures are the percentage of individuals with FRDA in that study who have that clinical feature Harding (12) (N ¼ 115) Gait ataxia Limb ataxia Lower limb muscle weakness Diminished vibration sense Pes cavus Dysarthria Extensor plantar response Cardiomyopathy Diabetes Scoliosis Reﬂexes LL Sphincter disturbance Decreased visual acuity Hearing loss

Durr et al. (13) Delatycki et al. Lamont et al. (N ¼ 140) (18) (N ¼ 51) (15) (N ¼ 56)

– 99 88

100 99 67

100 100 –

100 100 53

73

78

88

87

55 97 89

55 91 79

74 95 74

– 91 96

66 10 79 0.9 –

63 32 60 12 23

65 8 78 2 41

– – – 13 –

18

13

–

–

8

13

–

–

have proven to be highly speciﬁc but lacking in sensitivity (13,14). The frequency in percentages of common symptoms is shown in Table 1. The mean age of symptom onset was 10.5 years in Harding’s series of 115 patients (12), and was 15.5 years in a later series of 140 patients with molecularly proven disease (13). Ataxia is the presenting feature in the vast majority of cases (13–19). The natural history of FRDA is characterized by slow progression of neurological symptoms; however, detailed analysis of this has not been undertaken, and thus it remains an important area of future research (20). Lifespan is decreased with an average age of death of 37.5 years (based on Harding’s series published in 1981) (12), but the mean survival from disease onset in more recent studies has been reported as 36 years (21). The modest discrepancy in these numbers may reﬂect both improved therapy of the medical complications of FRDA in more recent times and the recognition of less severely affected individuals. The most common cause of death is cardiomyopathy, either by production of arrhythmias or by congestive heart failure. Neurological Features Ataxia results from a combination of spinocerebellar dysfunction and impaired proprioception. Central nervous system dysfunction results in

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extensor plantar reﬂexes, weakness, and corticospinal tract dysfunction (22). The ankle and knee jerks are absent in typical cases due to an axonal neuropathy (12,13,23) associated with reduced or absent sensory nerve action potentials (15,24). Optic atrophy is found in 30%, with or without decreased visual acuity (12). Eye signs include ﬁxation instability with square wave jerks and ﬂutter. Saccades are inaccurate with dysmetria and slowed pursuit (23,25,26). Hearing loss occurs in 13% of patients (13). A small number of studies have examined general cognition as well as visual and auditory reaction times in people with FRDA (27–29). While there is little impairment in general cognition, there is a signiﬁcant slowing in reaction and movement time when compared with controls. More complex cognitive function such as verbal span, letter ﬂuency, acquisition and consolidation of verbal information, and complex visuo-perceptual and visuo-constructive abilities may be affected based on a single study (29). Neuroimaging is usually normal early in disease. In advanced disease there may be atrophy of the cerebral hemispheres, cerebellum (mainly the dentate nucleus), brainstem, and cervical cord (30,31). These ﬁndings correlate with the clinical severity of the disease. Cardiac Features Echocardiography demonstrates concentric ventricular hypertrophy in 62–68% of individuals with FRDA (32,33) and diastolic dysfunction is commonly seen (34). The electrocardiogram is abnormal in the majority with T wave inversion, left axis deviation, and repolarization abnormalities (32,35,36). Dyspnea, palpitations, hypertension, and ankle edema are observed in 10–30% of patients (12,35). Diabetes Mellitus The incidence of diabetes mellitus is 8–30% and an additional proportion have impaired glucose tolerance (12–14,18,37). The mechanisms are poorly understood. Non-autoimmune loss of islet cells (38), abnormal regulation of insulin receptors (39), and lower insulin release (37) have been implicated. Other Features Scoliosis is present in about 66% of individuals with FRDA when assessed clinically (40) but is present in essentially all when assessed radiographically (41). Pes cavus is seen in about two-thirds of those with FRDA and equinovarus deformity becomes more common with disease progression (42). Differential Diagnoses The differential diagnoses for FRDA are listed in Table 2. Disorders that lead to derangement of vitamin E absorption and transport can present with features indistinguishable from FRDA. Head titubation, lower

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Table 2 Differential Diagnosis of FRDA Deﬁciency of vitamin E Malabsorption a Tocopherol transporter protein deﬁciency Abetalipoproteinaemia Early onset cerebellar ataxia with retained reﬂexes (EOCA) Hexosaminidase A deﬁciency L-2-Hydroxyglutaric acidemia Adrenoleukodystrophy

incidence of associated motor neuropathy, slower disease course, and retinitis pigmentosa are more common in low vitamin E states than FRDA (43). Ataxia with vitamin E deﬁciency (AVED) is due to mutations in the a-tocopherol transfer protein gene on chromosome 8 (44). b-hexosaminidase A deﬁciency that underlies Tay Sachs disease can also result in an FRDA phenocopy (45). PATHOLOGY The main sites of pathology in FRDA are the dorsal root ganglia, posterior columns of the spinal cord, the corticospinal tracts, and the heart. Macroscopically the spinal cord is small with the posterior and lateral columns particularly affected (6). Demyelination (presumptively secondary to axonal degeneration) occurs in the posterior columns and, in particular, the large ﬁbers arising in the dorsal root ganglia. There is striking involvement of Clarke’s columns. The corticospinal tract involvement explains the ﬁndings of up-going plantar responses in almost all patients and spasticity in some patients (46). There is loss of Purkinje cells in the cerebellar cortex and the deep cerebellar nuclei: in particular the dentate nuclei, from which efferent cerebellar ﬁbers originate, are affected by marked neuronal loss (47–49). Neuronal loss occurs in the gracile and cuneate nuclei of the brainstem where the dorsal column tracts terminate (49). A distal axonopathy affecting larger myelinated nerve ﬁbers is also present (50) and is due to a dying back process from the periphery (51). The most common cardiac lesion is hypertrophic cardiomyopathy, but hypokinetic cardiomyopathy has also been reported. This pattern usually occurs following cardiac hypertrophy (52). Iron deposits in the myocardium have been reported (53,54). Cardiac pathology consists mainly of cellular hypertrophy, diffuse ﬁbrosis, and focal myocardial necrosis (55,56). MOLECULAR GENETICS The gene mutated in FRDA was mapped to chromosome 9 in 1988 (57) and cloned in 1996 (58). Initially called X25 and later changed to FRDA, the

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gene contains seven exons (1–5a,5b,6). The most common transcript arises from exons 1–5a, and yields a 210 amino acid protein, frataxin. In 98% of mutant alleles there is a GAA triplet repeat expansion in the ﬁrst intron of FRDA (58); the other 2% of mutant alleles are point mutations (59). Normally there are 33 or fewer GAA triplets, while in pathological expansions there are from 67 to more than 1000 triplets (60). FRDA is expressed in heart, liver, skeletal muscle, and pancreas (61). FRDA expression studies in the central nervous system have revealed high levels of expression in the spinal cord, less in the cerebellum, and very little in the cerebral cortex. mRNA levels are very low or undetectable in patients compared to controls. Reduced mRNA levels result from the GAA repeat interfering with transcription elongation (62). This appears to occur from the formation of triplexes (resulting in sticky DNA) by DNA carrying expanded GAA repeats (63,64). The level of mRNA transcription is inversely related to the size of the GAA repeat (65,66). The GAA expansion of FRDA may cause gene silencing at least in part due to position effect variegation, and the degree of gene silencing may modify disease severity (67). Intergenerational Instability As in other trinucleotide repeat disorders, the GAA repeat that underlies FRDA is unstable in its transmission from parent to offspring (68). Maternal transmission may result in a larger or smaller allele in offspring. In contrast, the GAA repeat size almost always decreases when transmitted by a male (16,69–71). The size of the triplet repeat inﬂuences the direction of instability with smaller alleles being more prone to increase in size and larger ones to decrease. Studies on sperm from FRDA patients indicate that instability occurs pre- and post-zygotically (70,71). A small percentage of alleles are beyond the size found in the normal range but are smaller than alleles considered pathogenic for FRDA. These have been called premutation alleles (8,9,60,70). Such alleles are prone to very large expansions in one generation; however GAG interruptions appear to prevent expansions (9). Most expansions of this type do not cause FRDA because the allele inherited from the other parent is likely to be normal. Therefore, the incidence of such events is unknown. In vivo studies of replication fork progression of GAA repeats cloned into Saccharomyces cerevisiae plasmids demonstrate that premutation and disease-size repeats stalled the replication fork progression, while normalsize repeats do not affect replication (72). The stalling of replication fork progression predisposes to repeat expansion; this is hypothesized to be more likely with longer repeats.

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Genotype–Phenotype Correlation A number of studies have examined the correlation between the size of the GAA repeats and the presence and timing of various features of the disease (13–19). About 50% of the variation in the age of onset is accounted for by the size of the smaller allele. The GAA repeat length in each allele is important in predicting the age of onset and some features of FRDA. The smaller of the two alleles is more important in this regard. Features whose presence has consistently been shown to be associated with GAA repeat length include cardiomyopathy, diabetes mellitus, and scoliosis (13,14,17,18). As in other trinucleotide repeat disorders, repeat sizes cannot be used to accurately predict prognosis in an individual without a substantial degree of uncertainty (68). Disease severity is also inﬂuenced by the mitochondrial haplotype with the U mitochondrial haplotype being associated with an average delay of ﬁve years in disease onset and a reduced rate of cardiomyopathy when compared to other haplotypes (73). Most (but not all) cases of typical FRDA are linked to the FRDA locus (74–77). A second locus, FRDA2, has been found on chromosome 9p but no speciﬁc gene or disease-associated mutation has been identiﬁed (78,79). Late onset Friedreich ataxia (LOFA), deﬁned as onset of FRDA after 25 years of age, and FRDA with retained reﬂexes (FARR), are linked to the same locus as typical FRDA (80–83). The Acadian form of FRDA (FRDA-Acad) is characterized by slower progression than classical FRDA and is not associated with cardiomyopathy and diabetes mellitus. Spastic ataxia is also seen in the Acadian population (SPA-Acad). Both FRDAAcad and SPA-Acad are linked to the same chromosome 9 locus as classical FRDA (57,84). These linkage ﬁndings were all conﬁrmed by mutation analysis of FRDA (13–15,17).

PATHOGENESIS Although the precise function of frataxin is still a subject of intense study and debate, much has been learned about the pathogenesis of FRDA through the discovery of the human gene in 1996, the subsequent deletion of the yeast frataxin homolog (YFH1) and the development of mouse models. Soon after FRDA was cloned, it was shown that frataxin is localized to mitochondria. The N-terminal region of frataxin was predicted by detailed sequence comparison with known proteins, to contain a mitochondrial targeting sequence (85,86). Subsequently, using frataxin fused to a reporter protein, human frataxin was shown to be a nuclear encoded mitochondrial protein (86,87). Knockout (KO) of the YFH1 gene (yielding Dyfh1 cells) in S. cerevisiae causes an accumulation of iron in the mitochondria at the expense of cytosolic

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iron and increased sensitivity to oxidative stress (86–90). Dyfh1 cells show a deﬁciency in activity of complex IV of the respiratory chain and, to a lesser extent, reduction in the activity of complex V. Mitochondrial proteins containing iron–sulfur (Fe/S) clusters such as aconitase are also deﬁcient (88). Frataxin in yeast may have an iron storage function. Park et al. have suggested that frataxin catalyzes oxidation of Fe2þ to Fe3þ and also chaperones Fe2þ to proteins including ferrocheletase and ISU-type proteins to support heme and Fe/S cluster biosynthesis (91). They suggest that frataxin polymerizes in the presence of Fe2þ and O2 and the frataxin polymer then stores Fe3þ. Several further lines of evidence implicate Yfh1p involvement in the biosynthesis of Fe/S clusters in yeast. When the mitochondrial iron concentration in Dyfh1 cells is kept low by the addition of the iron chelator bathophenanthroline sulfonate (BPS), the activity of aconitase, which contains extremely labile Fe/S clusters, is reduced by more than 50% compared to a wild-type strain (92). This suggests that incorporation of the Fe/S clusters into aconitase may be impaired in a manner that is unrelated to iron toxicity. The depletion of Yfh1p by regulated gene expression leads to a primary reduction in activities of Fe/S cluster containing proteins in mitochondria with mitochondrial iron accumulation being a secondary event (93). The observation that mitochondrial iron accumulation is likely to be a secondary event resulting from the reduced activity of the Fe/S cluster containing proteins is supported by the observation that defects in the assembly of cellular Fe/S proteins in yeast are generally associated with the accumulation of mitochondrial iron (94,95). Bulteau et al. studied the etiology of the reduced activity of Fe/S cluster containing proteins in yeast with reduced frataxin production (96). They examined mitochondrial aconitase activity in this yeast and showed that frataxin protects the Fe/S cluster from disassembly and promotes enzyme reactivation. In mice, KO of the FRDA gene causes early embryonic lethality (97). This indicates an important role for frataxin in embryonic development and suggests the milder phenotype displayed by people with FRDA is due to residual frataxin expression (61). Through a conditional gene-targeting approach, mouse models have been created in which FRDA is selectively inactivated in neuronal and cardiac tissues (98). These ﬁrst mammalian models have progressive pathophysiological and biochemical features of the human disease; cardiomyopathy, a sensory nerve defect and Fe/S enzyme deﬁciency. These features precede intramitochondrial iron deposits, indicating that in mice, iron accumulation is a secondary phenomenon. A speciﬁc inducible neurological KO mouse has been developed which has progressive mixed cerebellar and sensory ataxia (99). A moderate decrease in succinate dehydrogenase, a Fe/S cluster containing enzyme, has been demonstrated while iron accumulation was shown to be a modest and late phenomenon.

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Frataxin appears to have the same function in humans as in yeast and mice (100). For example, human frataxin can complement the defect seen in Dyfh1 cells (101) and the insertion of the human frataxin gene in the mouse KO rescues these mice (102,103). In human FRDA ﬁbroblasts, the level of mitochondrial iron is signiﬁcantly higher than in control ﬁbroblasts (104). Furthermore, a generalized decrease in the activity of Fe/S cluster complex containing proteins (both mitochondrial and cytosolic aconitase and respiratory chain complexes I– III) has also been observed in heart tissue from two FRDA patients (105). Microarray studies on three human cell lines looking for frataxin-dependent transcripts have found that the only functional hypothesis supported by the identiﬁcation of frataxin-dependent genes is that frataxin is involved in Fe/S cluster synthesis (106). That antioxidant enzyme levels are signiﬁcantly different in blood in FRDA patients compared to controls is further evidence for the role of oxidative stress in the pathogenesis of FRDA (107). Additionally, there is evidence that oxidative stress induces actin glutathionylation and subsequent impairment of cytoskeletal function in FRDA ﬁbroblasts (108). In summary, it appears that the primary role of frataxin is related to Fe/S cluster biosynthesis resulting in a reduction of Fe/S cluster containing proteins. This leads to reduced cellular energy production and can result in increased production of free radicals (109). Mitochondrial iron accumulation appears to be a secondary event. It is likely that this contributes to the production of reactive oxygen species leaking from the respiratory chain, leading to the formation of free radicals through Fenton chemistry. These radicals are expected to further reduce Fe/S protein levels as these proteins are exquisitely sensitive to oxidative stress (110,111). These interactions, in addition to the disruption of energy production, ultimately lead to neuronal death (87,88). MANAGEMENT OF PATIENTS WITH FRDA There is little objective literature regarding management of FRDA. Intervention has been based on evidence gained from other neurodegenerative diseases or single case reports (112). Based on our experience in a dedicated FRDA clinic we have produced a protocol for clinical management of FRDA (Table 3). With ongoing natural history research, evidence-based protocols can be derived. There is sound evidence that people with chronic progressive neurological disease beneﬁt from long-term specialist multidisciplinary care (113–116). This approach incorporates a team of specialist clinicians including medical staff, allied health, and members of the patient population. A multidisciplinary model of care for people with FRDA ensures proactive, rather than reactive disease management. The signiﬁcant symptoms of FRDA impacting on functional capacity are sensory ataxia, muscle weakness, spasticity, and associated bony changes

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System

Assessment

Frequency

Possible ﬁndings

Intervention Pharmacological management of spasticity and bladder function. Splinting for upper and lower limbs. Stretching program. Prescription of adaptive equipment. Intervention for dysphagia including video-ﬂuoroscopy if indicated Pharmacological management of arrhythmias and cardiac failure. Pacemaker Pharmacological treatment of diabetes if present Visual aid prescription

Neurological

Neurological assessment. Physical therapy assessment. Occupational therapy assessment. Speech pathology assessment

Annual or symptomatic

Progressive ataxia, spasticity, impaired bladder function, sensory changes, dysarthria, dysphagia, reduction in capacity to participate in daily activities

Cardiac

Electrocardiogram and echocardiogram

Annual or symptomatic

Endocrine

Fasting blood sugar and hemoglobin A1C Ophthalmological examination

Annual or symptomatic

T wave inversion in inferolateral chest leads, concentric ventricular hypertrophy, and diastolic dysfunction Diabetes mellitus

Ocular

Three yearly or symptomatic

(Continued) Copyright 2006 by Taylor & Francis Group, LLC

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Decreased visual acuity, optic atrophy, oculomotor abnormalities, interference in daily activities

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Table 3 Clinical Management Protocol for FRDA

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Table 3

Clinical Management Protocol for FRDA (Continued )

System

Assessment

Frequency

Possible ﬁndings

Intervention Prescription of appropriate mobility, seating and adaptive equipment. Upper and lower limb splinting. Foot surgery. Botulinum toxin injection. Community based rehabilitation and maintenance program Hearing aid prescription

Musculoskeletal

Musculoskeletal examination. Physical therapy assessment. Occupational therapy assessment

Annual or symptomatic

Scoliosis, pes cavus, ankle equinovarus deformity. Reduced passive and active range of movement. Reduction in capacity to participate in daily activities

Auditory

Hearing assessment

Hearing impairment

Psychosocial

Psychosocial assessment

Three yearly or symptomatic Annual or symptomatic

Counseling (individual and group)

Delatycki et al.

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Response to initial diagnosis and disease process (both for patient and family). Grief regarding loss of function, roles, etc.

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such as scoliosis and foot deformity. From a functional perspective, these symptoms interfere with the ability to mobilize, transfer, and attend to personal daily tasks. The capacity to stand and transfer independently is the most signiﬁcant contributor to ongoing maintenance of independence. Many patients with FRDA experience progressive equinovarus deformity. This interferes with the capacity to transfer and therefore increases caregiver burden. Prevention of this deformity by stretching and exercise programs is imperative. Correction of this deformity by splinting and botulinum toxin followed by an ongoing rehabilitation program can result in signiﬁcantly improved functional performance (42). In cases where deformity is not reducible, surgery followed by an intensive rehabilitation program should be considered. Spasticity can have a signiﬁcant negative effect on the capacity to participate in daily activities and therefore quality of life (117). People with FRDA require proactive management of spasticity in both upper and lower limbs. Preventive interventions such as use of a standing frame, stretching, and splinting (for both upper and lower limbs) are recommended (117). Medications such as baclofen are often necessary and botulinum toxin injection can also be of beneﬁt (42). Scoliosis, if untreated, may be associated with pelvic obliquity, pain, restricted cardio-respiratory function, and uneven weight distribution on the seating surface (118). Appropriate wheelchair and seating prescription is essential to minimize progression of scoliosis. Surgery is recommended for scolioses greater than 60 and should be considered for curves between 40 and 60 (41). Swallowing difﬁculties are common in FRDA and may predispose to aspiration pneumonia. Dysarthria can also be severe and impact on quality of life. Proactive speech pathology assessment and management are essential to manage these issues. Cardiac complications should be monitored routinely and managed as these represent potentially treatable causes of morbidity and mortality. Dysrhythmias should be managed with antiarrhythmic agents and pacemaker insertion as indicated. While some individuals with FRDA have clinically signiﬁcant and progressive cardiomyopathy, many other individuals will have subclinical cardiac ﬁndings that remain stable over time; however annual screening is still recommended. As with any chronic neurodegenerative disease, signiﬁcant psychosocial morbidity is seen in individuals and families affected by FRDA. A proactive approach to these issues should be adopted from the time of diagnosis onwards. The presence of depression should be identiﬁed and appropriately managed. At all times, the autonomy of the individual with FRDA should be respected in management issues. Genetic counseling should be provided to families (40). Carrier, prenatal and preimplantation testing are possible utilizing molecular genetic testing.

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POTENTIAL PHARMACOLOGICAL THERAPIES FOR FRDA The rapid increase in knowledge regarding the pathogenesis of FRDA has led to a number of pharmacological therapies being considered, and some have been tried in people with FRDA. Antioxidants Antioxidants detoxify free radicals and therefore are of potential beneﬁt in FRDA because of the role of oxidative damage in FRDA (119). Idebenone is a short chain analogue of coenzyme Q10 (120–122). A preliminary study and open label trials of idebenone (5–10 mg/kg/day) showed a reduction of cardiac hypertrophy in FRDA patients; no serious side effects were reported (123–125). Additionally, idebenone delays the demise of the cardiac conditional FRDA KO mouse model by about 25% (126). A year long randomized placebo-controlled trial found idebenone treatment was associated with moderate, though persisting, effects on cardiac hypertrophy; however, no beneﬁt on neurological status was demonstrated (127). Besides a reduction in cardiac hypertrophy, Buyse et al. showed an improvement of regional myocardial function using quantitative cardiac strain and strain rate imaging (128). An editorial that accompanied these two publications recommended that idebenone should not yet be routine therapy for FRDA because the clinical signiﬁcance of the effects of idebenone is unclear (129). Coenzyme Q10 (400 mg/day) plus vitamin E (2100 IU/day) has been evaluated, but both neurological and echocardiographic evaluations did not show any beneﬁt after six months of treatment in an open-label trial (130).31P magnetic resonance spectroscopy demonstrated partial reversal of the surrogate biochemical markers cardiac phosphocreatine and skeletal muscle adenosine triphosphate after three months of treatment (130). Mitoquinone (MitoQ) is an antioxidant selectively targeted to mitochondria, resulting in a 100- to 500-fold accumulation of the construct in mitochondria (131). The ubiquinone derivative is embedded in the inner membrane of the mitochondrion where it is reduced by the respiratory chain to ubiquinol, which is an effective antioxidant that prevents lipid peroxidation and regenerates vitamin E (132–134). MitoQ is 800 times more potent than idebenone in protecting FRDA ﬁbroblasts from death due to endogenous oxidative stress generated by glutathione synthesis inhibition (135). This drug has not yet been tested in people with FRDA. Iron Chelators Since excess mitochondrial iron is likely to play a role in the pathology of FRDA, iron chelators have potential in the treatment of FRDA. Desferioxamine (DFO), a parenteral iron chelator used in the treatment of iron overload conditions (such as transfusion dependent hemoglobinopathies), has

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been tried in FRDA (136). The results have not been published, however, DFO is not likely to be beneﬁcial as it cannot efﬁciently mobilize iron from iron loaded mitochondria (137,138). Deferiprone (1,2-dimethyl-3-hydroxypyridin-4-one; L1) is an orally active iron chelator that is more effective than DFO in the removal of myocardial iron in patients with b-thalassemia (139). Even though no toxic effects from L1 were seen in healthy volunteers (140), the use of L1 in FRDA must be studied very carefully, since signiﬁcant toxicity is possible. A group of iron chelators, 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) analogs, have been speciﬁcally designed to target mitochondrial iron pools (141). Several PCIH analogs show very high activity in mobilizing 59Fe from 59Fe-loaded mitochondria in reticulocytes. These agents are therefore being studied as potential therapies for FRDA (141). Glutathione Peroxidase Mimetics By blocking the rate-limiting enzyme in glutathione (GSH) synthesis, g-glutamyl cysteine synthase, with L-buthionine (S,R)-sulfoximine (BSO), cells become partly depleted of GSH and more susceptible to endogenous oxidative stress. FRDA ﬁbroblasts are more sensitive to BSO treatment than control ﬁbroblasts by a viability assay (142). FRDA ﬁbroblasts treated with glutathione peroxidase (GPX) mimetics (ebselen, monoselenide, diselenide) showed increased viability. Further studies are needed to address the toxicity of GPX mimetics in humans before human FRDA trials can be considered. Increase Expression of Frataxin FRDA is due to reduction of frataxin expression to a few percent of normal levels, with a correlation between disease severity and residual levels of frataxin expression (61). A mouse model with 25–30% of normal frataxin levels did not show any phenotypic abnormalities (143). Therefore, it is estimated that a 5–10 fold increase in frataxin expression could be therapeutic, while lower levels of induction could still reduce disease severity and progression. A low level of enhancement of FRDA gene expression by hemin and butyric acid was initially demonstrated in BHK21 stable cell lines (144). Subsequently, resistance to cisplatin in a cancer cell line was associated with an increase in frataxin expression (145), and 3-nitropropionic acid was found to increase frataxin expression in human lymphoblasts and in transgenic rat PC12 cells (146). Gene and Cell Therapy Supplementing the mutant FRDA gene with a normal copy of the gene by gene therapy or by the transplantation of stem cells with normal frataxin

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production may be the most direct ways of overcoming the frataxin deﬁciency. However, there are limitations in applying such techniques safely and with high efﬁciency to many hard-to-reach tissues. CONCLUSION FRDA was initially characterized as a childhood onset disorder and now is recognized to have a variable phenotype that includes onset in middle adulthood. The severity of neurological features and involvement of other organ systems varies based on genetic mutation as well as other factors. During the last decade there has been great progress toward understanding the pathophysiology of FRDA and development of therapeutic targets that are now entering clinical trials.

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15 Autosomal Dominant Ataxias Susan L. Perlman Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A.

INTRODUCTION The autosomal dominant (AD) ataxias are a group of disorders affecting the cerebellum and its afferent and efferent pathways. Their pattern of inheritance can be explained on the basis of mutation in a single gene. The trait controlled by this gene is deemed dominant if it manifests in the heterozygote – an AD ataxia is caused by a mutant allele that produces the ataxic phenotype in the affected individual, despite the presence of a normal allele on the second homologous chromosome. This can occur in a number of different ways. Most gene mutations result in an inactive gene product. Recessive disorders require both alleles to be defective, as one functioning normal allele will usually produce enough gene product to maintain normal function. If the function of the remaining normal allele is not sufﬁcient to maintain normal cellular function, the resulting haploinsufﬁciency will cause dominant disease by ‘‘loss of function.’’ This mechanism may manifest later in life, when the activity of the normal gene may be lost in certain cell types due to an acquired problem, but most often occurs when the gene involved controls a critical rate-limiting step in a metabolic pathway or a regulatory or structural process that is sensitive to gene dosage effects. The mutant protein might also interfere with the action of the corresponding normal protein (dominant-negative effect), producing a similar loss of function. Most identiﬁed mutations to-date in the dominant ataxias, however, cause disease primarily by a toxic ‘‘gain of function.’’ The most common

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example is the CAG/CTG triplet repeat expansion in a coding region of the gene, which produces a mutant protein with an expanded polyglutamine sequence that triggers oxidative stress or premature apoptosis; interferes with normal cellular protein interaction, processing, or degradation; or affects other genes or gene products. There is evidence that suggests that the protein region containing the polyglutamine repeat is cleaved by caspases to produce fragments that enter the neuronal nucleus. These fragments aggregate in the cytoplasm and the nucleus, where chaperone proteins and proteosomal complexes act to refold or dispose of the fragments, to reduce aggregate formation. Aggregates that do form sequester other proteins, protein complexes (including proteosomes), and transcription factors to form inclusion bodies. Neuronal dysfunction ensues (possibly through activation of apoptotic cascades/glutamate receptor mechanisms, free radical production/ mitochondrial dysfunction, and axon transport defects), and cell death results. Recent studies in SCA2 have shown that the presence of cytoplasmic inclusions, in the absence of nuclear inclusions, is sufﬁcient to cause neuronal death, possibly by disruption of the Golgi complex (1). These pathologic molecular mechanisms are seen as potential sites for therapeutic intervention and have been recently reviewed by several authors (2–11). The CAG/CTG triplet repeat expansion in the gene may be ‘‘unstable,’’ with a risk of expanding further during generational transfer (meiosis). Longer repeat lengths are associated with earlier age of onset and more severe phenotype, and triplet repeat disorders can show ‘‘anticipation’’ of upto 10–20 years earlier onset in succeeding generations. Similarly, an asymptomatic individual with a high normal or borderline expansion may pass on a fully expanded/disease-causing allele, a pattern of inheritance now known to underlie some forms of ‘‘new dominant’’ mutation (12). Advances in molecular genetic technology have focused on the CAG/ CTG expansion process and utilized the presence of larger than normal CAG/CTG sequences as possible markers for candidate genes for neurologic disease (13,14). The use of the repeat expansion detection (RED) methodology and other CAG/CTG dependent techniques may have preferentially identiﬁed the SCAs now attributable to triplet repeat dysfunction [12 of 27 proposed spinocerebellar ataxias (SCA) have had causative genes and mutations identiﬁed, six of which are CAG/CTG expansions]. Newer technologies may well be required to fully uncover all the molecular pathologies involved in causing autosomal dominant ataxia. The same mutation in a known genotype may result in a phenotype (age of onset, rate of disease progression, disease severity, and neurologic features) that varies from individual to individual. In the polyglutamine disorders, age of onset inversely correlates with CAG repeat length, and the size of the triplet repeat may inﬂuence other aspects of the phenotype as well (15,16). Phenotypic variability may also be due to a number of other genetic and non-genetic factors, including tissue mosaicism, other ‘‘modifying’’

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genetic loci/promoters/polymorphisms, and lifestyle or environmental factors [e.g., the origin and effects of anti-gliadin antibodies found in patients with genetic ataxias (17–19)]. While some progress has been made in identifying the role of mosaicism in certain inherited ataxias [SCA1 (20), SCA2 (21), SCA3 (22,23)], research in the pursuit of modifying genes has only just begun [SCA3 (15,24)]. Epidemiologic factors are almost completely unexplored in these disorders. There is also no agreement as to which neurologic features of an SCA are directly due to the effects of the mutant gene and which might be related to other factors inﬂuencing disease progression, as the molecular basis of brain regional susceptibility within and between the SCAs is not fully understood (25–28). Transgenic animal models (Caenorhabditis elegans, Drosophila, Danio rerio/zebraﬁsh, and mice) have been generated to further research into these molecular genetic mechanisms. There are now mouse models for SCA1 (29,30), 2 (31), 3 (32), 7 (33), 8 [Drosophila (34)], and DRPLA (35,36), which are serving to deﬁne the subcellular localization of pathogenesis and to act as templates for therapeutic intervention [chaperone overexpression (37,38), histone deacetylase inhibition (39), small inhibitory RNA action (10)] (40). An excellent introduction to medical genetics and methods of DNA testing can be found in a recent review by Pulst (41). The dominantly inherited ataxic disorders include the typical SCA, which now number 27 (including SCAs 1–26 and SCA with tremor and dyskinesia, due to mutation in the gene for ﬁbroblast growth factor 14); the episodic ataxias (EA 1–5); and the atypical spinocerebellar ataxias, which may have prominent features other than ataxia. Of this latter group, only Dentatorubral– Pallidoluysian Atrophy/DRPLA) and Gerstmann–Straussler–Schienker/GSS disease will be mentioned here. There are excellent recent clinical reviews of the episodic ataxias (42–49). DRPLA has also recently been reviewed (50–52). Detailed overviews of the inherited prion diseases are continuing to evolve (53–58). Continuing updates can be searched at www.ncbi.nlm.nih.gov (PubMed, OMIM) and www.neuro.wustl.edu/neuromuscular/ataxia. Pathogenetic classiﬁcation would group SCA1–3, 6, 7, 17, and DRPLA as polyglutamine disorders; SCA14, SCA due to FGF14, and GSS as resulting from point mutations; SCA6 and EA1 and 2 as channelopathies; and SCA8, 10, and 12 as repeat expansions outside the coding region that result in decreased gene expression. The molecular bases of SCAs 4, 5, 11, 13, 15, 16, and 18–25 are still unknown. THE TYPICAL DOMINANT ATAXIAS These SCAs have an incidence of 1–5 per 100,000 (SCA3 being the most common, followed by SCAs 1, 2, 6, 7, and 8; the others being quite rare) and an average age of onset in the third decade. They are difﬁcult to distinguish on clinical grounds, but all share features of gait ataxia and slurred

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speech, on the basis of loss of cerebellar and brainstem neurons. The presence of certain associated features might be helpful in choosing the most likely diagnosis (Table 1), and neuroimaging may aid in that choice (59,60), but only genetic testing will be deﬁnitive. Gene testing is currently commercially available for only 10 of the SCAs, but will identify a mutant gene in about 50% of familial cases. Individuals with no family history, but an otherwise consistent clinical presentation, may be found to have a genetic cause 1–5% of the time, most commonly SCA6, fragile X-tremor ataxia syndrome (FXTAS), or late-onset Friedreich’s ataxia (an autosomal recessive disease) (61). SCA gene mutations may also be found in apparently sporadic cases when the family history is unclear or a parent is found to have an unstable triplet repeat expansion in the borderline range. A careful work-up should be done in all apparently sporadic cases, to also rule out acquired causes of ataxia. Some testing should be done even in individuals with a conﬁrmed genetic cause, as the presence of a secondary factor (nutritional deﬁciency, thyroid dysfunction) can contribute to the phenotype. The diagnostic evaluation of patients with ataxia has been extensively reviewed in a recent publication (62). On-line resources to ﬁnd commercial laboratories performing SCA testing can be found at www.geneclinics.org. These tests may cost $250–500 apiece—and the entire battery of available tests could run over $3000, posing a ﬁnancial barrier to exact diagnosis in many cases. Harding’s seminal phenotypic classiﬁcation of the AD cerebellar ataxias (ADCAI—Cerebellar syndrome plus pyramidal signs, supranuclear ophthalmoplegia, extrapyramidal signs, and dementia; ADCAII—Cerebellar syndrome plus pigmentary maculopathy; and ADCAIII—‘‘Pure’’ cerebellar syndrome, mild pyramidal signs) (63) may provide some guidance in prioritizing choices for gene testing (Table 2). It is increasingly recognized that access to genetic testing for the inherited ataxias can signiﬁcantly improve patient management and family counseling (64). The molecular genetic features of the SCAs are summarized in Table 3. SCA1 Clinical Features Onset has been reported as young as four years of age and into the eighth decade, but is most common in the fourth decade. The initial course is one of pancerebellar dysfunction (gait ataxia, limb ataxia, dysarthria, and gaze-evoked nystagmus), but progressive pontine involvement leads to slowing of saccades and ophthalmoparesis. Pyramidal changes underlie the commonly seen spasticity, hyperreﬂexia, and extensor plantar responses. Mixed axonal peripheral neuropathies are seen in about half the patients, usually in those with higher CAG repeats. Amyotrophy is also reported. Frontal subcortical executive dysfunction is also found in about half. With disease progression, bulbar dysfunction with dysphagia, stridor, and vocal

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Table 1 Associated Features in the Differential Diagnosis of the Spinocerebellar Ataxias Ataxic disorder SCA1 SCA2 SCA3

SCA4 SCA5 SCA6 SCA7 SCA8 SCA9 (reserved) SCA10 SCA11 SCA12 SCA13 SCA14 SCA15 SCA16 SCA17 SCA18 SCA19 SCA20 SCA21 SCA22 SCA23 SCA24 (reserved) SCA25 SCA26 SCA due to FGF14 EA-1

Typical associated clinical features beyond ataxia and dysarthria Hyperreﬂexia/spasticity, cerebellar tremor, dysphagia, optic atrophy Slow saccades, hyporeﬂexia, cerebellar tremor, Parkinsonism, dementia Nystagmus, spasticity (onset <35 yr), neuropathy (onset >45 yr), basal ganglia features, lid retraction, facial fasciculations Sensory axonal neuropathy, pyramidal signs Bulbar signs, otherwise ‘‘pure cerebellar’’ Nystagmus (often downbeat), otherwise ‘‘pure cerebellar,’’ onset >50 yr Macular pigmentary retinopathy, slow saccades, pyramidal signs Nystagmus, cerebellar tremor Nystagmus, seizures Nystagmus, hyperreﬂexia Nystagmus, arm tremor, hyperreﬂexia Nystagmus, hyperreﬂexia, mental & motor retardation, childhood onset Head tremor or myoclonus Nystagmus, hyperreﬂexia Nystagmus, head and hand tremor Dementia, psychosis, extrapyramidal features, hyperreﬂexia, seizures Nystagmus, Babinski sign, sensory-motor axonal neuropathy Cognitive impairment, nystagmus, tremor, myoclonus Palatal tremor, dysphonia Cognitive impairment, extrapyramidal features, hyporeﬂexia Nystagmus, hyporeﬂexia Slow saccades, pyramidal signs, sensory neuropathy Nystagmus, sensory neuropathy, gastric pain and vomiting Pure cerebellar Limb tremor, orofacial dyskinesia, cognitive/behavioral/ mood changes Brief episodes of ataxia or choreoathetosis, interictal neuromyotonia Phenytoin or carbamazepine responsive (Continued)

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Table 1 Associated Features in the Differential Diagnosis of the Spinocerebellar Ataxias (Continued ) Ataxic disorder EA-2

EA-3

EA-4

EA-5

DRPLA GSS

Typical associated clinical features beyond ataxia and dysarthria Episodes of ataxia lasting hours, interictal nystagmus, fatigue/weakness Acetazolamide responsive Episodes of ataxia with diplopia and vertigo, defective smooth pursuit Not acetazolamide responsive Kinesigenic episodes of ataxia and vertigo, with diplopia and tinnitus Acetazolamide responsive Similar to EA-2, but later-onset; generalized, absence, and myoclonic seizures Acetazolamide responsive Epilepsy, myoclonus (onset < 20 yr); Dementia, psychosis, choreoathetosis (onset > 20 yr) Dementia, pyramidal signs

cord paralysis; chorea or dystonia; and optic atrophy occur (65,66). The ﬁrst decade of symptoms is one of progressive disability, while the second decade has greater morbidity and mortality, a general rule for most of the typical dominant ataxias. Laboratory Testing Brain MRI shows pontine and cerebellar atrophy. Magnetic resonance spectroscopy (MRS) may reveal early pontine change (67). Clinical severity has been found to correlate with quantitative volumetric, diffusion MRI and proton MR spectroscopy ﬁndings in the brainstem, suggesting possible use as a biomarker (68). Electronystagmography conﬁrms this physiologically (69,70). Motor evoked potentials are abnormal, with slowed peripheral and central motor conduction times (71). Pathology Loss of neurons and neuronal connections occurs in the cerebellum (dentate nucleus, Purkinje cells), brainstem (inferior olive, red nucleus, and substantia nigra), spinal cord (spinocerebellar tract, Clarke’s nucleus, posterior columns, and anterior horn), and basal ganglia (globus pallidus externa). In the mouse model, there is early loss of Purkinje cell dendritic spines and branches, and symptomatology occurs before visible nuclear aggregates or cell loss are seen (72). (text continues on page 326)

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Characteristic feature Pure cerebellar phenotype and MRI Complex phenotype, but pure cerebellar atrophy on MRI Brainstem involvement or atrophy on MRI Pyramidal involvement, hyperreﬂexia Extrapyramidal involvement Peripheral nerve involvement or hyporeﬂexia on the basis of spinal long tract changes Supratentorial features or MRI ﬁndings

Ocular features

Prominent postural/action tremor

Episodic features Early-onset (<20 yr) Most can have rare cases with early-onset

Genetic syndromes to consider ADCAIII—SCA5, 6, 8, 10, 11, 14, 15, 16, 22, 26 ADCAI—SCA4,18, 21, 23, 25, SCA due to FGF14 ADCAI—SCA1, 2, 3, 7, 13, DRPLA ADCAI—SCA1, 3, 4, 7, 8, 11, 12, 23 ADCAI—SCA1, 2, 3, 12, 21, DRPLA, FGF14 SCA1, 2, 3, 4, 8, 12, 18, 19, 21, 22, 25, FGF14 Cerebral atrophy—SCA2, 12, 17, 19 Subcortical white matter changes – DRPLA Dementia—SCA2, 7, 13, 17, 19, 21, DRPLA; or milder cognitive defects—SCA1, 2, 3, 6, 12; FXTAS Mental retardation—SCA13, 21, FGF14 Seizures—SCA7, 10, EA-5, DRPLA Psychosis—SCA3, 17, DRPLA, FGF14 Slow saccades—SCA1, 2, 3, 7, 23 Downbeat nystagmus—SCA6, EA-2 Maculopathy/ADCAII—SCA7 SCA2, 8, 12, 16, 19, 21, FGF14; FXTAS Palatal tremor—SCA20 (dentate calciﬁcation) Myoclonus—SCA1, 2, 3, 6, 7,14,19, DRPLA EA1-5, SCA6 Childhood—SCA2, 7,13,25, DRPLA, FGF14 Young adult—SCA1, 2, 3, 21

317

(Continued)

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Autosomal Dominant Ataxias

Table 2 Prioritizing Genetic Testing, as Tests Continue to Become Available

Prioritizing Genetic Testing, as Tests Continue to Become Available (Continued ) Genetic syndromes to consider

Characteristic feature Late-onset (>50 yr) Most can have rare cases with late-onset Rapid progression (death in <10 yr) (Average progression to disability is 5–10 yr, to death 10–20 yr) Slow progression over decades Anticipation/intergenerational DNA instability [usually paternal > maternal; maternal > paternal indicated by (m)] Variable phenotype

318

Table 2

SCA6 Early-onset SCA2, 3, 7, DRPLA SCA4, 5, 8, 11, 13, 14, 15, 16, 18, 20, 21, 22, 23, FGF14 Normal lifespan—SCA5, 6, 11, 18, FGF14 SCA1, 2, 3, 4, 5 (m), 6 (not due to repeat size), 7, 8 (m), 10, ?19, 20, ?21, 22, DRPLA SCA2, 3, 4, 5, 7, 14, 15, 17, GSS

Note: See www.geneclinics.org for test availability. Copyright 2006 by Taylor & Francis Group, LLC

Perlman

Ataxic disorder

Gene locus

SCA1

6p23

SCA2

SCA3/MachadoJoseph disease

SCA4

12q24

Gene/Product Ataxin-1

CAG expansion/coding exon

Ataxin-2

Normal < 39 repeats Disease-causing > 44 If not CAT interruption, diseasecausing 39–44 CAG expansion/coding exon

14q24.3-q31 Ataxin-3

16q22.1

Mutation

Unknown

Prevalence 6–27% of dominant ataxias worldwide

13–18% of dominant ataxias worldwide

Autosomal Dominant Ataxias

Table 3 Molecular Genetics of the Spinocerebellar Ataxias

Normal < 33 repeats, with CAA interruption. Disease-causing 33, with no CAA interruption (two patients with interrupted 34 expansion) CAG expansion/coding exon 23–36% of dominant ataxias Normal < 41 repeats worldwide Disease-causing 45 Homozygous mutant genes cause earlier-onset, more severe disease. Families in Utah & Germany; 6 Linkage studies with DNA families in Japan with laterpolymorphisms point to location, no onset pure cerebellar CAG expansion found despite syndrome evidence of anticipation. (Continued) 319

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Table 3

Molecular Genetics of the Spinocerebellar Ataxias (Continued )

Ataxic disorder

Gene locus

SCA5

11p11-q11

SCA6

SCA7

SCA8

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Unknown

Mutation

Linkage studies with DNA polymorphisms point to location, maternal anticipation suggests gene instability of unknown mechanism. CAG expansion/coding exon 19p13 CACNa1A/P/Qtype Normal < 19 repeats calcium channel Disease-causing 19 subunit (disease mechanisms may result Homozygous mutant genes cause earlier-onset, more severe disease. from both CAG repeat Allelic with EA-2 (gene truncations) and channelopathy and hemiplegic migraine (missense processes) mutations) CAG expansion/coding exon 3p21.1-p12 Ataxin-7 Component of Normal < 28 repeats TFTC-like Disease-causing 37 transcriptional Intermediate 28–36, may expand into complexes (disease disease range, especially with mechanisms may result paternal transmission from both CAG repeat and transcriptional dysregulatory processes) CTG expansion at 30 end 13q21 Normal product is an untranslated RNA that Normal < 80 repeats Disease-causing 80–300, although functions as a gene expansions in this range occur in regulator non-ataxic persons and in other neurologic diseases Expansions >300 may not cause disease in SCA8 pedigrees

Prevalence Lincoln family in US; families in Germany & France

10–30% of dominant ataxias worldwide

2–5% of dominant ataxias worldwide; may be more common in Sweden & Finland

2–4% of dominant ataxias worldwide; genetic testing results may be open to interpretation

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SCA9(reserved)

Gene/Product

SCA11

SCA12

SCA13 SCA14

Ataxin-10 Gene product essential for cerebellar neuronal survival

Mexican families (ataxia and epilepsy); 5 Brazilian families (no epilepsy)

2 British families

German-American family; may account for up to 7% of ADCA in India.

French family—7 of 8 affected members were women Japanese (axial myoclonus), English/Dutch, Dutch, and French (broader age of onset, cognitive impairment) families described. Incomplete penetrance. 321

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Pentanucleotide repeat (ATTCT) expansion in intron 9, probable loss of function mutation Normal 22 repeats Disease 800–4500 Intergenerationally more likely to contract than expand 15q14-q21.3 Unknown Linkage studies with DNA polymorphisms point to location, possible evidence of anticipation in one family suggest intergenerational instability. 5q31-q33 PPP2R2B/brain speciﬁc CAG expansion in 50 untranslated region of gene, possibly upstream regulatory subunit of from transcription start site and protein phosphatase 2A affecting gene transcription. Minimal (serine/threonine intergenerational instability. phosphatase) 19q13.3Unknown Linkage studies with DNA q13.4 polymorphisms point to location 19q13.4PRKCG/Protein kinase Missense mutations in conserved qter Cg (serine/threonine residues of C1/exon 4—regulatory kinase) domain and in catalytic domain of the enzyme. Increased intrinsic activity of mutant enzyme moves intraneuronal distribution from cytosol to plasma membrane. May reduce expression of ataxin-1 in Purkinje cells, and mutant ataxin-1 may reduce expression of PRKCG 22q13

Autosomal Dominant Ataxias

SCA10

(Continued)

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Table 3

Molecular Genetics of the Spinocerebellar Ataxias (Continued )

Ataxic disorder

Gene locus

SCA15

3p24.2-pter Unknown Region may contain gene(s) for three linked or allelic disorders

SCA16 SCA17/ Huntington disease-like 4

Unknown

Mutation Linkage studies with DNA polymorphisms point to location

Linkage studies with DNA polymorphisms point to location CAG/CAA expansion TATA box-binding protein (DNA binding Normal 42 repeats Disease-causing 45 subunit of RNA Intermediate 43–48, with incomplete polymerase II penetrance. transcription Minimal intergenerational instability. factorD (TFIID), Homozygous mutant genes cause essential for the earlier-onset, more severe disease. expression of all protein-encoding genes; Variable phenotypes include similarities to Huntington’s disease, disease mechanisms Parkinson’s disease, Alzheimer’s may result from both disease, and variant JacobCAG repeat and Creutzfeldt disease transcriptional dysregulatory processes)

Prevalence One Australian family (pure cerebellar), 2 Japanese families (with tremor/myoclonus), and one family with autosomal dominant congenital nonprogressive cerebellar ataxia One Japanese family Japanese, German, Italian, and French families

Perlman

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8q22.1q24.1 6q27

Gene/Product

7q22-q32

Unknown

SCA19

1p21-q21

Unknown

SCA20

11p13-q11

Near SCA5 locus, gene/ product unknown

SCA21

7p21–15

Unknown

SCA22

1p21-q23

Unknown

SCA23

20p13–12.3 Unknown

SCA24(reserved) SCA25

2p15–21

Unknown

SCA26

19p13.3

Unknown

SCAduetoFGF14

13q34

Fibroblast growth factor 14

Linkage studies with DNA polymorphisms point to location Linkage studies with DNA polymorphisms point to location; possibly allelic with SCA22 Linkage studies with DNA polymorphisms point to location; repeat expansion detection did not show CAG/CTG or ATTCT/ AGAAT repeat expansions Linkage studies with DNA polymorphisms point to location; evidence of anticipation suggests intergenerational instability. Linkage studies with DNA polymorphisms point to location; possibly allelic with SCA19, but without cognitive impairment Linkage studies with DNA polymorphisms point to location Linkage studies with DNA polymorphisms point to location Linkage studies with DNA polymorphisms point to location Missense and frameshift mutations

One Irish-American family One Dutch family

Anglo-Celtic family in Australia

One French family

One Chinese family

One Dutch family

One southern French family. Incomplete penetrance. One family of Norwegian ancestry Dutch, German, and French families 323

(Continued)

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Autosomal Dominant Ataxias

SCA18

Ataxic disorder

Gene locus

EA-1

12p13

EA-2

19p13

EA-3

Not identiﬁed Not identiﬁed 2q22-q23

EA-4 EA-5

KCNA 1/Potassium voltage-gated channel component CACNa1A/P/Qtype voltage-gated calcium channel subunit

Unknown Unknown CACNB4b4/P/Qtype voltage-gated calcium channel subunit; two domains interact with a1 subunit.

Mutation

Prevalence

Rare families worldwide. Missense mutations cause altered neuronal excitability in CNS and PNS. Point mutations in exons and introns Rare families worldwide. De novo mutations in 25% of (nonsense, missense) and small cases. deletions; mutations cause reduced calcium channel activity in CNS and PNS. Allelic with familial hemiplegic migraine and SCA6; 2 families with CAG expansion and phenotype of episodic ataxia. Linkage excluded to EA-1 and EA-2 North Carolina families Linkage excluded to EA-1 and EA-2. Clinically different from EA-3. Point mutations leading to aminoacid substitution or premature stop codon; mutations cause altered calcium channel activity in CNS.

Canadian Mennonite family French-Canadian family (phenotype similar to EA-2 with later-onset, incomplete penetrance). German family with seizures. Michigan family with phenotype of juvenile myoclonic epilepsy (premature stop codon).

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Gene/Product

324

Table 3 Molecular Genetics of the Spinocerebellar Ataxias (Continued )

GSS

12p13.31

20p12

Atrophin-1 Required in diverse developmental processes; interacts with even-skipped homeobox 2 repressor function.

PrP/prion protein

1–5% of dominant ataxias CAG expansion/coding exon worldwide; 10–20% of ADCA Normal < 26 in some areas of Japan. Disease-causing 49 Intermediate 37–48, may expand into disease range, especially with paternal transmission. Homozygous mutant genes cause earlier-onset, more severe disease; homozygous intermediate genes may cause a recessive predominantly spinal syndrome. Allelic with Haw River syndrome (no seizures). Rare families worldwide. Point mutations causing aminoacid substitutions in PrP or octapeptide insertions, resulting in proteinase K resistant form of protein which accumulates in CNS.

Autosomal Dominant Ataxias

DRPLA

Note: See www.ncbi.nlm.nih.gov (PubMed, OMIM) and www.neuro.wustl.edu/neuromuscular/ataxia for continuing updates.

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Molecular Genetic Features (See also Table 3) The SCA1 gene was identiﬁed and cloned in 1994 by the collaborative efforts of the laboratories of Zoghbi and Orr (73). Disease results from a CAG trinucleotide repeat expansion in the reading frame of the gene, producing a polyglutamine expansion in the protein product. The normal CAG range is from 6 to 38 repeats. The intermediate range, 39–44 repeats, will cause disease if CAT interruptions in the sequence are lacking. Disease has been associated with repeat expansions from 41 to 83. Lack of CAT interruptions in the CAG sequence predisposes to meiotic enlargement of the triplet repeat disease-causing range or to expansion from a non-disease causing size to a disease-causing size. Increased numbers of CAG repeats correlate with earlier onset, greater severity, and the appearance of upper motor neuron features, but not with increased rapidity of progression. Earlier onset with a generational increase in CAG length (anticipation) is seen more with paternal than maternal transmission and may lower age of onset by 10 years per generation. In homozygotes, disease severity corresponds to the size of the larger allele (65). The protein product (ataxin-1) is found in cytoplasm systemically and in the nucleus in neuronal cells. The intranuclear inclusions of SCA1 occur in neurons most affected by disease (Purkinje cells), stain for ubiquitinated ataxin-1, and interact with leucine-rich acidic nuclear protein (LANP), ubiquitin, and HSP-40 chaperone. LANP may play a key role in neuronal development and/or neurodegeneration by its interactions with microtubule associated proteins, so its sequestration in the inclusions could impair maintenance of dendritic structure (74), even before other polyglutaminemediated pathologic processes occur. SCA2 Clinical Features Onset usually is between ﬁve years of age and into the seventh decade, with 40% becoming symptomatic before the age of 25. Very small repeat expansions (33) are associated with onset in the ninth decade, and very large expansions (>200) are associated with onset before the age of one (75). The initial course is one of pancerebellar degeneration with early apparent slowed saccades, tremor and titubation, myoclonus, chorea, facial fasciculations, and reduced reﬂexes. Axonal sensory polyneuropathy affecting the lower extremities (especially vibratory sensation) is seen in 80% and increases with age. Amyotrophy, leg weakness, or upgoing toes occur in only 20%. With longer duration of illness, ophthalmoplegia (nuclear and supranuclear), dysphagia, reduced deep tendon reﬂexes, bladder/sphincter disturbance, and cognitive impairment become apparent in about 50% of patients, reﬂecting progressive pontine, neuropathic, and cerebral involvement. With earlier

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onset disease, more rapid progression, greater slowing of saccade velocity (76), more severe bulbar dysfunction, and more notable involuntary movements (myoclonus, dystonia, and myokymia) are seen. L-dopa-responsive Parkinsonism may occasionally be the presenting or predominant feature in a family or individual (77). Anticipation in families, more commonly with paternal transmission, can be up to 26 years per generation and averages 20 years. Laboratory Testing Brain MRI shows severe pontine and cerebellar atrophy. However, there was no correlation found between clinical severity and quantitative volumetric, diffusion MRI and proton MR spectroscopy ﬁndings in the brainstem (68). Pathology The most prominent neuronal loss is seen in the brainstem, affecting pontine nuclei and the inferior olives. Changes are also seen in Purkinje cells and white matter of the cerebellum, substantia nigra, and the cerebral subcortical white matter. Spinal cord pathology occurs in Clarke’s nucleus and spinocerebellar tracts, posterior columns and dorsal root ganglia, and corticospinal tracts. Molecular Genetic Features (See also Table 3) The SCA2 gene was identiﬁed and cloned by three separate research groups in three countries by three different methods and reported in back to back articles in 1996 (78–80). Disease results from a CAG trinucleotide repeat expansion in the reading frame of the gene, producing a polyglutamine expansion in the protein product, whose normal function is not known, but may be involved in translational regulation (81). Normal alleles (14–32 repeats, most common 22) often have stabilizing CAA interruptions in the CAG sequence, while > 95% of patients have no interruptions. An interrupted 34-CAG repeat allele has been reported in two sporadic patients (82). Disease-causing expansions range from 33 to 77 in the adult form of the illness (most common 37 repeats), while the infantile and childhood forms may have expansions exceeding 200 repeats. Intergenerational expansions are more common with paternal transmissions, and anticipation can be up to 26 years. The protein product (ataxin-2) is expressed in brain and systemic tissue, with the highest levels in ependyma and choroids plexus, followed by Purkinje cells, substantia nigra neurons, and trochlear nuclei. Levels of protein increase with age in SCA2, and there is increased perinuclear staining for the protein over time. Mutant ataxin-2 disrupts the Golgi apparatus (1) and induces cell death without nuclear aggregates (31), although aggregates can be seen in brainstem nuclei and are more likely with very large CAG expansions.

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SCA3 Clinical Features SCA3/Machado-Joseph disease, the most common of the typical dominant spinocerebellar ataxias, has several allelic clinical syndromes that vary in age of onset and symptomatology, although within families the clinical presentation usually remains consistent, despite the occurrence of anticipation (paternal > maternal). An affected male parent will transmit the abnormal allele 73% of the time (83,84). Larger CAG repeat expansions contribute to earlier onset, more rapid progression, and more dystonia and spasticity. Homozygotes will have an earlier onset, more severe presentation. The most common presentation (type III) is a typical dominant ataxia, onset in the fourth to seventh decade, with spasticity, progressive axonal sensory (especially temperature discrimination) > motor polyneuropathy (54%), ophthalmoparesis with both vestibular and nuclear features, facial and lingual fasciculations, and autonomic dysfunction. Sleep disturbance due to restless legs syndrome and periodic leg movements of sleep is common. These features reﬂect spinopontine atrophy and are most commonly seen in patients of German or Dutch-African descent. The original discussions of ‘‘Machado-Joseph disease,’’ in the AzoreanPortuguese population, emphasized the ataxia, extrapyramidal features, and ‘‘bulging eyes’’ and noted additional phenotypes. Type I was the earliest onset (age 5–30 years), with a more spastic/dystonic presentation. Type II had onset in the fourth decade and shared features of Types I and III. Type IV was the oldest onset and more likely to present as L-dopa responsive Parkinsonism or as a peripheral neuropathy with fasciculations, amyotrophy, and sensory loss. A ﬁfth type with spastic paraparesis has been described in Japanese pedigrees. The clinical syndrome may overlap with DRPLA (extrapyramidal features) or multiple system atrophy (MSA) (extrapyramidal and autonomic features). Laboratory Testing Brain MRI shows an enlarged fourth ventricle and mild cerebellar and brainstem atrophy. Electronystagmography conﬁrms impaired vestibuloocular reﬂex gain, and nerve conduction velocity studies conﬁrm small motor and sensory action potential amplitudes, consistent with axonal loss. Pathology Basal ganglia pathology is prominent, with changes in globus pallidus interna, subthalamic nucleus, substantia nigra, and red nucleus. There is also notable change in cerebellar connections, in the dentate nucleus, spinocerebellar tracts, and pontine nuclei. Inferior olives are spared. Cranial nerve nuclei in the brainstem show involvement. Mild spinal cord changes are seen in Clarke’s nucleus, anterior horn, and posterior column.

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Molecular Genetic Features (See also Table 3) The SCA3 gene was cloned in 1994, revealing a CAG trinucleotide repeat expansion in the reading frame of the gene, producing a polyglutamine expansion in the protein product (ataxin-3), whose function is unknown (85). Ataxin-3 is located in the cytoplasm of the neuronal soma and processes. Ataxin-3-containing ubiquitinated intranuclear inclusions are found only with disease-causing mutations in affected brain areas, where conformationally altered protein is bound to the nuclear matrix. The presence of a polymorphism near the CAG expansion region has made possible research into the use of small inhibitory RNAs to selectively reduce the production of mutant ataxin-3 (11). SCA4 Clinical Features SCA4 has also been found to have at least two distinct phenotypes. In a Scandinavian-American family in Utah and a German family, a progressive cerebellar syndrome with sensory > motor axonal polyneuropathy, onset in the second to sixth decade, was described (86). Distal weakness is seen in about 20%. While tendon reﬂexes are typically absent in the lower extremities, plantar responses have been noted to be extensor in 10–20%. Anticipation of 5–7 years per generation is seen. Progression is typically slow over decades. A pure cerebellar syndrome has also been reported in Japanese families, onset in the ﬁfth to eighth decade (87), beginning with midline and lower extremity dysfunction and progressing to ﬁne ﬁnger incoordination, with saccadic pursuit, dysarthria, and hypotonia in over 50%. Laboratory Testing Brain MRI shows cerebellar atrophy. Electrodiagnostic studies conﬁrm the axonal neuropathy, with sensory action potential absence in >90% and motor action potential reduction in 38%. Pathology Atrophy and loss of Purkinje cells is seen in the cerebellum, with accompanying loss of dorsal root ganglion cells and of axons in the posterior columns of the spinal cord and axons in peripheral nerve. Molecular Genetic Features (See also Table 3) The gene has not yet been cloned and nothing is known about its protein product or the mechanism of damage caused by the mutant protein. Despite the presence of anticipation, analysis of nine CAG/CTG tracts in the candidate region revealed no evidence for a repeat expansion (88). No other candidate genes have been identiﬁed (87).

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SCA5 Clinical Features SCA5 was ﬁrst reported in an American pedigree descended from the paternal grandparents of Abraham Lincoln (89). It has also been reported in a German kindred (90). Onset is in the second to ﬁfth decade, with a mean age of 33 years. Anticipation is probable. The features of a predominantly cerebellar syndrome also include downbeat nystagmus, impaired smooth pursuit, and in some families horizontal gaze palsy, facial myokymia, and impaired vibration sense. Laboratory Testing Brain MRI shows atrophy of the cerebellar vermis and hemispheres, with sparing of brainstem and supratentorial structures. Nerve conduction studies are normal. Pathology This Pathology has not yet been reported. Molecular Genetic Features (See also Table 3) The gene has not yet been cloned and nothing is known about its protein product. SCA6 Clinical Features SCA6, a CAG repeat expansion disorder, has onset typically in the ﬁfth or sixth decade (60% are over the age of 50), with onset in the third or fourth decade occurring with larger CAG repeat expansions or homozygosity/ compound heterozygosity. There is intergenerational stability in the repeat size, so generational variation in age of onset or severity may relate to factors other than repeat size. Twenty-seven percent of cases may appear to be sporadic. SCA6 represents 10–30% of the dominant ataxias. The ﬁrst symptoms may be episodic sensations of vertigo or instability on turning, which slowly progress over decades to a typical cerebellar syndrome, affecting speech and truncal stability, more than limb incoordination. Progression to use of a wheelchair may occasionally occur in the ﬁrst decade. Two families with CAG expansions retained a phenotype of episodic ataxia. Downbeating nystagmus is a common oculomotor feature, but all cerebellar-mediated eye ﬁndings (horizontal gaze-evoked nystagmus, rebound nystagmus, impaired vestibule-ocular reﬂex, impaired smooth pursuit, but normal saccade velocities and VOR gain) can be seen. Vestibularmediated symptoms may cause a sense of imbalance with head movement, but other brainstem features occur only late in the disease (dysphagia).

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Some patients have a mild sensory neuropathy with loss of vibration and proprioception. Bradykinesia and frontal subcortical dementia may occur. Laboratory Testing Brain MRI shows cerebellar hemispheric and vermian atrophy, with slight atrophy of the red nucleus. Mild pontine and middle cerebellar peduncular atrophy have also been reported (91). Single-ﬁber and microelectrode electrophysiologic studies of the neuromuscular junction are normal, as opposed to jitter and blocking, reduced endplate potential and quantal content, and ultrastructurally smaller nerve terminals seen in EA-2, which has myasthenic weakness as an associated symptom. Pathology Pathology is localized to the cerebellum and its connections, with loss of Purkinje cells and mild changes in the inferior olivary nucleus and dentate nucleus. Intranuclear inclusions are not seen. Molecular Genetic Features (See also Table 3) In 1997, by genotype survey, Zhuchenko et al., found a CAG repeat expansion in the CACNA1A gene in eight unrelated families who showed a very similar clinical picture consisting predominantly of mild but slowly progressive cerebellar ataxia of the limbs and gait, dysarthria, nystagmus, and mild vibratory and proprioceptive sensory loss (92). Normal CAG lengths are 4–19. Disease-causing are 20–31 repeats. SCA6 is allelic to familial hemiplegic migraine and EA-2 (93). Familial hemiplegic migraine Type I is commonly caused by one of several reported missense point mutations in the CACNA1A gene—e.g., T666M which causes hemiplegic migraine (severe with coma in 50%) with nystagmus in 86%; R583Q where ataxia not nystagmus can be seen in 81%; and D715E with relatively fewer hemiplegic attacks (64%) and possible tremor (94). Familial hemiplegic migraine Type II, however, has similar features (including cerebellar dysfunction), but results from mutations in ATP1A2 on chromosome 1q23, encoding a Naþ/Kþ -ATPase subunit (95). EA-2 results from nonsense (R1281X, and R1549X) or missense (C27Y, G293R, F1406C, F1493S, R1666H, and E1761K) point mutations in the CACNA1A gene, or occasionally small deletions. It is characterized by acetazolamideresponsive attacks of ataxia (with vertigo or generalized weakness in 50%) interictal nystagmus, and a slowly progressive baseline cerebellar deﬁcit (44). Both of these allelic disorders have an average age of onset before 20 years, and the episodes (hemiplegic migraine or ataxia with nystagmus) may be triggered by emotional or physical stress. The attacks of hemiplegia, however, can last days, whereas the attacks of ataxia last just a few hours. SCA6 pedigrees may show increased numbers of individuals with vascular headaches, vertigo, motion sickness, or epilepsy, without ataxia. The

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episodic features of SCA6 may respond to acetazolamide, as do the episodic features of EA-2. Ongoing research has put in place a detailed understanding of the molecular genetics and pathophysiology of the voltage-gated calcium channel in progressive cerebellar ataxia. Some debate remains about whether SCA6 is primarily a glutamine expansion disorder or a channelopathy (96). A loss-of-function model has been proposed, whereby the mutant protein interferes with the ability of the calcium channel to reduce Ca2þ inﬂux and abolishes the cell-deathpreventing effect of the normal Ca2þ channel (97). SCA7 Clinical Features The only conﬁrmed genetic cause of ADCAII (ataxia with retinal degeneration), SCA7 is also a CAG trinucleotide repeat expansion and has a broad age of onset (1 month to 76 years), dependent on the size of the CAG repeat expansion. Earlier onset disease is more severe and may lead to death in as little as two years, while the later onset presentations may progress slowly over decades. Paternal transmission is associated with anticipation of 20 or more years per generation. Expansions from the intermediate range of repeats in an asymptomatic parent to the symptomatic range in an affected child may also occur. The typical onset is in adolescence with repeats in the mid-range. Ataxia may be the ﬁrst symptom, but with larger CAG expansions macular degeneration may precede ataxia by 10 years. Early blue–green color blindness is reported, followed by macular degeneration and visual loss in 83%. Blindness occurs in about 28% of those affected. Ophthalmoplegia (supranuclear and with slow saccades and ptosis) develops in 70%. Pyramidal signs and hearing loss are also reported. The infantile form includes features of microcephaly, cardiac failure, hepatomegaly, hemangiomas, and capillary leak syndrome, as well as developmental delay, hypotonia, and a rapidly fatal course. With the smallest expansions, patients may become only mildly symptomatic at an advanced age. Laboratory Testing Brain MRI shows early pontine and cerebellar atrophy (98). Ophthalmologic studies reveal pigmentary retinopathy in 43% and optic atrophy in 69%. There is no peripheral nerve change. Pathology Purkinje cell, inferior olive, and dentate nuclear changes are prominent. Subthalamic nucleus and substantia nigra show involvement, in addition to cerebral atrophy, loss of anterior horn cells, and reduced axons in posterior columns, spinocerebellar tracts, and pyramidal tracts.

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Intranuclear inclusions are seen, most frequently in the inferior olivary neurons, where 60% are ubiquitinated. They do not contain LANP. Some protein may also accumulate in the cytoplasm of affected neurons. Mitochondrial abnormalities are occasionally reported in muscle and liver, with reduced Complex IV activity. Molecular Genetic Features (See also Table 3) The SCA7 gene was cloned in 1997, revealing a CAG trinucleotide repeat expansion in the reading frame of the gene, producing a polyglutamine expansion in the protein product (ataxin-7) (99). Normal CAG lengths are 4–27 repeats, with 10 being the most common. Disease-causing expansions range from 37 to >200. The average expansion in typical late-adolescent onset disease is 54–55 repeats. The infantile form is associated with expansions >200 repeats. Shorter expansions (38–43 repeats) may remain asymptomatic until later adulthood. Expansions in the intermediate range (28–36 repeats) are rare in the general population, but occur with increased frequency in SCA7 pedigrees—leading to the risk of de novo expansion (usually in paternal transmission) into the disease-causing range. Parent to child transmission can increase the repeat size four- to nine-fold. Ataxin-7 is localized in the nucleus (matrix and nucleolus). It is an integral component of the mammalian SAGA-like complexes TFTC (TATAbinding protein-free TAF-containing complex) and STAGA (SPT3/TAF9/ GCN5 acetyltransferase complex), which are involved in transcription (100). Over-expression of mutant ataxin-7 in neuronal and extra-neuronal cells causes ﬁbrillar caspase-3 containing inclusion formation and changes in proteosomes, components of the toxic gain of function proposed for triplet repeat disorders. It also inhibits function of the cone-rod homeobox protein (CRX) in photoreceptors, an innocent bystander effect that may be the cause of the associated maculopathy (101). SCA8 Note—The name SCA8 was previously assigned to the recessively inherited infantile onset spinocerebellar ataxia (IOSCA), mapped to 10q24 by a Finnish group in 1995 (102), but has been reassigned to reﬂect the classiﬁcation system for the dominant ataxias accepted and monitored by the HUGO Nomenclature Committee (103–105). Clinical Features SCA8, the result of a CTG expansion, has a uniform presentation in families, but may vary from family to family or in apparent sporadic cases. It may present from the ﬁrst year of life until the eighth decade, the average age of onset being in the ﬁfth decade. Incomplete penetrance may make the dominant transmission difﬁcult to recognize in an isolated individual presenting with

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ataxia. Gait, trunk, and limb ataxia, dysarthria, and impaired smooth pursuit are seen in 100% of cases, with horizontal nystagmus in 67%, sensory neuropathy (vibratory sense reduction) in 25%, and spastic dysarthria with increased deep tendon reﬂexes and extensor plantar responses in those most severely affected. Progression is slow, with ambulatory aids often not being required during the ﬁrst 20 years of illness. Symptom severity correlates with repeat length and age (106,107). One case with congenital onset presented with severe cerebellar symptoms in the ﬁrst year, myoclonic epilepsy at three years, and mental retardation. Presentation typical of idiopathic Parkinson’s disease with late onset of disease, resting tremor in the limbs, rigidity, bradykinesia, and a good response to levodopa has also been noted (108). Laboratory Testing Brain MRI shows cerebellar vermis and hemispheric atrophy. Pathology Pathology has not yet been reported. Molecular Genetic Features (See also Table 3) The clinical syndrome of SCA8, ﬁrst reported in 1999, is associated with an expanded CTG repeat in the 30 untranslated region of transcribed RNA from the SCA8 gene, which is felt to act as a gene regulator (109). CTG expansions in transcribed but untranslated DNA are also associated with myotonic dystrophy Type I (19q13.2-q13.3) (CTG) (110) and Type II (3q13.3-q24) (CCTG) (111). The SCA8 transcript is found in brain and at low levels in lung. Its CTG repeat has ﬁve ranges: a very short normal length (15–21 repeats) in 19%; a short normal length (22–37 repeats, 24 the most common) in 80%; an intermediate ‘‘normal’’ length (40–91 repeats) in 0.7%, although 80 repeats has caused symptoms in some families; a disease-causing length (100–155 repeats, occasionally up to 300 repeats); and a very large asymptomatic range (300–800 repeats), which with intergenerational transmission can contract back into the symptomatic range. The CTG expansion is very unstable and may expand or contract, with maternal anticipation (11 to þ600 repeats) more prominent than paternal (86 to þ16 repeats). Affected SCA8 individuals have usually inherited the CTG expansion from the mother. There is no correlation of age of onset or disease severity with CTG size. Somatic mosaicism is reported in normal and expanded transcripts. Individuals from SCA8 pedigrees with diseasecausing expansions may be asymptomatic, and CTG expansions have been reported in normal and disease control populations (Parkinson’s disease, Alzheimer’s disease, SCA1, SCA3, and SCA6). The pathophysiologic behavior of this CTG expansion is still not completely understood.

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SCA9 This section is reserved. SCA10 Clinical Features SCA10 is also the result of a disease-causing expansion in transcribed but untranslated DNA, in this case a pentanucleotide repeat (ATTCT). It is a rare SCA, being originally described in a set of Mexican families (112) and more recently in ﬁve Brazilian families (113). The phenotypes differ between the two populations. In the Mexican pedigrees, onset is between the ages of 10 and 40, with gait ataxia (which may progress to wheelchair use), followed by limb ataxia, dysarthria, gaze-evoked nystagmus, and smooth pursuit fragmentation. Complex partial seizures with/without secondary generalization contribute to morbidity and mortality and are seen in 50% of patients, either at onset or with disease progression. Personality changes are reported in some (apathy, and depressed affect). The Brazilian families did not develop seizures. Anticipation is seen, especially with paternal inheritance, and may move up disease onset by 20 years. Laboratory Testing Brain MRI shows cerebellar atrophy. EEG shows epileptiform discharges in about 50%. Nerve conduction studies are normal. Pathology Pathology has not yet been reported. Molecular Genetic Features (See also Table 3) The clinical syndrome of SCA10, ﬁrst reported in 2000, is associated with an expanded ATTCT repeat in intron 9 of the SCA10 gene (112). The normal range of repeats is 10–22 uninterrupted ATTCT sequences. The diseasecausing range is from 800 to 4500 repeats. The gene product, ataxin-10, is predominantly cytoplasmic and perinuclear in localization and restricted to olivocerebellar regions. Blocking of SCA10 in neuronal cells by small interfering RNAs results in increased apoptosis of cerebellar neurons, suggesting a loss-of-function process in SCA10 patients, although the associated epiloptogenesis has not yet been explained (114). Family-dependent factors may alter the frequency of the seizure phenotype (115). The ATTCT expansion is unstable and may expand or contract by hundreds of repeats, more so with paternal transmission. There is somatic mosaicism, observed in leukocytes, lymphoblastoid cells, buccal cells, and

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sperm. A larger number of repeats correlates with younger age of onset, but other factors may inﬂuence the rest of the clinical phenotype. SCA11 Clinical Features This SCA has been reported in two British families (116). Its age of onset is 17–33 years, presenting with a slowly progressive gait disorder, dysarthria, saccadic pursuit, horizontal > vertical gaze-evoked nystagmus, and hyperreﬂexia. Limb ataxia develops in about 93%. Life expectancy is normal. Anticipation was possible in one family. Laboratory Testing Brain MRI shows cerebellar atrophy. Nerve conduction studies are normal. Pathology Pathology is unknown. Molecular Genetic Features (See also Table 3) The gene has not yet been cloned and nothing is known about its protein product. SCA12 Clinical Features SCA12 was reported in a German-American family with a progressive gait ataxia with dysarthria and head/arm tremor (117). It showed onset between the ages of 8 and 55 years, with average onset in the fourth decade. Onethird of patients had eye movement abnormalities, with slow saccades, saccadic pursuit, and nystagmus. Tendon reﬂexes were increased in 80% and mild axonal sensory > motor polyneuropathy was noted in 80%. Thirtythree percent had facial myokymia. Axial dystonia and bradykinesia were reported to occur. Dementia was noted in the oldest patients. More recent surveys have shown a notable incidence in India, where SCA12 may account for 7% of all ADCA (118). Laboratory Testing Brain MRI shows cortical and cerebellar atrophy. Nerve conduction velocities conﬁrm axonal loss. Pathology Pathology has not yet been reported.

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Molecular Genetic Features (See also Table 3) SCA12 is associated with an expansion of a CAG repeat in the 50 untranslated region of the gene PPP2R2B, possibly upstream from the transcription start site. PPP2R2B encodes a brain-speciﬁc regulatory subunit of the protein phosphatase PP2A. Normal repeat length is 7–32. Disease-causing repeat lengths are from 55 to 93 uninterrupted repeats. There is minor repeat size instability over generations. It is possible that an expansion mutation in PPP2R2B may inﬂuence PPP2R2B expression, perhaps altering the activity of PP2A, an enzyme implicated in multiple cellular functions, including cell cycle regulation, tau phosphorylation, and apoptosis (119). This would be another example (see SCA6, SCA10, and SCA17) of a repeat expansion possibly causing a loss of function process, rather than a toxic gain of function. SCA13 Clinical Features SCA13 has been reported in one French family, where seven or eight affected members were women (120). The clinical picture was one of a slowly progressive, childhood-onset gait ataxia, with dysarthria in 88%, moderate mental retardation (IQ, 62–76), and mild developmental delays in motor skills. Horizontal gaze-evoked nystagmus was present in 75%. Increased deep tendon reﬂexes were also seen. Loss of ability to walk could develop after four or more decades. Anticipation was not seen, although one patient had onset in the ﬁfth decade. Laboratory Testing Brain MRI shows cerebellar and pontine atrophy. Pathology Pathology has not yet been reported. Molecular Genetic Features (See also Table 3) The gene has not yet been cloned and nothing is known about its protein product. SCA14 Clinical Features SCA14 has been reported in several ethnic groups, with varying phenotypes. English/Dutch (121), Dutch (122), and French (123) studies have described a very slowly progressive gait ataxia onset in the fourth decade

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(range 10–60 years of age), with tremor of head and extremities in younger onset patients. Limb ataxia affected legs more than arms, with dysarthria, and brisk ankle jerks. Some family members developed cognitive impairment (French), dystonia (Dutch), or peripheral neuropathy. Limb myoclonus was most commonly seen in patients with onset <27 years, while the later onset patients had a more pure ataxia. The Japanese patients had a narrower age of onset, beginning with intermittent axial myoclonus progressing to ataxia. They did not develop dementia (124). Some patients with the mutation were asymptomatic, suggesting incomplete penetrance. Laboratory Testing Brain MRI shows cerebellar atrophy. Pathology Reduced staining for PRKCG and ataxin-1 in Purkinje cells was seen, while calbindin staining was preserved (125). Molecular Genetic Features (See also Table 3) SCA14 results from missense mutations in protein kinase Cg (PRKCG), either in conserved residues in C1, the cysteine-rich region of exon 4 (regulatory domain) (H101Y, Gly118Asp, S119P, and G128D) or in the catalytic domain of the enzyme (F643L). The protein product is a serine/threonine kinase, with high expression in the brain, especially cerebellar cortex and spinal cord. This mutation seems to increase the intrinsic activity of the enzyme and moves its intraneuronal distribution from cytosol to plasma membrane (126,127). Mutant PRKCG may reduce expression of ataxin-1 in Purkinje cells, and mutant ataxin-1 may reduce expression of PRKCG (128). The complete interaction of pathophysiologic mechanisms involved has not yet been determined. SCA15 Clinical Features SCA15 was initially described in one Australian family with a slowly progressive, often mild, pure cerebellar ataxia (129). Onset varied between ages 10 and 50 years, with average age of onset 26 years. Eighty-six percent of patients had limb ataxia and 71% gait ataxia. Gaze-evoked nystagmus, saccade dysmetria, and dysarthria were also seen. Patients usually remained ambulatory. Two Japanese families, also with a slowly progressive ataxia, but with the addition of postural and action tremor/myoclonus, seemed to link to a locus that overlapped the Australian locus (130). Most recently, another Australian family with a dominantly inherited nonprogressive congenital ataxia and cognitive disability (with variable features of dysarthria,

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dysmetria, dysdiadochokinesia, nystagmus, dystonic movements, and cerebellar hypoplasia on imaging) was also found to link to a locus that overlapped the same region (131). These three conditions may be allelic. Laboratory Testing Brain MRI shows superior vermian atrophy. Nerve conduction velocities are normal. Pathology Pathology has not yet been reported. Molecular Genetic Features (See also Table 3) The gene has not yet been cloned and nothing is known about its protein product. SCA16 Clinical Features SCA16 has been reported in one Japanese family with a slowly progressive cerebellar ataxia affecting gait, limb, and speech, and associated impaired smooth pursuit and nystagmus, with rotatory head tremor in 15%. Age of onset was between 20 and 66 years (average 40 years), with no anticipation (132). Laboratory Testing Brain MRI shows cerebellar atrophy. Nerve conduction velocities are normal. Pathology Pathology has not been reported. Molecular Genetic Features (See also Table 3) The gene has not yet been cloned and nothing is known about its protein product. SCA17 Clinical Features SCA17 is a polyglutamine disorder reported in several ethnic groups with varying phenotypes. Age of onset ranged from the ﬁrst year of life to the seventh decade (average onset in the third decade). SCA17 was ﬁrst reported in 1999 in a sporadic Japanese patient with ataxia and intellectual deterioration, associated with de novo expansion of the CAG repeat of the TATAbinding protein (TBP) gene (133). In 2001, two German families were reported with an AD degenerative multisystem disorder including ataxia and intellectual impairment, but also involvement of the pyramidal, extrapyramidal, and possibly autonomic system (134). Later that year, four Japanese families

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were reported with ataxia, bradykinesia, and dementia (135). In 2003, four German families were identiﬁed with features of ataxia, dysarthria, and dysphagia, with most showing psychiatric symptoms, including depression, disorientation, aggression, paranoia, and dementia. Dystonia and extrapyramidal movements were occasionally present (136). In a 4-generation Italian family, the earliest onset case was reported, apparently reﬂecting a 13-repeat expansion from the affected parent. The child had onset at age three with ataxic gait with foot intrarotation and dysarthria. At the age of 13, she had developed loss of sphincter control, seizures, tremor, spasticity, hyperreﬂexia, extensor plantar responses, and mental retardation (137,138). Huntington’s disease-like phenotypes (in addition to cerebellar ataxia, patients exhibited psychiatric disturbances, dementia, and chorea) were reported in Germany (139) and Italy (140), while in Japan a homozygous case was also noted to have an HD-like phenotype, without an earlier age of onset (141). Incomplete penetrance was identiﬁed in France, in a pedigree that manifested severe psychiatric features, dementia, axial rigidity, and seizures, reminiscent of juvenile HD (142). In the latest stages of disease, patients were bedridden, anarthric, dysphagic, and incontinent. Laboratory Testing Brain MRI shows cerebellar and cerebral atrophy. Pathology Postmortem brain tissue from one patient from the Japanese pedigrees showed shrinkage and moderate loss of small neurons with gliosis predominantly in the caudate nucleus and putamen, with similar but moderate changes in the thalamus, frontal cortex, and temporal cortex (135). Moderate Purkinje cell loss and an increase of Bergmann glia were seen in the cerebellum. Immunocytochemical analysis performed with anti-ubiquitin and anti-TBP antibodies showed neuronal intranuclear inclusion bodies. Most neuronal nuclei were diffusely stained with 1C2 antibody, which recognizes expanded polyglutamine tracts (135). The immunoreactive inclusion bodies were much more widely distributed throughout the brain gray matter than in other SCAs. In the homozygous HD-like phenotype, there was mild neuronal loss with compaction of the neuropil in the cerebral cortex, mild loss of neurons in the striatum, and moderate loss of Purkinje cells in the cerebellum (141). Many 1C2-positive neuronal nuclei were present in the deep layers of the cerebral cortex, as well as in the putamen and cerebellum. Diffuse intranuclear polyglutamine aggregate accumulation was found in a wide range of CNS regions beyond those affected by neuronal loss. In the juvenile HD-like phenotype, the brain showed diffuse atrophy in all brain regions, with the cerebellum most affected (140). There was severe neuronal loss and gliosis in the striatum, the dorsomedial thalamic nucleus, and inferior olive. Neuronal intranuclear inclusions stained with anti-TBP and 1C2.

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Molecular Genetic Features (See also Table 3) SCA17 is caused by a CAG trinucleotide repeat expansion in the reading frame of the gene for the TATA box-binding protein, producing a polyglutamine expansion in the protein product (a DNA binding subunit of RNA polymerase II transcription factor D/TFIID). TFIID is essential for the expression of all protein-encoding genes, so the disease mechanism of SCA17 could involve both polyglutamine-mediated toxic gain of function (suggested by the intranuclear aggregates) and transcriptional dysregulation. The normal CAG repeat length is 25–42 repeats. Disease-causing expansions are in the 45–66 repeat range, the largest expansions causing childhood-onset disease. Intermediate CAG repeats (43–48) have incomplete penetrance. There is no predictable meiotic instability, but there may be occasional large variations, as indicated above. SCA18 Clinical Features SCA18 has been reported in one Irish-American family, with progressive gait and limb ataxia, head tremor, panmodal sensory loss, and neurogenic muscular atrophy (143). Weakness was present in 70%, the Romberg test was positive, and deep tendon reﬂexes were reduced or absent. Some patients had nystagmus or extensor plantar responses. Hearing loss was present in 20%. There was no cognitive involvement. Onset was between 13 and 27 years of age. Disease progression was slow, leading to use of a wheelchair in the later stages. Lifespan was normal. Laboratory Testing Brain MRI shows mild cerebellar atrophy. Nerve conduction studies show a sensory axonal neuropathy. Electromyogram shows denervation, and muscle biopsy is consistent with neurogenic atrophy. Pathology Outside of neurogenic atrophy on muscle biopsy, pathology has not yet been reported. Molecular Genetic Features (See also Table 3) The gene has not yet been cloned and nothing is known about its protein product. SCA19 Clinical Features SCA19 was identiﬁed in a Dutch family with a mild, progressive ataxic gait disorder, postural tremor, saccadic pursuit, and mild cognitive impairment

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for abstract reasoning (144,145). Onset was between 20 and 45 years of age. Myoclonus (cortical or spinal) was present in 30%. Vibratory and joint position senses were reduced. Deep tendon reﬂexes were reduced in 60%, but increased in 20%. Laboratory Testing Brain MRI shows marked cerebellar hemisphere atrophy, but mild vermian and cerebral atrophy. Transcranial magnetic stimulation shows slightly increased central conduction times. Nerve conduction velocities have occasional mild reduction. Pathology Pathology has not yet been reported. Molecular Genetic Features (See also Table 3) SCA19 has a genetic locus that overlaps the locus for SCA22 and may be allelic. The gene has not yet been cloned and nothing is known about its protein product. SCA20 Clinical Features SCA20 has been described in one Anglo-Celtic family in southeastern Australia (146). Onset was between age 19 and 64 years, average 46 years, but with anticipation of 10 years in the next generation. The most common presenting symptom was dysarthria, followed by gait ataxia and upper limb ataxia. There was slow disease progression, with minimal risk of becoming wheelchair-dependent. Other variable features included palatal tremor or myoclonus, mild pyramidal signs, hypermetric saccades, and mild nystagmus. Laboratory Testing Brain CT scan shows calciﬁcation of the dentate Nucleus and brain MRI scan shows pan-cerebellar atrophy with low signal in the dentate. Nerve conduction studies are normal. Pathology Pathology has not yet been reported. Molecular Genetic Features (See also Table 3) SCA20 maps to a locus near the SCA5 locus. The gene has not yet been cloned and nothing is known about its protein product.

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SCA21 Clinical Features SCA21 has been described in a French family (147). Age of onset was between 6 and 30 years, average 17 years. Anticipation appeared to be present. There was a slowly progressive cerebellar syndrome, affecting gait, limbs (85%), and speech (85%). Extrapyramidal features were seen (akinesia in 80%, rigidity in 30%, and tremor in 60%, non-L-dopa responsive). Deep tendon reﬂexes were reduced in 90%. Cognitive impairment was present in some patients, ranging from mild to severe. Laboratory Testing Brain MRI shows cerebellar atrophy with normal brainstem and basal ganglia. Pathology Pathology has not yet been reported. Molecular Genetic Features (See also Table 3) The SCA21 locus was mapped in 2002 (148). The gene has not yet been cloned and nothing is known about its protein product. SCA22 Clinical Features Reported in a Chinese Han family with a pure cerebellar ataxia (149), this SCA may be allelic to SCA19 (150). Age of onset was similar, with slightly earlier onset reported (age 10–46 years) and more clear evidence for anticipation. It was more purely cerebellar, with slowly progressive gait and truncal ataxia, limb incoordination, scanning speech, dysphagia, and gaze-evoked nystagmus (67%). Hyporeﬂexia was present to a greater extent (89%). Myoclonus, tremor, sensory loss, and cognitive impairment were not seen. Laboratory Testing Brain MRI shows homogeneous cerebellar atrophy with a normal brainstem. Electromyogram and nerve conduction studies are normal. Brainstem auditory and somatosensory evoked potentials are abnormal. Pathology Pathology has not yet been reported. Molecular Genetic Features (See also Table 3) SCA22 has a genetic locus that overlaps the locus for SCA19 and may be allelic.

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The gene has not yet been cloned and nothing is known about its protein product. SCA23 Clinical Features Reported in a Dutch family, SCA 23 is a slowly progressive typical cerebellar ataxia, involving gait, limb, and speech (in 60%), with slow saccades, ocular dysmetria, pyramidal signs (hyperreﬂexia in 80%, extensor plantar responses in 40%), and vibratory sense loss in the legs (60%) (151). Age of onset is 43–56 years, average 50 years. Laboratory Testing Brain MRI shows cerebellar atrophy. Pathology Neuropathological examination in one affected subject showed neuronal loss in the Purkinje cell layer, dentate nuclei and inferior olives; thinning of cerebellopontine tracts; demyelination of posterior and lateral columns in the spinal cord; and ubiquitin-positive intranuclear inclusions in nigral neurons that were considered to be Marinesco bodies. Molecular Genetic Features (See also Table 3) The gene has not yet been cloned and nothing is known about its protein product. SCA24 This section is reserved. SCA25 Clinical Features SCA25 was reported in one family in southern France (152). Age of onset is usually in childhood, but ranges from 17 months to 39 years. Anticipation has not been seen, but incomplete penetrance is reported. Symptom onset is variable within families, but includes progressive ataxia of gait (leading to wheelchair dependence), hypotonia, and nystagmus or slow saccades in some patients. Dysarthria is not seen. However, sensory neuropathy is universal, with vibration loss at the ankles and loss of pain sensitivity in some patients. Deep tendon reﬂexes are absent at the ankles and may be reduced elsewhere. Episodic gastric pain and vomiting are common, possibly on a neuropathic basis.

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Laboratory Testing Brain MRI shows global, severe cerebellar atrophy, with normal brainstem. Nerve conduction velocities show absent sensory action potentials, but normal motor nerve velocities. Pathology Pathology has not yet been reported. Molecular Genetic Features (See also Table 3) The SCA25 locus has been mapped to a region with several candidate genes, none of which yet has been found to have a disease-associated mutation. No pathological expansions were found at the protein or at the DNA level using the 1C2 antibody and the RED method, respectively. The gene has not yet been cloned and nothing is known about its protein product. SCA26 Clinical Features SCA26 was reported in a six-generation family of Norwegian ancestry (153). Age of onset is from 26 to 60 years. No signiﬁcant anticipation has been observed. Symptoms include pure cerebellar ataxia of trunk and limbs, dysarthria, and irregular visual pursuit movements. No motor or sensory deﬁcits, extensor plantar responses, fasciculations, seizures, or cognitive dysfunction have been seen. Laboratory Testing Brain MRI shows cerebellar atrophy, with normal pons and medulla. Pathology Pathology has not yet been reported. Molecular Genetic Features (See also Table 3) The SCA26 locus has been mapped to a region (19p13.3) with approximately 100 known and predicted genes and adjacent to the genes for Cayman ataxia and SCA6. RED analysis did not detect abnormal CAG repeats. The gene has not yet been cloned and nothing is known about its protein product or mechanism of neurodegeneration. SCA Due to FGF14 Clinical Features Described in a Dutch family with ataxia, tremor, and dyskinesia, this SCA was found to result from mutation in the gene for ﬁbroblast growth factor

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14 (FGF14) (154). Onset was in childhood, with tremor of limbs and head, exacerbated by stress and exercise. Ataxia of arms > legs developed in the late teens, then gait ataxia between the ages of 27 and 40. Dysarthria and nystagmus were present, as well as orofacial dyskinesias. Vibratory sensation was reduced at the ankles in some patients. Cognitive impairment was present, and psychiatric episodes (depression, and aggression) occurred in 50% of affected individuals. Gait ataxia was disabling by the seventh or eighth decade. Anticipation was not seen. Laboratory Testing Brain MRI may show cerebellar atrophy. Nerve conduction studies show mild axonal neuropathy. Pathology Pathology is not yet reported. Molecular Genetic Features (See also Table 3) This SCA results from missense or frameshift mutations (e.g., F145S) in the gene for ﬁbroblast growth factor 14, which belongs to a subclass of ﬁbroblast growth factors that are expressed in the developing and adult central nervous system (155). FGF14-deﬁcient mice developed ataxia and a paroxysmal hyperkinetic movement disorder and had reduced responses to dopamine agonists, suggesting a function for FGF14 in neuronal signaling, axonal trafﬁcking, and synaptosomal function (156). CONCLUSION Autosomal dominant cerebellar ataxias constitute one of the most clinically, neuropathologically, and genetically diverse groups of neurodegenerative disorders. Currently available genetic testing can identify the responsible gene mutation in approximately 50–80% of the families with spinocerebellar ataxia. Many new loci have been mapped, often in single families, but the responsible genes have not yet been identiﬁed. Currently, the variety of genetic mutations and pathogenetic mechanisms involved in causing cerebellar degeneration in the SCAs makes it difﬁcult to think toward the design of universally applicable disease-modifying therapies (157). REFERENCES 1. Huynh DP, Yang HT, Vakharia H, Nguyen D, Pulst SM. Expansion of the polyQ repeat in ataxin-2 alters its Golgi localization, disrupts the Golgi complex and causes cell death. Hum Mol Genet 2003; 12:1485–1496. 2. Taroni F, DiDonato S. Pathways to motor incoordination: the inherited ataxias. Nat Rev Neurosci 2004; 5:641–655.

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16 Huntington’s Disease: Mechanisms of Pathogenesis and Development of New Therapies Blair R. Leavitt Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics, and Division of Neurology, Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada

Lynn A. Raymond Department of Psychiatry, Division of Neurology, Department of Medicine, and Brain Research Centre, University of British Columbia, Vancouver, British Columbia, Canada

CLINICAL, PATHOLOGIC, AND GENETIC FEATURES OF HUNTINGTON’S DISEASE Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disorder with an estimated prevalence of 0.5–1 in 10,000 individuals of European descent (1). HD is characterized by the insidious development of motor, cognitive, and psychiatric disturbances with an inexorable progression towards complete disability and eventual death averaging 18 years after the onset of symptoms (2). George Huntington ﬁrst characterized the cardinal features of the disease that now bears his name in a report published in 1872 (3). This concise treatise outlined the distinctive choreiform movement disorder, the hereditary nature of the chorea, and the frequent association of the chorea with psychiatric disease (‘‘a tendency to insanity and suicide’’). In 1983, the genetic defect in HD was mapped to chromosome

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4p16.3 (4), and 10 years later the causative mutation was identiﬁed following a massive effort by a collaborative research consortium (5). HD is caused by an expanded CAG trinucleotide repeat sequence in the HD gene encoding for the huntingtin protein. The movement disorder in HD consists of both abnormal involuntary movements such as chorea or dystonia, and abnormalities of voluntary movement, such as abnormal saccades, bradykinesia, dysphagia, dysarthria, impaired ﬁne motor coordination, and gait disturbance. Functional disability in HD is better correlated with the severity of the voluntary movement impairments than with the severity of the classic ‘‘Huntington’s chorea’’ (6). Early in the course of HD, chorea, hypotonia and hyper-reﬂexia usually predominate, but as the disease progresses, rigidity and bradykinesia become progressively severe (2). Cognitive defects in HD usually progress at a similar rate to the motor disturbances and usually begin with a subtle slowing of intellectual processes, dis-inhibition and reduced mental ﬂexibility, eventually developing into a slowly progressive ‘‘subcortical dementia’’ (7). Aphasia and agnosia are uncommon, whereas a prominent loss of cognitive speed, ﬂexibility, and concentration are the hallmark cognitive changes. Memory recall is generally affected more than memory storage in HD (8). Depression, apathy, and irritable or impulsive behavior are the most common psychiatric manifestations of HD, and the expression of these symptoms varies widely both in onset and in progression (9). Obsessive-compulsive symptoms and frank psychosis also occasionally develop during the course of the illness. Cachexia is an almost invariant feature of HD as the disease progresses, with a marked loss of body weight occurring over time despite maintenance of a previously adequate caloric intake suggesting that an altered metabolic state may be associated with progression of the disease (10). Juvenile-onset HD, sometimes termed the Westphal variant of HD, occurs in about 5% to 10% of patients and is characterized by a predominantly akinetic-rigid presentation with spasticity, bradykinesia, and dystonia (11). Unlike adult-onset HD, the juvenile form is often complicated by myoclonus and seizures, with seizures occurring in up to one-third of juvenile HD patients. In contrast, there is no increased frequency of seizures in adult-onset HD cases compared to the general public (12). Chorea is a variable ﬁnding and may occur early or not at all; when present, it is often gradually superseded by spasticity and rigidity. Parkinsonian features may be the primary abnormalities in those affected under the age of 20 years and can make diagnosis difﬁcult if the family history is not available. Rapid intellectual deterioration, apathy, personality changes, and academic problems at school are common presenting complaints. In general, the larger the CAG repeat expansion and earlier the age of onset of HD, the more likely that rigidity and bradykinesia will be the major neurologic features (13).

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Figure 1 HD is caused by an expanded CAG trinucleotide repeat sequence in exon 1 of the HD gene that encodes for an expanded polyglutamine stretch near the N-terminus of the huntingtin protein. The mutant huntingtin protein is believed to have altered physical and functional properties, which lead to a novel toxic gain of function (and possibly an additional loss of huntingtin’s normal function), resulting in selective neurodegeneration in speciﬁc brain regions. The brain regions which demonstrate the earliest and most extensive neuronal loss in HD include the striatum, and to a lesser extent, the cerebral cortex, globus pallidus, and thalamic and subthalamic nuclei.

The hallmark neuropathologic feature of HD is early neuronal loss in the caudate and putamen (striatum). The medium-sized spiny neurons of the striatum are selectively vulnerable to neurodegeneration in HD (14) (Fig. 1). These neurons receive massive excitatory (glutamatergic) inputs from wide areas of neocortex and thalamus, and make efferent inhibitory projections (GABAergic) to the globus pallidus and substantia nigra (15). The other major cell type of the striatum, the aspiny interneurons, are relatively resistant to degeneration in HD. Loss of pyramidal projection neurons in layers

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V and VI of the cerebral cortex (16) and the CA1 region of hippocampus (17) is also a feature of the selective neuronal cell death in HD (Fig. 1). As the disease progresses, degenerative changes become more generalized and include other brain regions such as the globus pallidus, subthalamic nucleus, substantia nigra, cerebellum, and thalamus (14) (Fig. 1). Nuclear and cytoplasmic intracellular inclusions are also a pathologic feature of HD and the other polyglutamine diseases; these inclusions are insoluble, ubiquitinated protein aggregates that sequester a variety of cellular proteins including full-length and truncated fragments of huntingtin. The presence of neuronal intranuclear huntingtin inclusions was ﬁrst deﬁnitively recognized in transgenic mice (18). Subsequent immuno-histochemical analyses demonstrated the presence of neuronal huntingtin inclusions in post-mortem brain material from HD patients (19) and patients with other polyglutamine disorders (20). Despite the presence of aggregates in these disorders, their role in the pathogenesis of HD is unclear. In juvenile onset HD, the neuropathology is much less speciﬁc, and the degenerative changes occur earlier and are more widespread than in adult-onset cases (including loss of neurons within the cerebellum and brainstem) (1). Intranuclear inclusions are much more common and present from an earlier stage than in adult-onset HD (19). The underlying genetic defect in HD is the expansion of a CAG trinucleotide repeat in the ﬁrst exon of the HD gene that produces a huntingtin protein with an expanded polyglutamine tract (5) (Fig. 1). Expanded HD alleles containing greater than 35 CAG repeats are associated with the eventual development of the clinical phenotype of HD, with earlier age of onset occurring with larger repeat sizes (21). Due to the inverse correlation of CAG repeat length with the age of onset of HD symptoms, CAG repeat sizes between 36 and 39 are associated with very late onset leading to the appearance of reduced penetrance for this repeat range (22). The CAG repeat expansion in the HD gene is a dynamic mutation, and the length of the CAG repeat is unstable during somatic development and during vertical transmission of the gene from parent to child. Larger CAG repeat sizes exhibit greater instability, and paternal transmission of the CAG repeat allele is more likely to result in expansion rather than contraction of the CAG repeat length (23). The unstable nature of the CAG expansion in HD provides the molecular basis for the clinical phenomenon of anticipation, deﬁned as decreasing age of onset, or increasing severity of disease in successive generations. From this brief review of the clinical manifestations, genetics, and neuropathology of HD, it is clear that any proposed mechanisms of HD pathogenesis must include explanations for the earlier age of onset that occurs with larger CAG expansions, age-related factors contributing to the late onset of the disease, the role of huntingtin aggregation and inclusion formation, and the selective neuronal degeneration seen in HD.

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ANIMAL MODELS OF HD Prior to the cloning of the HD gene, the only animal models of HD available were generated by the administration of speciﬁc neurotoxins. Intrastriatal injection of kainate, a glutamate agonist, in rodents induces neurodegeneration of medium spiny neurons similar to that observed in HD (24,25). More speciﬁc striatal lesions that spare the large cholinergic interneurons of the striatum and are also associated with behavioral features characteristic of HD are produced by intrastriatal injections of the selective NMDA receptor agonist quinolinic acid into rodents and primates (26–30). Similar striatal lesions are induced by inhibition of mitochondrial function with toxins such as 3-nitroporoprionic acid or malonate (31,32). Importantly, the neurodegenerative effects of these mitochondrial toxins can be blocked by pretreatment with NMDA receptor antagonists such as MK801 or APV leading to the hypothesis that they induce neuronal death by a mechanism of ‘‘indirect excitoxicity’’ (33). In general, HD models generated by administration of neurotoxins have provided important insights into the basic mechanisms of neuronal cell death; however, more genetically accurate transgenic models of HD now exist. Genetic Mouse Models of HD Following the identiﬁcation of the causative CAG repeat expansion mutation in the HD gene, three independent groups created ‘‘knock-out’’ mice with targeted disruption of Hdh, the mouse HD gene (34–36). The huntingtin knock-out was embryonic lethal, clearly not a model for HD, and suggested that huntingtin is essential for proceeding through gastrulation during embryonic development. Interestingly, one group reported that mice heterozygous for targeted disruption of the Hdh gene and expressing only half the normal levels of wild-type htt, develop late-onset neuronal degeneration in the basal ganglia in adulthood (35). This result was later conﬁrmed in these mice based on a detailed sterological anaylsis (37). Targeted partial disruption of the Hdh gene leading to dramatically reduced huntingtin levels leads to reduced body size, movement abnormalities, and increased ventricular volume in mice (38). Furthermore, post-natal conditional deletion of the Hdh gene in mice caused neurodegeneration and a motor phenotype that is similar to that described in transgenic mouse models of HD (39). These studies and a variety of other evidence suggest that the loss of wild-type htt function may play a role in the selective pattern and timing of neurodegeneration in HD (40). The CAG expansion in HD and other polyglutamine disorders is generally accepted to primarily result in a toxic gain of function of the resultant proteins. The proposed gain of function mechanism for the HD mutation suggests that the creation of an accurate mouse model replicating the phenotype and neuropathology of HD requires the stable introduction of

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a mutant copy of the HD gene into the genome of mice. Various lines of transgenic mice expressing different forms of the mutant HD gene have been generated and will be discussed below: (i) mice expressing a fragment of transgenic mutant htt, (ii) mice expressing full-length transgenic mutant htt, and (iii) mice with a modiﬁed Hdh gene containing an expanded CAG repeat region (‘‘htt knock-in’’ mice). A summary of the current mouse models of HD is updated regularly and maintained on the website of the Hereditary Disease Foundation: http://www.hdfoundation.org/ PDF/hdmicetable.pdf. Fragment HD Transgenic Mice The HD gene contains 67 exons and spans an enormous genomic distance of over 210 kb, making it very difﬁcult to manipulate the full-length gene using standard transgenic techniques (41). The CAG trinucleotide repeat is present in exon 1 of the HD gene, and several groups have produced HD mouse models that are transgenic for small fragments of the HD gene, including exon 1. One of the most important breakthroughs in HD research came with the development of transgenic mice expressing exon 1 of the HD gene with 115–156 repeats under control of the ﬁrst 1 kb of HD regulatory gene sequences (42). These R6 lines of mice express the ﬁrst 69 amino acids of human htt, representing 3% of the total protein at various levels (R6/1, R6/2, and R6/5), and develop a progressive neurological phenotype—with the highest levels of transgenic protein corresponding to earliest onset of phenotype in the R6/2 line. The most extensively studied line (R6/2) has motor deﬁcits (gait and rotarod testing) as early as ﬁve weeks of age followed by progressive weight loss, clasping, tremor, convulsions, and diabetes with death occurring at 10–14 weeks (43). Despite the severe behavioral phenotype, the brains of R6/2 mice do not have evidence of selective neuronal loss in the striatum or cortex, although overall brain size is reduced by 20% with some striatal atrophy. Pathological studies of these mice using an N-terminal anti-huntingtin antibody identiﬁed the presence of immunoreactive neuronal intranuclear inclusions (18). This ﬁnding led directly to the ﬁrst identiﬁcation of intranuclear htt inclusions in human HD brain tissue (19). A cDNA construct encoding an N-terminal fragment (171 residues) of htt with 82 glutamine repeats under the prion protein promoter was used to create another fragment HD model (44). These transgenic mice also develop motor dysfunction, htt inclusions, diabetes and early death, although they express lower levels of the mutant htt fragment than R6/2, the phenotype is more variable, and the progression more protracted (45). A conditional transgenic model was developed by using an exon 1 construct with 94 CAG repeats under the control of the tetracycline-regulatable system, which enables the expression of the transgenic protein to be turned off (46). When the mutant exon 1 htt transgene is expressed, these mice develop nuclear

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aggregates and motor anomalies similar to the other exon 1 models, but these changes are reversible when transgene expression is shut down. This model provides convincing evidence that some elements of the motor phenotype and htt inclusions are dynamic, and further suggest that decreasing levels of mutant protein will improve or even reverse symptoms of HD. Full-Length HD Transgenic Mice Yeast artiﬁcial chromosomes (YACs) containing the entire genomic region of the full-length human HD gene including all endogenous regulatory elements were used to generate a YAC transgenic mouse model of HD (47). These mice express human transgenic htt with 18, 46, or 72 polyglutamine repeats in a normal developmental and cell-speciﬁc regulation pattern. Unlike other transgenic HD mice, YAC transgenic mice rescue the embryonic lethality of the htt knock-out, providing deﬁnitive evidence that the mutant protein retains the developmental function of wild-type htt (48). Over a prolonged 12-month timeline, YAC72 mice develop a progressive motor phenotype, neuronal dysfunction, and selective striatal neurodegeneration similar to that seen in HD (47). The phenotype and neuronal loss precede the development of classic nuclear htt inclusions in these mice, providing evidence that inclusions are not required for initiation of the HD pathogenic process. Mice were also generated expressing full-length mutant htt from a cDNA construct with 48 or 89 CAG repeats under control of the CMV promoter, developing a slow motor and neuropathologic phenotype (49). More recently, a YAC128 mouse model has been developed that expresses high levels of mutant htt with 128 polyglutamines, and develops a more rapid and consistent phenotype, making this line of transgenic mice extremely useful for pre-clinical experimental therapeutic trials (50). Knock-in HD Transgenic Mice In theory, transgenic mouse models of HD generated by the selective introduction of CAG expansions into the mouse Hdh gene (HD knock-in models) should represent the ideal genetic model of HD. To date, four knock-in mouse models of HD have been published (51–54). Unfortunately, while being accurate genetic models of HD, the various knock-in HD mouse models have modest and inconsistent behavioral phenotypes. Neuropathological changes in knock-in models include robust nuclear htt inclusion formation with aging, but none of these models have convincing evidence for selective striatal or cortical neurodegeneration. Invertebrate Models of HD In addition to mammalian transgenic HD animal models, a number of groups have made ﬂy (Drosophila melanogaster) and worm (Caenorhabditis elegans) models of HD (55–61). These models share some of the basic

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features identiﬁed in mammalian models of HD, including evidence of abnormal behavioral phenotypes, neuronal dysfunction, neuronal death, and inclusion formation. Genetic screens can be rapidly performed in these lower model organisms and have the potential to identify novel pathways and mechanisms involved in basic polyglutamine pathogenesis (61). However, the signiﬁcant differences between the nervous systems of these organisms and mice and man suggest that caution be employed before results from these approaches are applied to mammalian systems. Any ﬁndings from ﬂies, worms, or other non-mammalian model systems will need extensive further characterization and conﬁrmation in mammalian HD models.

PATHOGENESIS OF HD Role of Gain/Loss in Function of Huntingtin Data suggest that HD pathogenesis results from the combined effects of loss-of-function of wild-type htt and a gain-in-function of huntingtin (htt) with the expanded polyglutamine (polyQ) tract, referred to hereafter as mutant htt. Wild-type htt demonstrates anti-apoptotic properties in vitro (62), as well as in mouse peripheral tissues in vivo (63). Some of these effects can be explained by wild-type htt’s positive regulation of brain-derived neurotrophic factor (BDNF) expression, via sequestration of the transcriptional repressor REST/NRSF (64,65). Since BDNF is a pro-survival factor for striatal neurons, the loss of wild-type htt expression in HD mice models may contribute to disease pathogenesis (66). Consistent with this hypothesis, depletion of wild-type htt in adult mouse forebrain results in neurodegeneration (39). On the other hand, humans with deletions in the HD gene (67), as well as mice deﬁcient in htt expression (34–36), do not exhibit an HD-like phenotype; moreover, a single copy of mutant HD can rescue embryonic lethality in htt-deﬁcient mice (68). These genetic data suggest that loss of wild-type htt function cannot fully explain HD pathogenesis. A toxic gain-in-function in mutant htt may result from a conformational change induced by the polyQ expansion, leading to novel interactions with intracellular proteins and/or facilitating self-association (69,70). Aggregates of short (40 kDa) N-terminal fragments of polyQ-expanded htt have been demonstrated in the brains of humans dying with HD, as well as in transgenic mice models of HD, and in cell cultures of mutant htt-transfected primary neurons and cell lines (18,19,71–73). These aggregates can be found in both cytoplasm and nucleus, and are often ubiquitinated in neurons of the cortex and neostriatum (19). Neuritic aggregates have been shown to impair neurotransmitter release (74,75) as well as axonal transport (76,77), although one study suggested that mutant htt fragments were sufﬁcient, in the absence of aggregatation, for impairment of axonal transport (78). As well, other cellular proteins can be found associated with these aggregates,

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including chaperone proteins and constituents of proteasomes (79), and depletion of such proteins from the cytosol may contribute to cellular dysfunction. Whereas evidence suggests that cytoplasmic aggregates impair neuronal function, nuclear localization of mutant htt fragments is particularly toxic to cells (80,81). Such nuclear fragments can sequester transcription factors (82), altering the balance of gene transcription (83,84). Proteolysis and Aggregation of Huntingtin Although the discovery of mutant htt aggregates has triggered a high interest in developing therapies to slow or prevent aggregation, the role of aggregates in HD pathogenesis is far from clear. For example, the distribution of htt aggregates visible at the light microscope level in HD human and mouse brain is not well correlated with neuronal loss (47,85,86). In fact, certain manipulations that decrease mutant htt ubiquitination, and/or the appearance of aggregates visible at the light microscope level, actually promote cellular toxicity (80,87). On the other hand, these results may be explained by the proposal that aggregation at the submicroscopic level, in the form of soluble oligomers, is more toxic to cells (88,89). As well, two studies show a clear correlation between approaches that decrease brain aggregate load and improved survival in the R6/2 mouse model (46,90). Evidence suggests that mutant htt must ﬁrst be cleaved to shorter fragments in order to translocate to the nucleus, and that cleavage also facilitates aggregate formation (73,91,92). Huntingtin proteolysis is mediated in part by activation of the proapoptotic cysteine proteases caspases-2, -3, -6, and -7 (93–97) and by activation of the calpains, a Ca2þ-dependent family of enzymes (96,98,99). Other studies also support a critical role for caspases in HD pathogenesis. For example, caspase-1 inhibition slows progression in the R6/2 mouse model of HD (100,101), and caspase-8 is activated by interactions with expanded polyQ tracts in vitro and is found in the activated form in the brains of patients with HD (102). These studies indicate that caspase and calpain activitation, as well as huntingtin cleavage and/or aggregation, may play important roles in HD pathogenesis. Protein Interactions and Cellular Dysfunction in HD Mice Models The molecular triggers of caspase and calpain activations, as well as huntingtin proteolysis, are not well understood. Moreover, since huntingtin is widely expressed in the brain, as are the proteases that cleave it, the basis for selective neuronal vulnerability remains unexplained. It is possible that proteins interacting with huntingtin in a polyglutamine-dependent manner confer selectivity due to differential expression patterns. Huntingtinassociated protein 1 (HAP1) and huntingtin-interacting protein 1 (HIP1) show increased and decreased strength, respectively, of association with huntingtin as polyQ length increases (103,104). Both are enriched in brain

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but their distribution does not correlate with selective neuronal vulnerability in HD. HAP1 is associated with motor proteins involved in microtubuledependent intracellular transport (105) and synaptic vesicle release (75), functions found to be altered in HD mice models (74–77). HIP1 is involved in clathrin-mediated endocytosis, suggesting that surface protein and receptor trafﬁcking may also be altered in HD (106,107). In addition to HAP1 and HIP1, huntingtin interacts with a ubiquitin conjugating enzyme (108) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (109), as well as several members of the WW domain family of proteins (110); the latter are involved in regulation of cellular signaling pathways, channel function, and protein processing. Together, these ﬁndings suggest that mutant htt may disrupt a wide variety of cellular functions but still do not explain the pattern of neuronal degeneration found in HD. Excitotoxicity and Selective Neuronal Degeneration in HD Excitotoxicity is a term used to describe excessive neuronal activation by the transmitter glutamate, leading to cell stress and/or death from inﬂux of sodium, calcium and water; the N-methyl-D-aspartate type glutamate receptor (NMDAR) plays a major role in mediating this process (for review see Ref. 111). Before the HD gene was identiﬁed, it was discovered that excitotoxins could reproduce many of the neurochemical, neuropathological and behavioural features of HD in rodents and non-human primates (see section on ‘‘Animal Models of HD’’). Systemic injection of the mitochondrial toxins 3-nitropropionic acid or malonate produced a similar pattern of striatal neuronal degeneration that was dramatically attenuated by NMDAR antagonists (112,113) (see section on ‘‘Animal Models of HD’’). These data suggest that mitochondria in striatal MSNs are more susceptible to injury than those in other neuronal populations and brain regions, consistent with recent data (114), and that reduced mitochondrial function—including less buffering capacity for calcium and free radicals, as well as lower energy stores—exacerbates toxicity induced by NMDAR activation. Moreover, in autopsy material from brains of humans with HD, there is a disproportionate loss of striatal NMDARs, suggesting that neurons with high expression of these receptors are more vulnerable to degeneration (115,116). Recent studies in the YAC transgenic mouse model expressing fulllength human htt with 46Q or 72Q (YAC46 and YAC72, respectively— see section on ‘‘Genetic Mouse Models of HD’’) support the notion that over-activation of NMDARs contributes to the selective neuronal degeneration found in HD. Increased susceptibility to NMDAR-mediated neuronal death was demonstrated in vivo by intrastriatal injection of the NMDAR agonist quinolinate, and in vitro in dissociated striatal neurons cultured from neonatal animals exposed to NMDA (117). Moreover, electrophysiological recordings showed increased NMDAR current density, and NMDA

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application produced enhanced intracellular calcium responses as well as mitochondrial depolarization (a marker of cell stress) for striatal neurons from YAC72 and YAC46 mice compared to wild-type controls (117,118). NMDA also induced dramatically higher levels of caspase-3 activity in striatal neurons from YAC72 and YAC46 mice compared with wild-type mice (117,118), and glutamate has been shown to trigger cleavage of htt via calpain activation in a clonal striatal cell line (99). These results suggest a link between NMDAR-mediated neuronal stress and htt proteolysis. Notably, striatal MSNs express a higher proportion of NR2B-type NMDARs compared with other brain regions (119–122), and mutant htt has been shown to selectively potentiate current and toxicity mediated by NMDARs containing NR2B and not NR2A in a transfected non-neuronal cell line (123,124). Together, these data suggest that an interaction between mutant htt and NR2B-containing NMDARs could explain, in part, why striatal MSNs are targeted in HD. In contrast to results in the YAC transgenic mice, some of the other mice models of HD that express only short fragments of htt have not shown enhanced vulnerability to NMDAR-mediated excitotoxicity (125,126). However, at least in one model (R6/2), striatal neurons were also more resistant to oxidative stress induced by a variety of other stimuli (127,128), and the development of resistance to NMDAR-mediated toxicity in these mice was well correlated with changes in intracellular calcium handling and aggregate load (129). Consistent with the lack of enhanced vulnerability to excitotoxicity, striatal neurons from the R6/2 mice showed no signiﬁcant difference in NMDAR-mediated current density compared with wild-type mice when recordings were made in the absence of magnesium, although a subgroup of neurons exhibited reduced magnesium sensitivity, suggesting changes in NMDAR subunit composition in this mouse model (130). These data suggest that enhanced NMDAR-induced excitotoxicity is associated with expression of the full-length human HD gene. The results are also consistent with the idea that excitotoxicity is involved in selective neuronal degeneration, since R6/2 mice exhibit widespread htt aggregates and brain atrophy but no selective neuronal degeneration (18), whereas the YAC transgenic mice show selective degeneration of mainly striatal neurons with few aggregates, similar to the neuropathology observed in human adult-onset HD (47,50). In addition to the strong evidence for enhanced NMDAR function, there are data to indicate that other pathways contribute to altered neuronal calcium homeostasis. Mitochondria isolated from ﬁbroblasts of humans with HD or the brains of mutant YAC transgenic mice exhibit more depolarized resting membrane potentials and lower thresholds for the calcium-induced permeability transition pore formation associated with apoptosis (131). These changes in mitochondrial function suggest that the toxic effects of stimuli that increase neuronal intracellular calcium, including excitotoxicity,

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will be enhanced in the presence of mutant htt. As well, it has recently been demonstrated that mitochondria isolated from rat striatum show increased sensitivity to calcium-induced depolarization compared with mitochondria from rat cortex, suggesting an additional mechanism for selective striatal degeneration in HD (114). The high level of dopamine release in the neostriatum may also contribute to enhanced oxidative stress and selective neuronal degeneration (132). Furthermore, recent studies show evidence of increased glutamate release from cortical afferents to striatal neurons in the early symptomatic stage of disease in the R6/2 mice (133), as well as an interaction between mutant htt and inositol-3-phosphate receptors to increase receptor sensitivity for inducing calcium release from intracellular stores (134). Figure 2 illustrates many of the changes in neuronal function observed with expression of mutant htt, as discussed in this section. Clearly

Figure 2 Cartoon representing some of the changes observed in neuronal function with expression of mutant huntingtin. Many of the changes shown in the medium spiny striatal neuron converge to alter intracellular calcium homeostasis and promote proteolysis and initiation of cell death pathways. Additional changes are observed more generally, in a variety of neuronal and other cell types, including proteasomal dysfunction and impaired axonal transport (depicted in the cortical neuron), as well as transcriptional dysregulation (not shown).

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a better understanding of the factors inﬂuencing selective neuronal degeneration, as well as the signaling cascades involved in neuronal dysfunction/ death triggered by excitotoxicity, will facilitate future development of more effective therapies in HD. PRECLINICAL TRIALS IN MOUSE MODELS OF HD The development of transgenic mouse models of HD is a critical step in accelerating the development of new therapeutic approaches for this devastating neurodegenerative disease. The majority of therapeutic trials in mice to date have been performed in exon 1 htt fragment mouse models, (45,135). Pre-clinical trials have attempted to interfere with the pathogenic processes described above: htt proteolysis, htt aggregation and transcriptional dysregulation, excitotoxicity, and other less well-established mechanisms. Evidence that inhibiting caspase-dependent proteolysis and apoptosis in HD may be beneﬁcial was provided by a study on the treatment of R6/2 mice with either a broad-spectrum caspase inhibitor (zVAD-fmk) or by crossing these mice to mice with a dominant negative caspase 1 inhibitor which improved motor performance and survival (100). Minocycline, a tetracycline analogue that has been proposed to act as a caspase inhibitor in addition to potential therapeutic effects blocking mitochondrial apoptotic mechanisms and inﬂammation, was also reported to improve motor function and prolong survival in R6/2 mice. Several recent trials have failed to replicate this protective effect in this mouse model and in the N171– 82Q model, leaving the effectiveness of this agent in question (136,137). The bile acid taurosodeoxycholic acid inhibits apoptosis by unknown mechanisms, and was effective in improving motor performance and striatal atrophy in R6/2 mice, although no effect on survival was reported (138). Aggregation of htt, formation of intranuclear inclusions, and transcriptional dysregulation have also been identiﬁed as potential therapeutic targets in HD (83), and several trials in R6/2 mice have suggested that interference with this mechanism is protective in this model. Systemic treatment of R6/2 mice with congo red, a histochemical dye that blocks aggregation of htt and other misfolded proteins in vitro, resulted in modest increases in survival and decreased htt aggregate burden (90). A recent report of beneﬁt on rotarod performance, weight, and survival following addition of a disaccharide, trehalose, to the drinking water of R6/2 mice is also suggested to be via inhibition of htt aggregation (139). The histone deacetylase (HDAC) inhibitors suberoylanilide hydroxyamic acid (SAHA) and sodium butyrate, are thought to modulate gene transcription and possibly reverse the transcriptional dysregulation caused by intranuclear htt inclusions. Therapeutic trials of these agents have prevented neurodegeneration in a polyglutamine ﬂy model (58) and have also shown modest improvements in the motor

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phenotype and prolonged survival of R6/2 mice (140,141). Taken together these trials suggest that blockade of htt aggregation or the effects of intranuclear inclusions on transcription may be an effective therapeutic approach worth pursuing in HD. A number of agents which are proposed to improve mitochondrial function and/or block NMDAR-mediated excitotoxicity have been tested in mouse models of HD with varying degrees of efﬁcacy. Dichloroacetate treatment improved survival in both the N171–82Q and R6/2 mice, possibly by increasing respiratory chain efﬁciency (142). The respiratory chain cofactors creatine and Co-enzyme Q10 are two of the best studied agents in mouse models of HD and have shown improved motor performance and survival in both the R6/2 and N171–82Q mouse models, both alone and in combination with the NMDA receptor antagonist remacemide (143–145). Unfortunately, the CARE-HD trial failed to demonstrate a functional beneﬁt for this regimen over three years in a large cohort of HD patients (discussed further). Two other drugs currently approved for use in humans for other disorders have also shown promise in the R6/2 model; Riluzole—a glutamate release blocker (146), and lithium chloride—which protects from apoptosis by increasing Bcl-2 levels (147). Other potential mechanisms of HD pathogenesis that have been targeted in pre-clinical R6/2 mouse trials include: inﬂammation (148), transglutaminase activity (149,150), and oxidative stress (148,151).

CLINICAL TRIALS IN HD Treatments to Delay Onset or Slow Progression of HD Results of in vitro studies have suggested a variety of therapeutic targets for delaying onset and/or slowing progression of HD, including: suppressing glutamatergic neurotransmission and especially that mediated by subtypes of NMDA receptors, improving mitochondrial function and cellular energy levels, buffering free radicals to reduce oxidative stress, reducing inﬂammatory responses and protease activation, interfering with htt proteolysis and/ or aggregation, and restoring balance for transcriptional regulation. Several of these approaches have met with success in preclinical trials in HD mice and ﬂy models (see above). However, only a few agents have made it through the ﬁrst stages of human clinical testing. The largest randomized, placebo-controlled trial to date of agents that reduce excitotoxicity and improve mitochondrial function was CARE-HD, testing remacemide (a use-dependent NMDAR antagonist) and Coenzyme Q10, alone and in combination (152). This trial included 340 subjects followed over 2.5 years. Although Coenzyme Q10 appeared to slow progression by 13% in the early stages of HD as assessed by a functional scale, this difference was not statistically signiﬁcant; remacemide reduced chorea but

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produced no change in rate of functional decline. Further study of Coenzyme Q10 is planned, to include a higher dose, larger number of subjects and longer duration of study. Agents that suppress excitotoxicity by reducing glutamate release, such as lamotrigine and riluzole, have also been studied. In a relatively small, randomized controlled trial (RCT), lamotrigine slowed progression over a 30-month period by ~10% on a functional scale, but the result was not signiﬁcant (153). Riluzole reduced chorea in an 8-week tolerability study (154), and is currently under study in a larger RCT in Europe for efﬁcacy in slowing progression on a functional scale. Creatine (5–10 gm/day) and OPC-14117, which improve cellular energy stores and reduce oxidative stress, respectively, have been shown to be tolerated by subjects with HD (155–157), but in one-year trials creatine has not shown efﬁcacy in slowing disease progression (155,156). Finally, minocycline, which reduces inﬂammatory responses mediated by glia and also shows efﬁcacy in suppressing apoptotic neuronal death in vitro, has undergone a brief RCT for tolerability in subjects with HD (169), with plans for a larger RCT. Symptomatic Treatments Although no form of therapy has yet been shown to signiﬁcantly alter the natural progression of HD, many of the motor and psychiatric symptoms of HD can be ameliorated by judicious intervention. Currently, there are no effective drug interventions that improve cognitive impairment in HD. Most of the current symptomatic treatment recommendations for the practical symptomatic management of HD are not evidence-based, and are derived from clinical experience, case reports, or small randomized trials (158). While the gait disorder, incoordination and voluntary movement disorder in HD have no effective therapy at present, several agents are available which can suppress involuntary movements in HD. The pharmacologic treatment of chorea is best undertaken only when the involuntary movements become severe enough to signiﬁcantly impact functional activity. Drugs that act to decrease dopaminergic neurotransmission such as the neuroleptics (classic and atypical), and the dopamine-depleting agent tetrabenezine, are useful in reducing chorea. However, the risks of potential adverse side effects (such as depression induced by tetrabenezine) must be balanced against the potential beneﬁts of suppressing chorea. Haloperidol has long been considered the ﬁrst-line therapy for chorea in HD, and while no adequate clinical trials have been performed, it has empirically been found to supress choreiform movements in HD patients (159,160). However, in addition to dose-related side effects such as sedation, treatment of chorea with classic neuroleptics carries a signiﬁcant hazard of adverse effects, notably dystonia, parkinsonism, and tardive dyskinesia (161).

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The atypical neuroleptic olanzepine is our currently recommended agent for the suppression of chorea in HD. Symptomatic beneﬁt in HD with olanzapine therapy has been reported in several case reports (162–164), as well as in two small open-label trials (165,166), although high doses up to 30 mg per day may be required to adequately suppress chorea. The mechanism of action by which olanzapine has its anti-choreic effects is unknown, but it may act through a glutaminergic mechanism, or by action at adrenergic, muscarinic, seratonin, dopamine, or histamine receptors (167). Compared with conventional antipsychotics such as haloperidol, the use of olanzapine is associated with fewer and less severe adverse effects (161). In HD, the lack of signiﬁcant extrapyramidal side effects is the most important beneﬁt of olanzepine over the classic neuroleptics. Olanzepine does have dose-related sedative effects which can limit use, and causes increased appetite and weight gain in up to 30% of patients. In our clinical experience the increased weight gain is a common side effect of olanzepine in HD patients, and is beneﬁcial for those patients with cachexia. Based on our clinical experience, standard pharmacotherapy is generally very effective for the common neuropsychiatric manifestations in HD, although good clinical trial data is again lacking to support this indication (158). Most depressed or obsessive-compulsive HD patients respond well to standard doses of tricyclic, SSRI or atypical antidepressant medications. Benzodiazepines can be useful adjuncts in anxious or agitated patients, and psychotic symptoms generally respond well to standard doses of either typical or atypical neuroleptics (159). The approach to symptomatic treatment in HD ideally involves the coordinated efforts of a multidisciplinary team composed of medical, nursing, social service, and physical/occupational therapy modalities (159). Medications can be very helpful in suppressing speciﬁc symptoms at one stage of the illness, but may have a detrimental effect at later stages and need to be constantly re-evaluated as the natural history of the disease evolves. HD patients tend to be acutely susceptible to the cognitive and motor side effects of many medications. Ideally, symptomatic drug treatment should only be initiated when symptoms begin to impair function, initial doses should be low and titrated gradually based on clinical response, and in general polypharmacy should be avoided. Many patients also obtain beneﬁt from physical, speech, and occupational therapy. As dysphagia becomes more severe during the progression of the disease, a formal swallowing evaluation can yield practical suggestions for decreasing the risk of aspiration. Eventually, feeding via a gastrostomy tube will need to be presented as an option to the patient and family. Supportive family-based counseling is also a critical component of HD care. In addition to the affected patient, family members at risk and the unaffected spouse often have signiﬁcant emotional problems that require speciﬁc interventions and support.

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CONCLUSIONS Since the identiﬁcation of the HD gene in 1993, the establishment of cellular and animal models of HD has rapidly accelerated knowledge of the pathogenic mechanisms. Based on studies in these models, a variety of potential therapeutic agents have been identiﬁed. The challenge for the near future is to select the agents that will be brought forward to human clinical trials. It will be critical to choose well because of the large number of subjects, as well as amount of time and funding required to prove efﬁcacy in slowing progression of HD. Moreover, a wide variety of HD animal models are currently available, each with unique features that are similar as well as distinct from characteristics of human HD. Making decisions on therapeutic agents based on experimental therapeutic trials in individual animal models is difﬁcult, and new therapeutic agents should have proven efﬁcacy in several animal models prior to moving to human trials (168). To streamline this process, a centralized system for prioritizing agents for clinical testing has been developed (SET-HD; www.huntingtonproject.org). With this wealth of information and potential therapeutic agents, the future for therapy in HD looks promising. REFERENCES 1. Harper PS. Huntington’s Disease: London: WB Saunders Co, 1991. 2. Hayden MR. Huntington’s Chorea. Berlin: Springer, 1981. 3. Huntington G. On Chorea. The Medical and Surgical Reporter: A Weekly Journal 1872; 26(15):317–321. 4. Gusella JF, Wexler NS, et al. A polymorphic DNA marker genetically linked to Huntington’s disease. Nature 1983; 306(5940):234–238. 5. Huntington’s Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 1993; 72:971–983. 6. Feigin A, Kieburtz K, et al. Functional decline in Huntington’s disease. Mov Disord 1995; 10(2):211–214. 7. Zakzanis KK. The subcortical dementia of Huntington’s disease. J Clin Exp Neuropsychol 1998; 20(4):565–578. 8. Butters N, Wolfe J, Martone M, Granholm E, Cermak LS. Memory disorders associated with Huntington’s disease: verbal recall, verbal recognition and procedural memory. Neuropsychologia 1985; 23(6):729–743. 9. Anderson KE, Marder KS. An overview of psychiatric symptoms in Huntington’s disease. Curr Psychiatry Rep 2001; 3(5):379–388. 10. Sanberg PR, Fibiger HC, Mark RF. Body weight and dietary factors in Huntington’s disease patients compared with matched controls. Med J Aust 1981; 1(8):407–409. 11. Bittenbender JB, Quadfasel FA. Rigid and akinetic forms of Huntington’s chorea. Arch Neurol 1962; 7:275–288.

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119. Landwehrmeyer GB, Standaert DG, Testa CM, Penney JB Jr, Young AB. NMDA receptor subunit mRNA expression by projection neurons and interneurons in rat striatum. J Neurosci 1995; 15(7 Pt 2):5297–5307. 120. Kuppenbender KD, Standaert DG, Feuerstein TJ, Penney JB Jr, Young AB, Landwehrmeyer GB. Expression of NMDA receptor subunit mRNAs in neurochemically identiﬁed projection and interneurons in the human striatum. J Comp Neurol 2000; 419(4):407–421. 121. Christie JM, Jane DE, Monaghan DT. Native N-methyl-D-aspartate receptors containing NR2A and NR2B subunits have pharmacologically distinct competitive antagonist binding sites. J Pharmacol Exp Ther 2000; 292(3): 1169–1174. 122. Li L, Fan M, et al. Role of NR2B-type NMDA receptors in selective neurodegeneration in Huntington’s disease. Neurobiol Aging 2003; 24(8):1113–1121. 123. Chen N, Luo T, et al. Subtype-speciﬁc enhancement of NMDA receptor currents by mutant huntingtin. J Neurochem 1999; 72(5):1890–1898. 124. Zeron MM, Chen N, et al. Mutant huntingtin enhances excitotoxic cell death. Mol Cell Neurosci 2001; 17(1):41–53. 125. Petersen A, Chase K, Puschban Z, DiFiglia M, Brundin P, Aronin N. Maintenance of susceptibility to neurodegeneration following intrastriatal injections of quinolinic acid in a new transgenic mouse model of Huntington’s disease. Exp Neurol 2002; 175(1):297–300. 126. Hansson O, Peters n A, Leist M, Nicotera P, Castilho RF, Brundin P. Transgenic mice expressing a Huntington’s disease mutation are resistant to quinolinic acid-induced striatal excitotoxicity. Proc Natl Acad Sci USA 1999; 96(15):8727–8732. 127. Petersen A, Hansson O, et al. Mice transgenic for exon 1 of the Huntington’s disease gene display reduced striatal sensitivity to neurotoxicity induced by dopamine and 6- hydroxydopamine. Eur J Neurosci 2001; 14(9):1425–1435. 128. Hansson O, Castilho RF, Korhonen L, Lindholm D, Bates GP, Brundin P. Partial resistance to malonate-induced striatal cell death in transgenic mouse models of Huntington’s disease is dependent on age and CAG repeat length. J Neurochem 2001; 78(4):694–703. 129. Hansson O, Guatteo E, et al. Resistance to NMDA toxicity correlates with appearance of nuclear inclusions, behavioural deﬁcits and changes in calcium homeostasis in mice transgenic for exon 1 of the huntington gene. Eur J Neurosci 2001; 14(9):1492–1504. 130. Cepeda C, Ariano MA, et al. NMDA receptor function in mouse models of Huntington’s disease. J Neurosci Res 2001; 66(4):525–539. 131. Panov AV, Gutekunst CA, et al. Early mitochondrial calcium defects in Huntington’s disease are a direct effect of polyglutamines. Nat Neurosci 2002; 5(8):731–736. 132. Petersen A, Mani K, Brundin P. Recent advances on the pathogenesis of Huntington’s disease. Exp Neurol 1999; 157(1):1–18. 133. Cepeda C, Hurst RS, et al. Transient and progressive electrophysiological alterations in the corticostriatal pathway in a mouse model of Huntington’s disease. J Neurosci 2003; 23(3):961–969.

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134. Tang TS, Tu H, et al. Huntingtin and huntingtin-associated protein 1 inﬂuence neuronal calcium signaling mediated by inositol-(1,4,5) triphosphate receptor type 1. Neuron 2003; 39(2):227–239. 135. Schilling G, Savonenko AV, et al. Environmental, pharmacological, and genetic modulation of the HD phenotype in transgenic mice. Exp Neurol 2004; 187(1):137–149. 136. Smith DL, Woodman B, et al. Minocycline and doxycycline are not beneﬁcial in a model of Huntington’s disease. Ann Neurol 2003; 54(2):186–196. 137. Diguet E, Rouland R, Tison F. Minocycline is not beneﬁcial in a phenotypic mouse model of Huntington’s disease. Ann Neurol 2003; 54(6):841–842. 138. Keene CD, Rodrigues CM, Eich T, Chhabra MS, Steer CJ, Low WC. Tauroursodeoxycholic acid, a bile acid, is neuroprotective in a transgenic animal model of Huntington’s disease. Proc Natl Acad Sci USA 2002; 99(16): 10671–10676. 139. Tanaka M, Machida Y, et al. Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington’s disease. Nat Med 2004; 10(2): 148–154. 140. Ferrante RJ, Kubilus JK, et al. Histone deacetylase inhibition by sodium butyrate chemotherapy ameliorates the neurodegenerative phenotype in Huntington’s disease mice. J Neurosci 2003; 23(28):9418–9427. 141. Hockly E, Richon VM, et al. Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deﬁcits in a mouse model of Huntington’s disease. Proc Natl Acad Sci U.S.A. 2003; 100(4):2041–2046. 142. Andreassen OA, Ferrante RJ, et al. Dichloroacetate exerts therapeutic effects in transgenic mouse models of Huntington’s disease. Ann Neurol 2001; 50(1): 112–117. 143. Ferrante RJ, Andreassen OA, et al. Neuroprotective effects of creatine in a transgenic mouse model of Huntington’s disease. J Neurosci 2000; 20(12):4389–4397. 144. Ferrante RJ, Andreassen OA, et al. Therapeutic effects of coenzyme Q10 and remacemide in transgenic mouse models of Huntington’s disease. J Neurosci 2002; 22(5):1592–1599. 145. Andreassen OA, Dedeoglu A, et al. Creatine increase survival and delays motor symptoms in a transgenic animal model of Huntington’s disease. Neurobiol Dis 2001; 8(3):479–491. 146. Schiefer J, Landwehrmeyer GB, et al. Riluzole prolongs survival time and alters nuclear inclusion formation in a transgenic mouse model of Huntington’s disease. Mov Disord 2002; 17(4):748–757. 147. Wood NI, Morton AJ. Chronic lithium chloride treatment has variable effects on motor behaviour and survival of mice transgenic for the Huntington’s disease mutation. Brain Res Bull 2003; 61(4):375–383. 148. Andreassen OA, Ferrante RJ, Dedeoglu A, Beal MF. Lipoic acid improves survival in transgenic mouse models of Huntington’s disease. Neuroreport 2001; 12(15):3371–3373. 149. Karpuj MV, Becher MW, et al. Prolonged survival and decreased abnormal movements in transgenic model of Huntington’s disease, with administration of the transglutaminase inhibitor cystamine. Nat Med 2002; 8(2):143–149.

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150. Dedeoglu A, Kubilus JK, et al. Therapeutic effects of cystamine in a murine model of Huntington’s disease. J Neurosci 2002; 22(20):8942–8950. 151. Klivenyi P, Ferrante RJ, Gardian G, Browne S, Chabrier PE, Beal MF. Increased survival and neuroprotective effects of BN82451 in a transgenic mouse model of Huntington’s disease. J Neurochem 2003; 86(1):267–272. 152. Huntington Study Group. A randomized, placebo-controlled trial of coenzyme Q10 and remacemide in Huntington’s disease. Neurology 2001; 57(3):397–404. 153. Kremer B, Clark CM, et al. Inﬂuence of lamotrigine on progression of early Huntington’s disease: a randomized clinical trial. Neurology 1999; 53(5): 1000–1011. 154. Huntington Study Group. Dosage effects of riluzole in Huntington’s disease: a multicenter placebo-controlled study. Neurology 2003; 61(11):1551–1556. 155. Verbessem P, Lemiere J, et al. Creatine supplementation in Huntington’s disease: a placebo-controlled pilot trial. Neurology 2003; 61(7):925–930. 156. Tabrizi SJ, Blamire AM, et al. Creatine therapy for Huntington’s disease: clinical and MRS ﬁndings in a 1-year pilot study. Neurology 2003; 61(1):141–142. 157. Huntington Study Group. Safety and tolerability of the free-radical scavenger OPC-14117 in Huntington’s disease. The Huntington Study Group. Neurology 1998; 50(5):1366–1373. 158. Bonelli RM, Hofmann P. A review of the treatment options for Huntington’s disease. Expert Opin Pharmacother 2004; 5(4):767–776. 159. Rosenblatt A, Ranen, N.G., Nance, M.A., Paulsen, J.S. A Physician’s Guide to the Management of Huntington’s Disease. 2nd ed. New York: Huntington’s Disease Society of America, 1999. 160. Grimbergen YA, Roos RA. Therapeutic options for Huntington’s disease. Curr Opin Invest Drugs 2003; 4(1):51–54. 161. Tollefson GD, Beasley CM, Jr, Tamura RN, Tran PV, Potvin JH. Blind, controlled, long-term study of the comparative incidence of treatment-emergent tardive dyskinesia with olanzapine or haloperidol. Am J Psychiatry 1997; 154(9):1248–1254. 162. Dipple HC. The use of olanzapine for movement disorder in Huntington’s disease: a ﬁrst case report. J Neurol Neurosurg Psychiatry 1999; 67(1):123–124. 163. Bogelman G, Hirschmann S, Modai I. Olanzapine and Huntington’s disease. J Clin Psychopharmacol 2001; 21(2):245–246. 164. Bonelli RM, Niederwieser G, Tribl GG, Koltringer P. High-dose olanzapine in Huntington’s disease. Int Clin Psychopharmacol 2002; 17(2):91–93. 165. Bonelli RM, Mahnert FA, Niederwieser G. Olanzapine for Huntington’s disease: an open label study. Clin Neuropharmacol 2002; 25(5):263–265. 166. Paleacu D, Anca M, Giladi N. Olanzapine in Huntington’s disease. Acta Neurol Scand 2002; 105(6):441–444. 167. Callaghan JT, Bergstrom RF, Ptak LR, Beasley CM. Olanzapine. Pharmacokinetic and pharmacodynamic proﬁle. Clin Pharmacokinet 1999; 37(3):177–193. 168. Bates GP, Hockly E. Experimental therapeutics in Huntington’s disease: are models useful for therapeutic trials? Curr Opin Neurol 2003; 16(4):465–470 169. Huntington Study Group. Minocycline safety and tolerability in Huntington’s disease. Neurology 2004; 63(3):547–549.

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17 Wilson’s Disease George J. Brewer Department of Human Genetics and Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, U.S.A.

INTRODUCTION AND HISTORICAL OVERVIEW An American neurologist, Samuel Alexander Kinnier Wilson, working in England, originally described the syndrome that bears his name (1). He noted the concomitant occurrence of a neurological movement disorder associated with liver disease and often associated with behavioral symptoms. At autopsy these patients had disease of the liver and of certain structures in the brain including the lenticular nuclei, leading to the name he used, hepatolenticular degeneration. Later, two ophthalmologists, Kayser and Fleischer, independently described the brownish-green corneal rings that are present in many patients with this syndrome (2,3). It took several observations of elevated copper in various anatomical locations before it was realized that copper accumulation was etiologically involved (4,5). After the demonstration that copper balance was controlled by biliary excretion of copper (6), it was shown that Wilson’s patients have defective biliary excretion of copper (7). Meanwhile it was observed that a copper containing serum protein, ceruloplasmin, was usually at quite low levels in Wilson’s disease patients (8,9). Later, the disease was shown to be inherited as an autosomal recessive disease (10). The ﬁrst really effective oral therapy was a chelator, penicillamine, developed by Walshe in 1956 (11), and Walshe followed up with a second chelator, trientine, for penicillamine-intolerant patients (12). Schouwink ﬁrst

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used zinc therapy, describing its use in two patients in his thesis in the Netherlands (13). His work was followed up with further zinc studies by Hoogenraad, also in the Netherlands (14). Independently, my group developed zinc therapy as a Wilson’s disease treatment in the United States, after observing zinc-induced copper deﬁciency in sickle cell disease patients (15,16). Zinc acts by reducing the intestinal absorption of copper (17). The causative gene, which produces a copper binding ATPase called ATP7B, has been identiﬁed (18–20). Already over 200 causative mutations have been identiﬁed, making it difﬁcult to use mutation screening for diagnosis in most populations (21). Several books and reviews on various aspects of Wilson’s disease have been published, and these can be consulted for more background on various aspects of the disease (22–33). PATHOPHYSIOLOGY The damage in Wilson’s disease is due to oxidant injury from copper (22–33). The key role of copper is shown by the association of liver and brain damage with copper accumulation in those organs, and by the cessation of injury and partial repair when drugs are used which reduce copper levels. This role is further conﬁrmed by ﬁnding that the causal gene codes for a copper binding enzyme that regulates copper excretion in the bile. The evidence for oxidant damage includes the demonstration that copper causes Heinz body type damage to erythrocytes and glutathione depletion, both known to be oxidant related events (34–36). There are several other types of evidence showing oxidant damage from copper to the cell membrane, mitochondria, and nuclear components of cells (32). The typical Western diet contains about 1.00.25 mg of copper/day (37–40). Adults have obligatory losses of about 0.75 mg/day, indicating an average excessive intake of about 0.25 mg/day (32). The liver is responsible for maintaining copper balance, and excretes the excess in the bile for loss in the stool (6). The ATP7B protein is a key step in that pathway, and when both copies of the gene are defective due to mutation, copper begins to accumulate, ﬁrst in the liver, where the major stores are held, and then elsewhere, such as in the brain. The ATP7B protein also plays a role in the incorporation of copper into ceruloplasmin. Apoceruloplasmin (the protein prior to copper attachment) is made by the liver, and in the absence of copper incorporation, is either not secreted normally into the blood and/ or has a shortened turnover time, causing the low blood ceruloplasmin in Wilson’s disease. GENETICS AND EPIDEMIOLOGY As already mentioned, the causative gene is ATP7B (18–20), which has considerable homology to the causative gene in Menke’s disease, ATP7A.

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ATP7B is expressed primarily in the liver, which ﬁts with the function already discussed, copper excretion into bile, while ATP7A is much more broadly expressed, which ﬁts with the widespread functional and copper accumulation abnormalities in Menke’s disease. In most populations, no single mutation, or small group of mutations, is sufﬁciently common to allow diagnosis by mutation screening using current technology (32). For example, in the United States, the most common mutation, His1069 Gln, accounts for about 30% of all mutations, but after that frequencies fall off rapidly into the 1% or lower range. Thus, most U.S. patients are compound heterozygotes. Worldwide, over 200 causative mutations have been described (21). In occasional populations, due to founder effects, one or a few mutations may be frequent enough to be useful diagnostically. On the other hand, once a patient is diagnosed, siblings can be evaluated very accurately by haplotype analysis. Polymorphic markers on both sides of the Wilson locus on chromosome 13 are analyzed, and it is determined if a given sibling shares two, one, or zero chromosome 13s with the patient, indicating affected, carrier, or clear, respectively. As we will discuss, Wilson’s disease can present clinically with either hepatic or neurologic symptoms predominating, and with considerable variation in age of onset. There have been attempts to carry out genotype–phenotype correlations, i.e., to see if certain mutations are correlated with speciﬁc phenotypic characteristics (41). In general, these attempts have not been very successful (32,42). They are hampered to some degree by most patients being compound heterozygotes. It has been stated that patients with the common mutation His1069 Gln are more likely to present later and with neurologic disease (41). However, it is always possible that the genetic background associated with having that mutation is the more important factor (32). In fact, it seems quite likely that numerous genetic factors determining such things as the strength of oxidant defense mechanisms in speciﬁc tissues, the level of metallothionein (which by sequestering copper can protect against toxicity) induction in speciﬁc tissues, and numerous other factors will strongly inﬂuence when and where toxicity will occur (32). Effects of the environment must not be forgotten. For example, copper is absorbed much more poorly from vegetable foods than from meat (43,44). Thus, the choice of eating less meat, or the economic forces causing less meat to be purchased, can probably delay onset of the disease. Wilson’s disease appears to be present in most populations, basically a result of the mutation rate, similar to most other rare recessive diseases. The frequency may vary a little, especially in relatively isolated populations where founder effects can play a signiﬁcant role. The general frequency number that is used is 1 in 40,000 (23,24,32). This number is soft, being determined in part by autopsy series, where under-diagnosis may occur unless postmortem copper studies are done. We attempted to strengthen the estimate

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of frequency by using the frequency of His1069 Gln in a series of U.S. Wilson’s patients, then determining the heterozygous frequency of His1069 Gln in newborn blood samples (45). This estimate of the frequency of Wilson’s disease was 1 in 55,000, but with a very wide conﬁdence interval. If we use 1 in 40,000 this results in a U.S. case load of about 6000, if they were all diagnosed and lived a normal life span, and a heterozygote frequency of about 1 in 100.

CLINICAL PRESENTATIONS Hepatic Presentation About half of Wilson’s disease patients present with liver disease, typically in their late teenage years, and this may become clinically apparent in three different forms. One form is hepatitis, usually with jaundice and elevated serum transaminase enzymes. If undiagnosed, the hepatitis often resolves, only to recur, perhaps several times. This often leads to the misdiagnosis of chronic active hepatitis, inferring a viral or autoimmune origin. The second form is that a hepatitis episode results in liver failure, meaning that the liver is not keeping up with its synthetic and metabolizing functions. Thus, the albumin will usually be low, the bilirubin elevated, coagulation factors low, and transaminase enzymes high. Ascites, peripheral edema, and hepatic encephalopathy may be present. The third form is the discovery of cirrhosis, perhaps by coincidence during workup for something else, or perhaps by one of the complications of the resulting portal hypertension, such as bleeding esophageal or gastric varices. Such a patient who drinks alcohol even moderately may be misdiagnosed as alcoholic cirrhosis. The clinical presentation of liver disease from Wilson’s disease tends to occur in the second and third decades of life in the U.S. and other Western countries, although the possible age of presentation is quite broad from childhood until the ﬁfties (32). In India and some other Far Eastern countries, the age of presentation tends to be much younger, often in children 10 years of age or younger (46–48). Psychiatric Presentation Behavioral abnormalities are quite common in the neurologic presentation of Wilson’s disease (49,50). About 25% of Wilson’s patients ﬁrst present with behavioral abnormalities, typically in late teen age years through early thirties in Western countries, and quite a bit younger in India and the Far East. Behavioral abnormalities can antedate neurologic symptoms by a year or two. However, our rule of thumb in seeing patients with movement disorder symptoms is that behavioral abnormalities antedating neurologic symptoms by more than three years may not be related to Wilson’s disease.

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The most common abnormality is personality change, which includes irritability, emotionality, bouts of anger, anxiety, hypermania, sexual preoccupation or loss of inhibitions, excessive hand washing, and others (49,50). Depression is also very common. Cognitive changes may occur but are often misdiagnosed because the patient has difﬁculty focusing on tasks, shortterm memory is affected, and motor skills are often defective. In their study, Dening and Berrios (51) describe incongruous behavior, irritability, aggression, and personality change as common. They paint a typical picture of odd, reckless, disinhibited behavior with loss of impulse control. Neuropsychologic tests that have been administered to patients at the time of diagnosis or early in therapy often reveal mild abnormalities, and can be abnormal even in presymptomatic patients (52). There are a number of publications on the psychiatric and behavioral disorder in Wilson’s disease other than those cited which the interested reader can consult (53–60). The paper by Lauterbach et al. (60) provides a good discussion of the complicated interconnections of the brain which may help explain why both neurologic and psychological symptoms can result from the injuries in Wilson’s disease. Later when we discuss treatment, we will discuss the effect of anticopper therapy on the behavioral aspects of this disease. Rarely do behavioral abnormalities alone lead to the correct diagnosis of Wilson’s disease. This is unfortunate because if it did, it would allow treatment before the onset of neurologic symptoms and prevent permanent neurologic deﬁcits. Rather, these patients are usually given other diagnoses. Often they are accused of substance abuse because of their young age. Even though these patients have not shown enough liver disease problems to come to clinical attention, by this time they almost all have underlying liver disease. Neurologic Presentation About 50% of Wilson’s patients ultimately develop neurologic symptoms. This includes the 25% who ﬁrst present with behavioral symptoms and then develop neurologic symptoms, and the 25% who present with neurologic symptoms without any obvious antecedent behavioral problems. The areas of the brain that are affected are those that coordinate movement, hence the classiﬁcation of Wilson’s disease as a movement disorder, alongside much more common movement disorders such as Parkinson’s disease, Huntington’s disease, essential tremor, and many other rarer disorders (26–30,61). The neurologic symptoms of Wilson’s disease can be explained by three basic defects: tremor, dystonia, and incoordination (32). Thus, dysarthria, which is very common, can be explained by dystonia and/or incoordination of the muscles involved in speech. The dysarthria is often hypokinetic (low volume) in addition to the problems making certain

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sounds. The dysarthria of Wilson’s disease is not pathognomonic, so it is not possible to make the diagnosis based on the speech pattern. Tremor occurs in about one-third of neurologic Wilson patients, and again is not of a pathognomonic nature. I once saw a patient whose Wilson’s disease had been supposedly ruled out by the tremor not being ‘‘wing beating’’; this is inappropriate. Wing beating tremor does occur, but so do a variety of other types of tremor. Incoordination begins with difﬁculty with ﬁne tasks—handwriting, typing, buttoning, etc., and gradually progresses to interfere with coordination of larger muscle groups, such as those involved in bringing food to mouth, handling larger objects, and walking. Classically, the handwriting in Wilson’s disease is said to be micrographic, possibly due to hypokinesia, or possibly an adaptation by the patient to keep their handwriting from being excessively sloppy (24). However, in my experience the handwriting is much more often simply very sloppy, not necessarily small. Dystonia may begin with what appear to be muscle cramps in ﬁngers, toes, hands, feet, arms, or legs. Later it may progress to the point where it pulls these body parts into increasingly unphysiologic positions, producing much pain in the process. Even the muscle groups of the neck and back may become involved, twisting the head and/or torso. Individual patients at the time they are ﬁrst diagnosed have various combinations of tremor, incoordination, and dystonia, and of varying severity. Thus, a given patient may have mild disease, perhaps with mild dysarthria, and a little tremor, to very severe disease producing an anarthric, bedridden patient with severe tremor, whose limbs and body are in ﬁxed positions. These differences are primarily related to disease progression. All patients start out with mild disease, but some are not diagnosed until they have progressed to severe disease. Hence the critical role of early recognition and diagnosis. Anticopper treatment can essentially prevent this progression. Other Types of Clinical Involvement Wilson’s patients often have additional clinical ﬁndings (32). These may be found coincidentally as the patient is being worked up because of one of the presentations already described, or may on occasion be a factor in triggering a Wilson’s workup and diagnosis in the ﬁrst place. Another eye abnormality, besides Kayser–Fleischer (KF) rings, is sunﬂower cataract. Patients may have diploplia, or abnormal eye movements. Disturbances of the autonomic nervous system are fairly frequent, and include postural hypotension, abnormal sweating, and bladder, bowel, and sexual dysfunction (62–64). Migraine-type headaches occur, as does epilepsy, both of which appear to be causally related to copper toxicity in some patients. Cholelithiasis and urolithiasis are more common in Wilson’s

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disease. Women usually stop menstruating sometime before presenting clinically with Wilson’s disease. A type of osteoarthritis, particularly of the knee, is fairly common. Cardiac abnormalities have been reported (65–67), but in my experience, the heart is not affected by Wilson’s disease. Abnormalities in the urine can occur (23,32). Presymptomatic Patients Usually as part of a family workup, but occasionally because of a chance ﬁnding (e.g., Kayser–Fleischer rings seen on a routine eye examination), patients will be diagnosed in an asymptomatic state. We call these presymptomatic patients under the assumption that the disease is 100% penetrant, and these patients are doomed to become ill unless treated prophylactically. Since the disease is inherited as an autosomal recessive, 25% of a newly diagnosed patient’s siblings are at risk for also being affected. Since prophylactic anticopper treatment will prevent these patients from ever becoming ill (68,69), it is important to screen and work up these siblings aggressively, as an important component of preventive medicine. Other more distant relatives also have an increased risk (children of a patient, 1:200; nieces and nephews, 1:600; cousins, 1:800, compared to the 1:40,000 population risk) and may also deserve screening.

RECOGNITION, SCREENING, AND DIAGNOSIS Recognition A major problem in Wilson’s disease is the recognition by clinicians that particular types of patients may have Wilson’s disease, and be very treatable. The occasional Wilson’s patient presenting with hepatitis is buried and often lost amongst hundreds of patients with viral or other types of hepatitis. The same with cirrhosis; the Wilson’s patient will be a rare patient among many with cirrhosis secondary to alcohol abuse, hepatitis C, hepatosteatosis, etc. On the neurology side, other causes of tremor, such as Parkinsonism, essential tremor, etc., are much, much more common than Wilson’s disease. The same is true of many of the other features of the movement disorder, often misdiagnosed for a while as due to Parkinsonism. The situation is even worse on the behavioral side. Given the frequency of abnormal behavior due to substance abuse, life’s stresses, or whatever, it is a rare psychiatrist who recognizes that the young person under evaluation with a six months history of odd and obnoxious behavior, might have Wilson’s disease. I think we can do better, and in hopes of helping the cause of early recognition, Table 1 provides a list of the types of patients various specialists and other health care workers should look for as possible Wilson’s candidates.

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Table 1 Patients That Should be Screened for Wilson’s Disease Neurologic area Patients 50 years or younger with recent onset (ﬁve years) with: Dysarthria Tremor or involuntary movements Dystonia Incoordination Diagnosis of Parkinsonism Diagnosis of essential tremor Psychiatric area Patients 50 years or younger with recent onset (three years) with: Loss of ability to focus on tasks Loss of emotional control Depression Loss of sexual inhibition or other bizarre behavior Hepatic area Patients 50 years or younger with: Viral negative hepatitis or elevated transaminase enzymes Chronic hepatitis Cirrhosis, without a deﬁnitive diagnosis as to cause Liver failure, without a deﬁnitive diagnosis as to cause

Screening Screening tests are listed in Table 2. The two most useful screening tests are Kayser–Fleischer (KF) ring examination by an ophthalmologist with a slit lamp, and 24 hours urine copper determination. KF ring examination is extremely useful in the neurologic/behavioral presentations. In our experience, 178/179 patients presenting in this manner were positive for KF rings. Only very rarely have patients been reported with what appeared to be KF rings who did not have Wilson’s disease. So by a simple referral to an ophthalmologist, a neurologist seeing a relatively young patient with tremor or Parkinson-like symptoms, or a psychiatrist seeing a relatively young patient with new and odd behavior, can essentially rule Wilson’s disease in or out. This examination is not as useful in patients with liver disease because the KF rings are present less often, perhaps only 50% of the time, particularly in younger patients. If present though, they help the gastroenterologist a great deal in getting to the right diagnosis. The 24 hours urine copper is also very valuable as a screening tool, but more cumbersome to obtain (Table 2). In my experience it is always elevated over 100 mg in untreated patients (normal 20–50). The procedure must be done with trace element free collecting materials to avoid contamination, and in a laboratory with the equipment and skills to measure the low levels of copper present in urine. The only other caveat is that long standing liver

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Table 2 Values or Findings in Screening Tests for Wilson’s Disease

Test

Normal Values

24-hour urine 20–50 mg copper

Symptomatic Affected Patients >100 mg

Carriers

Presymptomatic Affected Patients

<100 mg >65 mg 90% <65 mg

KayserFleischer ring exam

Not Present Not Present in patients present with neurologic and psychiatric symptoms Present in most hepatic patients unless they are very young

Present in 30–40%

Serum Cp

20–35 Often below 10. Low mg/dL normal to normal in about 15%

Same as symptomatic patients

80% > 20 20% < 20

Reprinted from: Kluwer Academic Publishers, Boston, Wilson’s Disease: A Clinician’s Guide to Recognition, Diagnosis, and Management. 2001, George J Brewer, page 31, Chapter 3. Simple approaches to screening and deﬁnitive diagnosis, Table 3.1, with kind permission of Springer Science and Business Media. (Reference 32).

disease, particularly with an obstructive component, can elevate liver and urine copper. Serum ceruloplasmin is often the ﬁrst and only screening test used by many physicians, but has serious drawbacks (Table 2). First, it is only abnormally low in about 80–85% of Wilson patients. Second, it abnormally low in about 15–20% of heterozygous carriers of the Wilson gene. Thus, it should only be used to affect index of suspicion. Other screening tests that have been suggested include a radiocopper test, but it does not differentiate carriers from affected individuals (22). Another test is a penicillamine ‘‘provocative’’ test, looking at copper excretion after a dose of penicillamine. This test, too, probably does not differentiate carriers and affected persons. We have already discussed why mutation screening is not useful in most populations. On the other hand, haplotype analysis is very effective for screening siblings of an affected patient (32). Definitive Diagnosis Some combinations of screening tests can be considered deﬁnitive, and these are listed in Table 3. For example, KF rings plus a urine copper over 100 mg in a neurologic/behavioral patient, or a sibling of an affected person, can be considered deﬁnitive. We usually recommend at least two positive tests, to mitigate against the occasional error in any test. Even ophthalmologists (usually those who have never seen KF rings) occasionally miss them, or

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Table 3 Diagnostic Scenarios not Requiring a Liver Biopsy Patient Has Wilson’s Disease Typical neurological symptoms KF rings and Elevated urine copper (over 100 mg/24 hours) Sibling of affected patient Elevated urine copper (over 100 mg/24 hours), or Haplotype analysis identical to affected sibling KF rings discovered by ophthalmologist Elevated urine copper (over 100 mg/24 hours) Hepatic presentation Elevated urine copper(over 100 mg/24 hours— in absence of long-standing liver failure or obstruction) and Cp less than 10 mg/dL Patient Does Not Have Wilson’s Disease Movement disorder type neurological symptoms No KF rings and Normal urine copper (50 mg or less/24 hours) Sibling of affected patient Normal urine copper (50 mg or less/24 hours—if age 15 years or older) Hepatic disease Near normal urine copper (60 mg or less/24 hours) Reprinted from: Kluwer Academic Publishers, Boston, Wilson’s Disease: A Clinician’s Guide to Recognition, Diagnosis, and Management 2001, George J Brewer, page 43, Chapter 3. Simple approaches to screening and deﬁnitive diagnosis, Table 3.4, with kind permission of Springer Science and Business Media. (Reference 32).

see them when they are not there. In the liver disease patient we usually prefer to get a liver biopsy because of the occasional occurrence of cholestatic syndromes that cause a high urine copper.

TREATMENT WITH ANTICOPPER DRUGS There are three commercially available anticopper drugs and a fourth well on its way to development. We will discuss them in the sequence in which they were introduced. Penicillamine Penicillamine was introduced in 1956 and was the ﬁrst orally effective anticopper drug (11). It is a reductive chelator which means that it reduces copper, in the process lowering copper’s binding afﬁnity to proteins, and then binding the copper itself, with excretion of the complex in the urine. The reductive step is important, because chelators that do not do this, such

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as EDTA, even though they have a high afﬁnity for copper, are ineffective in causing copper excretion. Penicillamine is given in doses of 1.0 g/day, often as 500 mg twice daily or 250 mg four times daily, avoiding food 1/2 hour before and two hours after each dose. It causes a great increase in urinary copper excretion in Wilson patients, often to several milligrams/day. It is generally effective in producing a negative copper balance, and with one exception noted below, to allow subsequent partial clinical recovery. It is also effective in preventing the onset of disease when used prophylactically in presymptomatic patients (68). There are two negative features of penicillamine use in Wilson’s disease. The ﬁrst is the long list of side effects of penicillamine therapy (22). These include a common (25%) hypersensitivity, bone marrow suppression including aplastic anemia, autoimmune disorders such as Goodpasture’s syndrome or lupus erythematosis, a variety of skin manifestations, hepatotoxicity, nephrotoxicity, and on and on. The second is neurologic deterioration when used as initial therapy in neurologically presenting patients (70). This may happen as often as 50% of the time, and half of the patients who worsen may never recover. The mechanism of this may be due to mobilization of hepatic copper with further elevation of brain copper. Trientine Trientine was introduced in 1982 as a replacement for penicillamine in penicillamine-intolerant patients (12). It is also a chelator but acts less aggressively than penicillamine. It is used in doses similar to penicillamine, with the same food avoidance, and causes a modest increase in urine copper, to 1.0 or 2.0 mg/day. It is also generally effective in producing a negative copper balance. Trientine shares some of the toxicities of penicillamine, but they appear to be much less frequent (32). Proteinuria, iron deﬁciency, lupus nephritis, and lupus erythematosis have been the main ones. The risk of neurologic worsening with trientine when used as initial therapy in neurologically presenting patients is less than with penicillamine, but still exists (71). Zinc After several years of work by our group (15–17,22,26–30,37,69) zinc was approved by the FDA in January 1997 for maintenance therapy in Wilson’s disease. Zinc acts by inducing intestinal cell metallothionein, which blocks absorption of food copper and endogenously secreted copper (17). The adult dose is 50 mg three times daily, each dose separated from food and beverages other than water by at least one hour. It is also generally effective in producing a negative copper balance.

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The only toxicity of zinc is gastric discomfort or burning in about 10% of patients, particularly with the ﬁrst morning dose (16). Such patients are advised to take their ﬁrst dose mid-morning rather than upon arising. They also may use a small amount of protein food (lunchmeat, cheese, jello) to mitigate the discomfort. Tetrathiomolybdate Tetrathiomolybdate (TM) is being developed as a fast acting anticopper drug for initial treatment of acutely ill Wilson’s disease patients (72–75). TM is not yet commercially available. It acts by forming a tripartite complex with protein and copper. Given with food, it complexes food copper and food protein and prevents the absorption of this bound copper. Given between meals, it is well absorbed and in the blood complexes available copper with albumin. The copper in this complex is unavailable for cellular uptake. The complex is metabolized in the liver and the components excreted in the bile. TM is rapidly effective in producing a negative copper balance and in mitigating further copper toxicity. The side effects of TM in Wilson’s disease are, ﬁrst, bone marrow suppression due to overtreatment, leading to anemia and/or leukopenia. This is quickly responsive to dose reduction. A second side effect is further elevation of transaminase enzymes, probably due to shifting copper pools from metallothionein in the liver. This, too, is responsive to dose reduction. Recommended Therapy My recommendations for therapy for different stages of Wilson’s disease are given in Table 4. I would like to emphasize that the days are gone when once the diagnosis is made, the physician simply prescribes penicillamine. We now have additional anticopper drugs, and they should be used differentially according to the patient’s status. 1. Initial therapy—neurologic/psychiatric presentation. It is important that penicillamine be avoided in this situation, if at all possible, because of its 50% risk of making the patient neurologically worse (70). Trientine seems to have an intermediate risk of neurologic worsening estimated currently at about 18% (71). Zinc, in our opinion is too slow acting to be optimal. Because none of these drugs are optimal for this type of patient, we have developed TM to ﬁll this therapeutic niche (72–75). Most of our work has been done with 20 mg three times daily with food and 20 mg three times daily between meals for eight weeks (120 mg/day). We usually start zinc concurrently at 50 mg two times daily, and increase this to three times daily as the patient goes off TM, for maintenance therapy.

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Table 4 Therapy Recommendations for Wilson’s Disease First choice Maintenance therapy (adult and pediatric) Treatment of presymptomatic patients (adult and pediatric) Treatment of pregnant patients Initial treatment neurologic/psychiatric Hepatic

Second choice

Third choice

Zinc

Trientine

Penicillamine

Zinc

Trientine

Penicillamine

Zinc

Trientine

Tetrathiomolyb- Trientine plus date plus zinc zinc Trientine plus Penicillamine zinc plus zinc

Zinc alone

This therapy has been very effective in preserving neurologic function. Only 2 of 55 patients (3.6%) have had neurologic worsening by our criteria (75), compared to the 50% estimate for penicillamine (70). Occasional patients may worsen irrespective of the drug used, probably due to the natural history of the disease. The 120 mg/day TM regimen causes anemia/leukopenia about 10–15% of the time, probably due to overtreatment and bone marrow depletion of copper. The anemia/leukopenia tends to come on after 4–5 weeks of treatment and resolves with halving the TM dose. This TM regimen also causes an elevation of serum transaminase enzymes in 10–15% of the patients. Again this side effect comes on after 4–5 weeks and is responsive to halving the dose. Because of the timing of these side effects, and their favorable response to halving the dose, we are now evaluating a TM regimen which gives a full dose (120 mg/day) for two weeks then a half dose (60 mg/day) for 14 weeks. TM is not yet commercially available and as of this writing the only practical way a patient can receive this therapy is to join a clinical trial protocol at the University of Michigan. Until TM is available commercially, other options for initial therapy of neurologic patients include trientine and accepting some risk of worsening, or zinc and accepting some risk of progression (Table 4). 2. Initial therapy—hepatic presentation. Here we are dealing with medical therapy. Some patients present with hepatic failure so severe that only hepatic transplantation will save their lives. The prognostic index of Nazer et al. (76) as well as other recent publications (32,77), provides guidance as to how to triage patients for transplantation versus medical therapy.

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The medical therapy of choice at this time, in our opinion, is a combination of trientine and zinc (Table 4) (77). Zinc is added because it induces hepatic metallothionein, which will bind up some of the excessive hepatic copper. Trientine is used because an additional faster acting agent is required, and we choose it over penicillamine because of a better safety proﬁle. We give the combination for 4–6 months, than continue with zinc alone. 3. Maintenance and presymptomatic therapy. Our ﬁrst choice is zinc, because of its superior safety, followed by trientine (Table 4). 4. Therapy in children. Initial therapy in children would be the same as discussed earlier under initial therapy, with dose reductions in younger children. For maintenance therapy we recommend zinc with dose reductions as previously published (78), with trientine as second choice. 5. Therapy during pregnancy. Pregnant women should be on anticopper therapy during pregnancy to protect their own health. Zinc is our ﬁrst choice, with trientine as second (Table 4). Penicillamine has some known teratogenic effects. We recommend not controlling copper levels too tightly during pregnancy, since copper deﬁciency is known to be teratogenic.

PROGNOSIS Patients who are neurologically and/or behaviorally symptomatic improve considerably during anticopper therapy. Usually real improvement does not begin until 5–6 months after therapy is started, and then continues for about 18 months (74,75). Most of the improvement occurs by 24 months after the initiation of therapy, assuming good compliance with therapy. Occasionally, patients will report some improvement in one or more symptom occurring after the two-year period. The degree of remaining disability depends usually upon the severity to begin with. Patients with mild disease often make a complete recovery, those with moderate disease usually have a little residual, and those with severe disease often end up with signiﬁcant disabilities. Throughout the 2-year primary recovery period, it is important that a hopeful prognosis be kept in front of the patient, and the prolonged period of recovery continually emphasized. In addition, the patient should maintain as much function as possible through exercise, physical therapy, speech therapy, and occupational therapy as appropriate and as available. Beginning during the second year, the patient and family should begin thinking about the future, and what type of school, occupation, etc., the patient might undertake.

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Improvement in behavioral problems generally follows the improvement in neurologic symptoms. While there is evidence that long treated patients test at least mildly abnormal in a number of psychosocial tests (79–83), they generally recover their functional skills and most are capable of living a productive life. Recovery of hepatic function in terms of normalizing liver function and liver status tests generally occurs during the ﬁrst year (16). Most patients have some degree of liver disease that remains, but in most cases, barring other insults to the liver, it will last patients the rest of their lives. The main risk factor over time in neurologic patients with residual damage, if they have some degree of dysphagia, is aspiration, producing aspiration pneumonia or chronic lung disease. If patients have some degree of incoordination, they are at some risk for falls and accidents of other types. Patients who have enough residual cirrhosis to produce portal hypertension are at some risk from variceal bleeding. REFERENCES 1. Wilson SAK. Progressive lenticular degeneration: a familial nervous disease associated with cirrhosis of the liver. Brain 1912; 34:295–509. 2. Kayser B. Ueber einen Fall von angeborener gru¨nlicher Verfa¨rbung der kornea. Klin Mbl Augenheilk 1902; 40:22–25. 3. Fleischer B. Zwei weitere Fa¨lle von gru¨nliche Verfa¨rbung der Kornea. Klin Mbl Augenheilk 1903; 41:489–491. 4. Glazebrook AJ. Wilson’s disease. Edinburgh Med J 1945; 52:83–87. 5. Cumings JN. The copper and iron content of brain and liver in the normal and in hepato-lenticular degeneration. Brain 1948; 71:410–415. 6. Ravestyn AH. Metabolism of copper in man. Acta Med Scand 1944; 118: 163–196. 7. Frommer DJ. Defective biliary excretion of copper in Wilson’s disease. Gut 1974; 15:125–129. 8. Scheinberg IH, Gitlin D. Deﬁciency of ceruloplasmin in patients with hepatolenticular degeneration (Wilson’s disease). Science 1952; 116:484–485. 9. Bearn AG, Kunkel HG. Biochemical abnormalities in Wilson’s disease. J Clin Invest 1952; 31:616. 10. Bearn AG. A genetical analysis of thirty families with Wilson’s disease (hepatolenticular degeneration). Ann Hum Genet 1960; 24:33–43. 11. Walshe JM. Penicillamine. A new oral therapy for Wilson’s disease. Am J Med 1956; 21:487–495. 12. Walshe JM. Treatment of Wilson’s disease with trientine (triethylene tetramine) dihydrochloride. Lancet 1982; 1:643–647. 13. Schouwink G. De hepatocerebrale degeneratie, me een onderzoek naar de zinktofwisseling. MD Thesis, University of Amsterdam 1961. 14. Hoogenraad TU, van Hattum J, van den Hamer CJA. Management of Wilson’s disease with zinc sulphate. Experience in a series of 27 patients. J Neurol Sci 1987; 77:137–146.

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15. Brewer GJ, Hill GM, Prasad AS, Cossack ZT, Rabbani P. Oral zinc therapy for Wilson’s disease. Ann Intern Med 1983; 99:314–320. 16. Brewer GJ, Dick RD, Johnson VD, Brunberg JA, Kluin KL, Fink JK. Treatment of Wilson’s disease with zinc: XV Long-term follow-up studies. J Lab Clin Med 1998; 132:264–278. 17. Yuzbasiyan-Gurkan V, Grider A, Nostrant T, Cousins RJ, Brewer GJ. The treatment of Wilson’s disease with zinc: X. Intestinal metallothionein induction. J Lab Clin Med 1992; 120:380–386. 18. Bull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW. The Wilson’s disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nat Genet 1993; 5(4):327–337. 19. Tanzi RE, Petrukhin K, et al. The Wilson’s disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nat Genet 1993; 5(4): 44–50. 20. Yamaguchi Y, Heiny ME, Gitlin JD. Isolation and characterization of a human liver cDNA as a candidate gene for Wilson’s disease. Biochem Biophys Res Commun 1993; 197:271–277. 21. Cox DW, Roberts EA. Wilson’s disease. GeneClinics, University of Washington, Seattle. Online. Available: http://www.geneclinics.org/proﬁles/wilson/details. html. 22. Brewer GJ, Yuzbasiyan-Gurkan V. Wilson’s disease. Medicine 1992; 71:139–164. 23. Scheinberg IH, Sternlieb I. Wilson’s disease. In: Smith LH Jr, ed. Major Problems in Internal Medicine. Philadelphia: W.B. Saunders, 1984:23. 24. Hoogenraad TU. Wilson’s disease. In: Warlow CP, Van Gijn J, eds. Major Problems in Neurology. London: W.B. Saunders, 1996:30. 25. Schilsky ML. Wilson’s disease: Genetic basis of copper toxicity and natural history. Semin Liver Dis 1996; 16:83–95. 26. Brewer GJ. Wilson’s disease. Curr Treat Options Neurol 2000; 2(3):193–203. 27. Brewer GJ, Fink JK, Hedera P. Diagnosis and treatment of Wilson’s disease. Semin Neurol 1999; 19(3):261–270. 28. Brewer GJ. Recognition, diagnosis and management of Wilson’s disease. Exp Biol Med 2000; 223(1):39–49. 29. Brewer GJ. Raulin Award Lecture: Wilson’s disease therapy with zinc and tetrathiomolybdate. J Trace Elem Exp Med 2000; 3:51–61. 30. Fink JK, Hedera P, Brewer GJ. Hepatolenticular degeneration (Wilson’s disease). Neurologist 1999; 5:171–185. 31. Steindl P, Ferenci P, Dienes HP, Grimm G, Pabinger I, Madl C, MaierDobersberger T, Herneth A, Dragosics B, Meryn S, Knoﬂach P, Granditsch G, Gangl A. Wilson’s disease in patients presenting with liver disease: A diagnostic challenge. Gastroenterology 1997; 113:212–218. 32. Brewer GJ. Wilson’s Disease: A Clinician’s Guide to Recognition, Diagnosis, and Management. Boston: Kluwer, 2001. 33. Brewer GJ. Wilson’s Disease for the Patient and Family: A Patient’s Guide to Wilson’s Disease and Frequently Asked Questions about Copper. Philadelphia: Xlibris, 2001. 34. Deiss A, Lee GR, Cartwright GE. Hemolytic anemia in Wilson’s disease. Ann Intern Med 1970; 73:413–418.

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35. Ishmael J, Gopinath C, Howell JMC. Experimental chronic copper toxicity in sheep. Biochemical and haematological studies during the development of lesions in the liver. Res Vet Sci 1972; 13:22–29. 36. Metz EN. Mechanism of hemolysis by excess copper (abstract). Clin Res 1969; 7:32. 37. Hill GM, Brewer GJ, Prasad AS, Hydrick CR, Hartmann DE. Treatment of Wilson’s disease with zinc: I. Oral zinc therapy regimens. Hepatology 1987; 7:522–528. 38. Holden JM, Wolf WR, Mertz W. Zinc and copper in self selected diets. J Am Diet Assoc 1979; 75:23–28. 39. Klevay LM, Reck SJ, Barcome DP. Evidence of dietary copper and zinc deﬁciencies. JAMA 1979; 241:1916–1918. 40. Reiser S, Smith JC Jr, Mertz W, Holbrook JT, Scholﬁeld DJ, Powell AS, Canﬁeld WK, Canary JJ. Indices of copper status in humans consuming a typical American diet containing either fructose or starch. Am J Clin Nutr 1985; 42:242–251. 41. Thomas GR, Forbes JR, Roberts EA, Walshe JM, Cox DW. The Wilson’s disease gene: spectrum of mutations and their consequences. Nat Genet 1999; 9:210–217. 42. Okada T, Shiono Y, Hayashi H, Satoh H, Sawada T, Suzuki A, Takeda Y, Yano M, Michitaka K, Onji M, Mauchi H. Mutational analysis of ATP7B and genotype–phenotype correlation in Japanese with Wilson’s disease. Hum Mutat 2000; 15:454–462. 43. Srikumar TS, Johansson GK, Ockerman P-A, Gustafsson J-A, Akesson B. Trace element status in healthy subjects switching from a mixed to a lactovegetarian diet for 12 mo. Am J Clin Nutr 1992; 55:1–6. 44. Brewer GJ, Yuzbasiyan-Gurkan V, Dick R, Wang Y, Johnson V. Does a vegetarian diet control Wilson’s disease? JACN 1993; 2:527–530. 45. Olivarez L, Caggana M, Pass KA, Ferguson P, Brewer GJ. Estimate of the frequency of Wilson’s disease in the U.S. Caucasian population: A mutation analysis approach. Ann Hum Genet 2001; 64:459–463. 46. Dastur DK, Manghani DK, Wadia NH. Wilson’s disease in India I. Geographic, genetic, and clinical aspects in 16 families. Neurology 1968; 18: 21–31. 47. Bhave S, Bavdekar A, Pandit A. Changing pattern of chronic liver disease (CLD) in India. Indian J Pediatr 1994; 61:675–682. 48. Bhave SA, Purohit GM, Pradhan AV, Pandit AN. Hepatic presentation of Wilson’s disease. Indian Pediatr 1987; 24:385–393. 49. Brewer GJ. Behavioral abnormalities in Wilson’s disease. In: Weiner WJ, Lang AE, Anderson KE, eds. Behavioral Neurology of Movement Disorders, 2nd ed. Philadelphia: Lippincott Williams & Wilkins 2005; 262–274. 50. Akil M, Brewer GJ. Psychiatric and behavioral abnormalities in Wilson’s disease. In: Weiner WJ, Lang AE, eds. Advances in Neurology. Vol. 65. Behavioral Neurology of Movement Disorders. New York: Raven Press, 1995:171–178. 51. Dening TR, Berrios GE. Wilson’s disease: Psychiatric symptoms in 195 cases. Arch Gen Psychiatry 1989; 46:1126–1134.

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71. Brewer GJ, Schilsky M, Hedera P, Carlson MD, Fink JK, Askari FK, Kluin K, Lorincz M, Shneider B, Velickovic M, Blitman S, Mojica J, Vila N. Double blind study of initial therapy of neurological Wilson’s disease. J Invest Med 2003; 51(suppl 2), S369. 72. Brewer GJ, Dick RD, Yuzbasiyan-Gurkan V, Tanakow R, Young AB, Kluin KJ. Initial therapy of patients with Wilson’s disease with tetrathiomolybdate. Arch Neurol 1991; 48:42–47. 73. Brewer GJ, Dick RD, Johnson V, Wang Y, Yuzbasiyan-Gurkan V, Kluin KJ, Fink JK, Aisen A. Treatment of Wilson’s disease with ammonium tetrathio"molybdate: I. Initial therapy in 17 neurologically affected patients. Arch Neurol 1994; 51:545–554. 74. Brewer GJ, Johnson V, Dick RD, Wang Y, Kluin KJ, Fink JK, Brunberg JA. Treatment of Wilson’s disease with ammonium tetrathiomolybdate: II. Initial therapy in 33 neurologically affected patients and follow-up with zinc therapy. Arch Neurol 1996; 53:1017–1025. 75. Brewer GJ, Hedera P, Kluin KJ, Carlson MD, Askari F, Dick RB, Sitterly JA, Fink JK. Treatment of Wilson’s Disease with tetrathiomolybdate III. Initial therapy in a total of 55 neurologically affected patients and follow-up with zinc therapy. Arch Neurol 2003; 60:378–385. 76. Nazer H, Ede RJ, Mowat AP, Williams R. Wilson’s disease: clinical presentation and use of prognostic index. Gut 1986; 27:1377–1381. 77. Askari FK, Greenson J, Dick RD, Johnson VD, Brewer GJ. Treatment of Wilson’s disease with Zinc XVIII. Initial treatment of the hepatic decompensation presentation with trientine and zinc. J Lab Clin Med 2003; 142(6):385–390. 78. Brewer GJ, Dick RD, Johnson VD, Fink JK, Kluin KJ, Daniels S. Treatment of Wilson’s disease with zinc: XVI. Treatment during the pediatric years. J Lab Clin Med 2001; 137(3):191–198. 79. Portala K, Westermark K, et al. Psychopathology in treated Wilson’s disease determined by means of CPRS expert and self-ratings. Acta Psychiat Scand 2000; 101:104–109. 80. Portala K, Westermark K, et al. Personality traits in treated Wilson’s disease determined by means of the Karolinska Scales of Personality (KSP). Eur Psychiatry 2001; 16:362–371. 81. Portala K, Westermark K, et al. Sleep in patients with treated Wilson’s disease. A questionnaire study. Nord J Psychiatry 2002; 56(4):291–297. 82. Portala K, Levander S, et al. Pattern of neuropsychological deﬁcits in patients with treated Wilson’s disease. Eur Arch Psychiatry Clin Neurol 2001; 252(6): 262–268. 83. Portala K. Psychopathology in Wilson’s Disease Thesis. Acta Universitatis Upsaliensis, Uppsala, 2001.

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18 Neurogenetics of Dystonia and Paroxysmal Dyskinesias Jennifer Friedman Department of Neurology, Children’s Hospital and Health Center, San Diego, California, U.S.A.

David G. Standaert MassGeneral Institute for Neurodegenerative Disorders, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, U.S.A.

DYSTONIA Dystonia is a class of disorders in which the role of genetics has become increasingly apparent. Dystonia is not a diagnosis, rather it is a descriptive term which identiﬁes a class of symptoms. Dystonia is characterized by sustained muscle contractions, frequently causing twisting and repetitive movements, or abnormal postures (1). Dystonia may be seen in isolation or as the predominant symptom of disease. Isolated dystonias may be constant or task-speciﬁc, e.g., occurring only when writing or even exclusively when playing a particular musical instrument. Dystonia may also occur in association with a variety of other abnormalities of the nervous system, including neurodegenerative disorders, structural lesions of the basal ganglia, and after pharmacological blockade of dopaminergic receptors. Several speciﬁc genetic etiologies for dystonia have been identiﬁed, including disorders with autosomal dominant (AD) and recessive, and X-linked phenotypes. In addition, mitochondrial mutations producing predominantly dystonic symptoms have been described. Interestingly, most of

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the genetically determined dystonias have highly variable penetrance and considerable phenotypic variation even within single families. Moreover, there is also evidence for susceptibility genes for dystonia, which may interact with environmental triggers such as repetitive use in determining the development of dystonic disorders. Clinical Classifications of Dystonia Dystonias are classiﬁed in several different ways (Table 1). From a clinical perspective, the most useful classiﬁcations are by the age of onset and distribution of symptoms (2–5). These features have both diagnostic and prognostic signiﬁcance. Marsden et al. (6,7), ﬁrst pointed out that the age of onset of most dystonias is a predictor of the subsequent course: Early age at onset predicts a more severe course and higher likelihood of spread to multiple body parts (8). A subsequent study in the Ashkenazi Jewish population (which has a high prevalence of a genetically determined generalized dystonia, DYT1) demonstrated that there was a bimodal distribution of the ages at onset, with a nadir at 27 years (8,9). This age is often used as the dividing point between ‘‘early’’ and ‘‘late’’ onset dystonia, even in individuals who are not of Ashkenazi background. The distribution of symptoms is also an important and useful clinical descriptor. Current nomenclature divides the clinical forms of dystonia into focal, segmental (involving two or more contiguous body regions), multifocal (involving two or more non-contiguous body regions), and generalized (involving the legs and at least one other body part) (3,5). Hemidystonia, which might be considered segmental or multifocal, is classiﬁed separately from the other groups to reﬂect the fact that unlike the other forms it is often related to a demonstrable structural abnormality of the brain (5).

Table 1 Approaches to the Classiﬁcation of Dystonias By Age at Onset Early onset (<27 yr) Late onset (>27 yr) By Distribution Focal Segmental Multifocal Generalized Hemidystonia By Etiology Primary Primary-plus Secondary Heredodegenerative Psychogenic

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Classification of Dystonia by Etiology Recent efforts at classifying dystonias have attempted to move past clinical observation, and to classify dystonias by etiological mechanisms. This effort has not been simple or straightforward, largely because the understanding of the basic mechanisms underlying dystonia is incomplete. In addition, in many of these disorders no clear neuropathological abnormality has been identiﬁed. At present, the most widely accepted and useful approach divides dystonias into ﬁve categories by Etiology (Table 1) (3). In primary dystonia, dystonia is the only symptom and no underlying injury or disease can be identiﬁed. Primary dystonias include both apparent sporadic cases as well as deﬁned genetic disorders. Dystonia plus is the term applied to patients with dystonia and additional features, such as myoclonus, parkinsonism, or a paroxysmal course. This group includes patients with genetic biochemical defects, such as abnormalities of dopamine synthesis, but excludes those with neurodegenerative diseases. Secondary dystonia is used to describe dystonias arising after a wide variety of insults to the nervous system, including stroke, traumatic brain injury, or injury to the limbs. Heredodegenerative dystonia is used to describe the dystonia found in association with progressive degenerative neurological disorders, including Parkinson’s disease, Multiple Systems Atrophy, Wilson’s disease, and mitochondrial disorders (3). The ﬁfth group is apparent dystonias of psychogenic origin. The inclusion of this group reﬂects the fact that diagnosis of dystonia depends on the skills of the clinician, and in some cases the diagnosis may be in doubt. Further discussion in this chapter will focus on the genetics of the ﬁrst two categories of dystonia: the primary dystonias and dystonia-plus syndromes. These are the categories where the most progress has been made in deﬁning the underlying mechanism of the disease. It is important to acknowledge, however, that the forms of dystonia currently recognized as genetic in origin account for only a small fraction of the total number of cases of dystonia. In a clinical population, most cases are currently classiﬁed as idiopathic or sporadic primary dystonia (10). As the understanding of the underlying mechanisms continues to improve, this group should shrink in size with corresponding growth in the numbers of dystonia with an identiﬁable etiology. Genetic Classifications of Dystonia In addition to the clinically based classiﬁcations of dystonia, a genetically based nomenclature has arisen. Loci are assigned consecutive symbols preceded by ‘‘DYT.’’ Currently DYT1 to DYT15 are assigned. Some, but not all, of the gene symbols currently in use have been approved by the Nomenclature Committee of the Human Genome Organization (8,11,12). A confusing feature of this system is that when speciﬁc genes have been identiﬁed as the underlying etiology, some of the symbols have been reassigned. For example, the locus previously described as DYT5 is currently

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designated GCH1, reﬂecting the identiﬁcation of mutations in the gene for GTP cyclohydrolase I as the underlying defect in most cases. It should also be kept in mind that for most of the DYT subcategories, individuals or families with similar phenotypes but without linkage to described loci or mutations in known genes have been identiﬁed, suggesting locus heterogeneity. The Neural Mechanisms of Dystonia A mechanistic approach is an important framework for understanding the genetic origins of disease. Despite the recent progress in understanding how movements are controlled, the neural mechanism of dystonia remains largely a mystery, and an adequate model is lacking. It is important to recognize that dystonia is a disorder of the central nervous system, and not of the musculoskeletal system. This is illustrated dramatically in patients where dystonia has been treated by physical or chemical denervation of the overactive muscles (e.g., with botulinum toxin). Even if the involved muscles are treated successfully, the abnormal posture often reappears as additional muscle groups are recruited to the task. Thus, the problem is not in the muscles, but rather in the central representation of normal posture. This is not to say that the peripheral nervous system is unimportant. Indeed, the occurrence of dystonia after injury to peripheral nerves is a well-established phenomenon. Sensory input can also make an important contribution to the maintenance of dystonic postures, exempliﬁed by the ‘‘geste antagoniste,’’ a maneuver or position that through cutaneous stimulation at particular sites can greatly diminish the severity of dystonia in some patients. The search for the mechanism of dystonia has been difﬁcult because of the lack of clear structural abnormalities in the brain. Both in primary dystonias and in dystonia plus, routine neuropathological examination is normal. Two principal mechanisms have been implicated in the disease, which may be inter-related: abnormalities of dopaminergic neurotransmission in the basal ganglia and abnormalities of synaptic plasticity in the cerebral cortex. There is considerable evidence that dystonia can arise as a consequence of abnormal dopaminergic transmission in the basal ganglia. Acute dystonic reactions are often observed after treatment with potent dopamine D2-receptor antagonists such as prochlorperazine or haloperidol (13). In patients with secondary dystonia as a result of stroke, the lesions are often located in the putamen or pallidum, targets of dopaminergic inputs (14). A number of the dystonia-plus syndromes have been shown to result from biochemical abnormalities of dopamine synthesis and release (described further), and the gene responsible for DYT1 primary dystonia is strongly expressed by dopaminergic neurons (15). Although the connection to the dopaminergic system supports the view that dystonia is a basal ganglia disease, the current models of basal

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ganglia circuitry do not clearly account for the motor abnormalities seen in dystonia. The circuit diagrams were developed largely from observations in animals models of Parkinson’s and Huntington’s disease, and are built around ideas about the role of dopamine in regulating the activity of neurons in the striatum (caudate and putamen), the principal input structure of the basal ganglia (16). In recent years, it has become increasingly clear that a ‘‘wiring diagram’’ approach is overly simplistic, and it is important to consider not only the connections of the basal ganglia, but also the nature of the information ﬂowing through these pathways (17,18). Only relatively limited direct studies of the electrical activity of brain neurons in dystonia have been conducted, but these have revealed in many cases abnormal activity of output neurons in the basal ganglia (19,20). Studies in humans with dystonia by using neuroimaging and electrophysiological approaches have pointed to abnormalities in synaptic connectivity and plasticity outside the basal ganglia. Positron emission tomography (PET) and functional magnetic resonance imaging studies (fMRI) have revealed that dystonia is associated with abnormal activity in multiple regions of the brain, including motor cortex, supplementary motor areas, and cerebellum as well as structures of the basal ganglia (21–26), but in most cases it is not clear which of these is the primary defect and which are downstream modiﬁcations of activity. Using transcranial magnetic stimulation, it has been demonstrated that one feature of many focal task-speciﬁc dystonias is a ‘‘remapping’’ of the motor cortex, so that the area of cortex with direct inﬂuence over movements of the affected body part is greatly enlarged (27–29). There is also a prominent abnormality of cortical inhibition (30). Both of these observations suggest an underlying abnormality of synaptic plasticity, but the speciﬁc etiology of these functional abnormalities remains unclear. Recent morphometric studies have demonstrated abnormalities in the thickness of the cortical mantle in focal dystonia. Interestingly, these are present bilaterally, suggesting that in these individuals there is abnormal cortical structure even when no dystonia is detected clinically (31). An important focus of current research is ‘‘bridging the gap’’ between these mechanistic approaches to dystonia and the genetic etiologies as they are uncovered. In some cases, particularly the disorders of dopamine biosynthesis, considerable progress has been made. In other cases, the connection is much less apparent at present, and will require further understanding of the mechanisms of dystonia on a systems as well as cell biological level.

GENETICS OF PRIMARY DYSTONIAS DYT1: Early Onset Generalized Dystonia; Oppenheim’s Dystonia The most common cause of early onset generalized dystonia is DYT1 dystonia (or ‘‘Oppenheim’s Dystonia’’) caused by three base pair deletion

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(GAG deletion) in the DYT1 gene (4,32–36). This mutation accounts for approximately 74% of early onset limb dystonia in the general population and for roughly 90% of early onset limb dystonia in those of Ashkenazi Jewish descent (35). The increased incidence in the Ashkenazi Jewish population is due to the occurrence of a founder mutation in this population (32). DYT1 dystonia is inherited in an AD fashion, although the penetrance is only about 30% (9,37). The average age of onset is about 12 years, and onset after age 30 is very uncommon, although in rare cases symptoms have developed as late as 65 years (12,32). In those manifesting DYT1 dystonia, the severity can vary considerably, even within the same family (38–41). The dystonia typically begins in a limb (50% leg and 50% arm) and subsequently generalizes. Those with onset in the arm tend to begin later and are somewhat less likely to generalize than those with onset in the lower limb. Craniocervical or axial onset does occur but is less common (12,32,39). Rare cases with atypical features have been described (42). The dystonia can be quite severe and disabling, and the response to treatment is not completely satisfactory. Anticholinergic therapy, baclofen, and surgical approaches are often useful. The lack of phenotypic expression in 60–70% of individuals who carry the DYT1 mutation suggests the existence of modifying factors, either genetic or environmental, although the nature of these are unknown at present. Recently, a second mutation in the DYT1 gene, an 18 bp deletion of the carboxy terminus, has been identiﬁed in a pedigree with familial dystonia and myoclonus (43). The DYT1 gene encodes the protein torsinA (44,45). The encoded protein is 332 amino acids in length, corresponding to a predicted mass of 37,800 Da. There is a predicted amino terminal hydrophobic leader sequence, as well as potential sites for both glycosylation and phosphorylation. TorsinA is a member of the class of ‘‘ATPases associated with a variety of cellular activities’’ (AAAþ proteins, also see Chapter on hereditary spastic paraparesis) (45,46). Many of these proteins are chaperones that mediate protein folding or conformational change. All share a Mgþþ/ATP binding domain, an AAA-speciﬁc region, and form six-membered oligomeric rings. Other members of this class are N-ethylmalimide-sensitive factor (NSF) that is involved in vesicle fusion and release, dynein, and the Clp ATPase/HSP 100 proteins, which are stress-induced molecular chaperones and proteases. The mRNA for torsinA is expressed by dopamine neurons in the substantia nigra, as well as by neurons in other regions including the hippocampus and cerebellum (15). The functions of torsinA are largely unknown. The hydrophobic leader sequence suggests it is likely to be membrane-associated. In cellular models, over-expressed wild-type and mutant torsinA are found in the endoplasmic reticulum (47,48). A binding partner of torsinA is the molecular motor protein, kinesin, suggesting it has a role in vesicular transport (49). Over-expression of mutant torsinA results in multiple abnormal ER-derived

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cytoplasmic membrane inclusions. TorsinA is also found in association with the nuclear envelope, which is contiguous with the cytoplasmic ER (50–52). In cellular models as well as transgenic animals, the presence of the mutation that caused dystonia is reported to enhance the localization of the protein to the nuclear envelope (53). All of these mechanisms, of course, represent the effects of expressing torsinA at very high levels. Whether such abnormalities may occur at more physiologic levels of expression is unknown. Pathological studies of human brain from DYT1 dystonia are few. No deﬁnite evidence for neurodegeneration has been described thus far. There is no obvious alteration in the structure or density of dopaminergic neurons in the substantia nigra (54), but careful quantitative studies have not been performed yet. Studies of dopamine and its metabolites suggested an alteration of dopamine turnover, but the small number of cases available for these measurements limits the strength of this conclusion (55). Most studies have not observed any evidence for abnormal aggregation of torsinA in DYT1 cases, although a recent report has described ubiquitinated intraneuronal inclusions in three cases (56). Although the clinically apparent prevalence of the GAG deletion mutation in DYT1 is only about 30%, recent evidence from functional imaging studies suggests that patterns of neural activity may be abnormal even in apparent asymptomatic carriers (57). In two cohorts of clinically nonmanifesting carriers, Eidelberg et al. (58) have identiﬁed an abnormal pattern of regional cerebral metabolism, with increases in the posterior putamen and globus pallidus, cerebellum, and supplementary motor area. Clinically unaffected carriers also demonstrate abnormalities on motor sequence learning tasks, along with patterns of activation during the tasks that differ from normal subjects, most notable for enhanced activity of cerebellar structures (57). These data suggest that the DYT1 carrier state is associated with abnormal functional organization of motor learning and control, even when dystonia is not apparent. DYT6: Mixed Focal and Generalized Dystonia DYT6 is a form of mixed focal and generalized primary dystonia. The locus was identiﬁed in two Mennonite families comprising 220 individuals by Almasy et al. (59). The features were similar to DYT1 dystonia, except that the age of onset was later (18.9 years) and there was more prominent cranial, rather than limb, involvement. The locus has been linked to 8p21-q22, and subsequent investigation has traced the disorder in the two families to a common ancestor. The obligate linkage region has been narrowed to 23 cM, but the gene responsible remains unknown (60). Interestingly, both manifesting and nonmanifesting carriers from these families demonstrate abnormal patterns of cerebral metabolism very similar to those seen in DYT1 dystonia (57).

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DYT7: Adult-Onset Focal Dystonia The majority of cases of focal dystonia are classiﬁed as sporadic, but 25% of patients with focal dystonia have similarly affected relatives (61). This suggests that the underlying genetic contributions may be more signiﬁcant than is currently appreciated. In a single family from Northern Germany, a gene responsible for AD focal dystonia with reduced penetrance has been localized to chromosome 18p (62). In this family, average age of onset is 43 years (range 28–70). Family members present with a variety of forms of dystonia including; spasmodic dysphonia, craniocervical dystonia, writer’s cramp, and/or torticollis. Symptoms remain focal or multifocal in all cases. Several studies described linkage disequilibrium for several chromosome 18p markers in additional sporadic cases of focal dystonia in patients from Northwest Germany (63) and in members of affected families of Central European origin (64). Subsequent investigations have failed to replicate this observation (60,65,66). In other families with similar phenotype, linkage to 18p has been excluded, (67) suggesting that most cases of focal dystonia are not due to mutations on chromosome 18.

DYT13: Early Onset Focal and Generalized Dystonia The locus for DYT13 was identiﬁed in a large Italian kindred with 11 deﬁnitely affected family members (68). The phenotype in this family is similar to that of DYT6, with cranial, cervical and upper limb dystonia, with early onset (mean age 15.6 years). The locus has been linked to a region on the short arm of chromosome 1 (60,69).

GENETICS OF DYSTONIA PLUS SYNDROMES The dystonia plus disorders are characterized by the presence of dystonia together with additional symptoms, and the absence of neurodegeneration or other structural abnormality of the brain. The additional symptoms may include parkinsonism, myoclonus, or a paroxysmal expression of the dystonic movements. Many of the dystonia plus disorders are genetic in origin (Table 2), and several arise from genetic mutations that alter the biochemical synthesis or release of dopamine (5,12). Dopa-Responsive Dystonia; Segawa’s Syndrome Dopa-responsive dystonia with diurnal ﬂuctuation, or Segawa’s syndrome, is characterized clinically by childhood-onset dystonia with dramatic and sustained response to relatively low doses of levodopa (70,71). Inheritance

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Table 2 Hereditary Forms of Primary and Primary Plus Dystonias Genetic designation Inheritance Primary dystonia Early onset DYT1 dystonia— generalized DYT6 Adolescent— onset dystonia— mixed DYT7 Adult—onset dystonia— focal DYT13 Early onset dystonia— mixed Primary plus dystonia GCH1 Dopa(formerly responsive DYT5) dystonia— Segawa’s TH Doparesponsive dystonia— TH deﬁciency DopaDYT14 responsive dystonia 6-PTPS 6-pyruvoyltetrahydropterin synthase deﬁciency Sepiapterin SPR reductase deﬁciency Dihyropteridine DHPR reductase deﬁciency Aromatic LAADC amino acid decarboxylase deﬁciency

Chromosomal location

Gene product

OMIM identiﬁers

AD

9q34

TorsinA

605204, 128100

AD

8p21-8p22

Unknown

602629

AD

18p

Unknown

602124

AD

1p36.32p36.13

Unknown

607671

AD

14q22.1q22.2

GTP cyclohydrolase I

600225, 128230

AR

11p15.5

Tyrosine hydroxylase

605407, 191290

AD

14ql3

Unknown

607195

AR

11q22.3q23.3

261640

AR

2p14-p12

6-pyruvoyltetrahydropterin synthase Sepiapterin reductase

AR

4p15.31

AR

7p11

182125

Dihyropteri- 261630 dine reductase Aromatic L- 107930 amino acid decarboxylase (Continued)

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Table 2 Hereditary Forms of Primary and Primary Plus Dystonias (Continued ) Genetic designation Inheritance MyoclonusDYT11 AD dystonia MyoclonusDYT15 AD dystonia Rapid onset DYT12 AD dystonia parkinsonism Paroxysmal dystonias and dyskinesias DYT8 AD Paroxysmal nonkinesigenic dyskinesia (PNKD) DYT9 AD Choreoathetosis/ spasticity episodic (CSE) DYT10 AD Paroxysmal kinesigenic choreoathetosis (PKC)

Chromosomal location 7q21-31 18p11

Gene product e-Sarcoglycan Unknown

OMIM identiﬁers 159900 607488

19ql3

128235 Naþ/KþATPase a3 subunit

2q33-q35

Unknown

118800

1p21-p13.3 Unknown

601042

16p11.2q12.1

128200

Unknown

Listed are the name of the disorder, genetic designation (symbols approved by the Human Genome Organization are used where available), the pattern of inheritance (AD—autosomal dominant, AR—autosomal recessive), chromosomal location of the gene, the product of the gene, and the identiﬁers listed in Online Mendelian Inheritance in Man (OMIM; http://www.ncbi.nlm.nih. gov/Omim/).

is usually AD with variable penetrance. Patients with this disorder typically present with a dystonic gait abnormality and subsequently develop parkinsonian features (70,72) though wide phenotypic variability has been reported (73–78). The average age of onset is six years though it can range from infancy to adulthood (79). Penetrance is 2–4 times higher in females (87–100%) than in males (38–55%) (73,80). Classically, dystonic symptoms are most severe later in the day and are improved or absent in the morning (diurnal ﬂuctuation) or after a nap (sleep beneﬁt) (79). Sustained and complete response to levodopa without adverse effects of long-term treatment distinguishes Segawa’s syndrome clinically from other types of early onset parkinsonism with dystonia (70,81). About half of the patients with the clinical characteristics of Segawa’s syndrome have identiﬁable genetic defects in the gene for the enzyme GTP

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cyclohydrolase I (GCH1) (75,78,82–84). GCH1 is required for the biosynthesis of tetrahydrobiopterin, an essential cofactor for phenylalanine, tyrosine and tryptophan hydroxylases. Patients with a mutation in this gene have lowered tetrahydrobiopterin levels and thus impaired production of dopamine, other catecholamines and serotonin. Administration of levodopa bypasses the defect in dopamine synthesis, and can provide a long-lasting curative treatment for affected patients (70,81). Genetic testing for mutations in GCH1 is available, and may be useful in conﬁrming the diagnosis. However, as the currently available tests will identify only about half of the cases, all patients with a suggestive clinical phenotype should have a therapeutic trial of levodopa treatment (300–1000 mg daily). In those cases where the diagnosis remains unclear, an oral phenylalanine loading test or analysis of CSF pterins and neurotransmitter metabolites may be helpful (82,85). Measurement of neopterin and biopterin levels as well as GTPCH activity in cytokine-stimulated ﬁbroblasts may also be useful in establishing a diagnosis (86). Other genetic mechanisms may produce a Segawa’s syndrome phenotype. Although the inheritance of disease caused by GCH1 mutations is usually AD, recessive forms with mutations in both alleles have also been described (87). In addition, another autosomal recessive form of this disorder has been described in association with mutations in the tyrosine hydroxylase (TH) gene. Typically, however, mutations in the TH gene lead to a more severe phenotype (discussed further below). Another locus for dopa responsive dystonia has been identiﬁed in a single family. This disorder has been designated DYT14 and maps to chromosome 14q13, clearly outside the GCH1 gene (88).

Other Abnormalities of Dopamine Biosynthesis Associated with Dystonia and Parkinsonism A number of other genetic abnormalities of catecholamine biosynthesis can produce dystonia. A dramatic phenotype is observed in infants with defects in tyrosine hydroxylase, which converts tyrosine to levodopa. Only a very small number of these cases have been identiﬁed (89–91). Affected infants may present with parkinsonian and dystonic features early in life. The phenotype is variable, and may also include axial hypotonia, ptosis, developmental motor delay, hypokinesia, and appendicular rigidity without clearly associated dystonia (91–93). Treatment with levodopa bypasses this defect and may produce marked improvement. A similar clinical phenotype may be observed with defects in aromatic amino acid decarboxylase, which converts levodopa to dopamine, but this defect is much more difﬁcult to treat because there is a combined deﬁcit in both dopamine and serotonin production and the enzyme defect renders levodopa ineffective (94–100).

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Other disorders of catecholamine biosynthesis which may present with dystonia include 6-pyruvoyltetrahydropterin synthase deﬁciency (101); sepiapterin reductase deﬁciency; and dihydropteridine reductase deﬁciency (102). Further discussion of these rare disorders can be found in Hyland et al. (85) DYT11: Myoclonus-Dystonia Myoclonus-dystonia is a non-progressive disorder characterized by dystonic movements and postures in association with involuntary, lightning-like jerks (103). In most cases, this disorder is inherited in an AD fashion with reduced penetrance (103), although apparently sporadic cases may also be observed. Interestingly, the gene undergoes maternal imprinting such that those who inherit the mutated gene from their fathers are far more likely to display symptoms than those who inherit the gene from their mothers (104). Symptoms generally begin in childhood or adolescence and in many cases respond favorably to small amounts of alcohol (105,106). Some individuals in families with myoclonus dystonia display myoclonus without associated dystonia, suggesting that hereditary essential myoclonus is the same disorder (103,105,106). In nine families linkage to chromosome 7q21-q31 has been established (107). Using positional cloning, ﬁve different mutations in the gene for e-sarcoglycan (SGCE) have been identiﬁed in six German families (108). SGCE is a component of the dystrophin–glycoprotein complex. Four other sarcoglycan family members are known (a,b,g, and d); mutations in these other family members have been linked to limb-girdle muscular dystrophies but not to CNS disease (109). All of the mutations in SCGE appear to result in loss of function of the protein. This is unusual for an AD disorder, but this feature as well as the incomplete penetrance may be accounted for by the maternal imprinting of the SCGE gene (110). The mechanism by which defects in SCGE lead to the clinical features of myoclonus and dystonia remains unclear. There are also other genetic mechanisms that can produce a clinical phenotype similar to DYT11. A number of cases, both sporadic and familial have been described without mutation in SGCE (111,112). In addition, single variants in the dopamine D2 receptor (DRD2) and DYT1 genes were found in combination with SGCE mutations in two myoclonus dystonia families (43,113). Also, another family with myoclonus dystonia without a mutation in either SGCE or DRD2 has recently been described (114,115) and mapped to chromosome 18p11. This syndrome is designated DYT15. DYT12: Rapid Onset Dystonia-Parkinsonism Rapid onset dystonia-parkinsonism is a disease with a striking phenotype. Affected individuals develop symptoms over a period of hours to days, with

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limb and cranial dystonia, bradykinesia and postural instability. Once they occur, the symptoms often persist throughout life. The disorder is inherited in an AD pattern. The onset is usually in young adults, and often is triggered by a stressful event such as fever, prolonged exercise or childbirth (116–118). The locus was linked to 19q13.2 (119). A recent analysis of candidate genes in the implicated region of chromosome 19 has revealed that the underlying mutation is a defect in the Naþ/Kþ-ATPase a3 gene (120). This is an electrogenic sodium pump responsible for the maintenance of ionic gradients. Six different mutations were identiﬁed in seven unrelated families with the DYT12 phenotype. All seem to inhibit the function of the Naþ/Kþ pump. Interestingly, mutations in another member of the Naþ/Kþ-ATPase family, a2, have been implicated in familial hemiplegic migraine (121,122). The mechanisms by which defects in an electrogenic sodium pump lead to the clinical phenotype of abrupt and persistent dystonia is unclear. One possibility is that the metabolic abnormality predisposes a critical population of neurons to injury, and in the face of a systemic stress they undergo permanent functional change or perhaps even cell death. Only a single brain from an individual with DYT12 has been examined, and no clear evidence for neurodegeneration was observed in this case (118). Clearly, further examination of this question is required.

GENETICS OF PAROXYSMAL DYSTONIAS AND DYSKINESIAS Paroxysmal dystonia is a unique clinical phenotype, where patients have discrete episodes of abnormal movements including hyperkinesias, dystonia, chorea, athetosis and ballism (123,124). Between these events, in most cases, clinical examination is normal. The abnormal movements may be triggered by activity (kinesigenic) or may occur at rest (non-kinesigenic). It is important to distinguish these disorders from other paroxysmal disease such as epilepsy or arrhythmias. Indeed, a nocturnal paroxysmal disorder originally described as hypnogenic dystonia has recently been shown to be a form of nocturnal frontal lobe epilepsy, caused by a mutation in the a4 subunit of the neuronal nicotinic acetylcholine receptor (125). Many of the paroxysmal dystonias are genetic, although both sporadic and secondary forms may occur. DYT8 and DYT9: Paroxysmal Non-kinesigenic Dyskinesias DYT8, or paroxysmal non-kinesigenic dyskinesia (PNKD), was ﬁrst described in a single large family where it was inherited in an AD fashion (126). Symptoms in this and subsequently identiﬁed families typically began shortly after birth but in some cases did not manifest until later childhood or adolescence. Attacks were precipitated by emotional stress, fatigue, and intake of chocolate

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or alcohol. Attacks were frequently preceded by aura and followed by uni- or bilateral dystonic or choreathetotic dyskinesias of two minutes to four hours duration. Frequency of episodes varied considerably from 20 per day to twice per year (127). The gene for this condition was mapped in four additional families to chromosome 2q33-q35 (128–131). This region of the chromosome contains a cluster of genes for sodium channels (e.g., anion exchanger SLC4A3) (128–131), but no mutation has been identiﬁed thus far (132). Phenotypically similar families not linked to chromosome 2 or 16 have also been described (67). A more complex paroxysmal phenotype is illustrated by DYT9, also known as choreoathetosis/spasticity episodic (CSE). A single family with this syndrome has been described (133). Individuals in this pedigree display episodic involuntary movements with dystonic posture of the limbs associated with dysarthria, paresthesias and double vision. Paroxysms are usually 20 minutes in duration with a frequency ranging from twice per day to twice per year. Episodes are precipitated by physical exercise, emotional stress, sleep deprivation and consumption of alcohol. In contrast to other forms of paroxysmal dystonia, ﬁve of eighteen affected members of this pedigree displayed spastic paraplegia between attacks. The gene for this disorder has been mapped to chromosome 1p (133). DYT10: Paroxysmal Kinesigenic Choreoathetosis Paroxysmal kinesigenic choreoathetosis (PKC) is the most frequent of the paroxysmal dyskinesias. Inheritance appears to be AD with reduced penetrance, although many cases appear sporadic. Symptoms begin in childhood to early adulthood. In contrast to the non-kinesigenic forms, dyskinesias are precipitated by sudden movements or startle, tend to have a later age of onset, and respond to anticonvulsant therapy (12,123,127,134). Attacks are of short duration (<5 min) and may occur frequently, often several times a day. Linkage has been established in eight Japanese families to 16p11.2-q12.1. Some families also have an increased incidence of infantile seizures. The overlap of clinical symptoms between this disorder and three other syndromes which all map to the same region on chromosome 16p— benign familial infantile convulsions, infantile convulsions and paroxysmal choreoathetosis (135), as well as rolandic epilepsy with paroxysmal exercise induced dystonia and writers cramp (136) suggests that these may be allelic disorders (137–139). Genetic Susceptibility to Dystonia Despite the substantial number of different genetic defects that are now known to cause dystonia, it is also clear that these account for only a very small fraction of the total number of cases of dystonia, and most forms are

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still classiﬁed as sporadic. Therefore the diagnosis is primarily based on clinical evaluation. Genetic testing based on clinical phenotype may be informative in some individuals to conﬁrm a diagnosis and provide genetic counseling to at-risk family members. Due to features of reduced penetrance and variable expressivity, which are common in many hereditary forms of dystonia, counseling families can be complicated. It is likely that there are genetic factors that increase or decrease the risk of dystonia, but few have been worked out in any detail. A family history of dystonia as well as a family history of tremor have been reported to increase the risk of dystonia (140,141). Co-occurrence of dystonia in a small number of twin pairs has also been reported (142,143). One gene which has been implicated in several independent case-control studies is the dopamine D5 receptor (144,145). It has also been reported that the e4 allele of apolipoprotein E, which has a strong effect on the incidence of Alzheimer’s disease, is associated with an early age of onset of dystonia (146). Clearly, more extensive study of genetic risk factors will be required to fully appreciate the contribution of genetics to the occurrence of dystonia. REFERENCES 1. Jankovic J, Fahn S. Dystonic Disorders. In: Jankovic J, Tolosa E, eds. Parkinson’s Disease and Movement Disorders. Baltimore: Williams and Wilkins, 1993: 337–374. 2. Marsden CD. Investigation of dystonia. Adv Neurol 1988; 50:35–44. 3. Fahn S, Bressman SB, Marsden CD. Classiﬁcation of dystonia. Adv Neurol 1998; 78:1–10. 4. Bressman SB, De Leon D, Raymond D, Ozelius LJ, Breakeﬁeld XO, Nygaard TG, Almasy L, Risch NJ, Kramer PL. Clinical-genetic spectrum of primary dystonia. Adv Neurol 1998; 78:79–92. 5. Fahn S. Concept and classiﬁcation of dystonia. Adv Neurol 1988; 50:1–8. 6. Marsden CD, Harrison MJ. Idiopathic torsion dystonia (dystonia musculorum deformans). A review of forty-two patients. Brain 1974; 97:793–810. 7. Marsden CD. Dystonia: the spectrum of the disease. Res Publ Assoc Res Nerv Ment Dis 1976; 55:351–367. 8. Bressman SB. Dystonia genotypes, phenotypes, and classiﬁcation. Adv Neurol 2004; 94:101–107. 9. Bressman SB, De Leon D, Brin MF, Risch N, Burke RE, Greene PE, Shale H, Fahn S. Idiopathic dystonia among Ashkenazi Jews: evidence for autosomal dominant inheritance. Ann Neurol 1989; 26:612–620. 10. Nutt JG, Muenter MD, Aronson A, Kurland LT, Melton LJ. Epidemiology of focal and generalized dystonia in Rochester, Minnesota. Mov Disord 1988; 3:188–194. 11. Muller U, Steinberger D, Nemeth AH. Clinical and molecular genetics of primary dystonias. Neurogenetics 1998; 1:165–177. 12. Klein C, Breakeﬁeld XO, Ozelius LJ. Genetics of primary dystonia. Semin Neurol 1999; 19:271–280.

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120. De Carvalho Aguiar P, Sweadner KJ, Penniston JT, Zaremba J, Liu L, Caton M, Linazasoro G, Borg M, Tijssen MA, Bressman SB, Dobyns WB, Brashear A, Ozelius LJ. Mutations in the Na(þ)/K(þ)-ATPase alpha3 Gene ATP1A3 Are Associated with Rapid-Onset Dystonia Parkinsonism. Neuron 2004; 43:169–175. 121. De Fusco M, Marconi R, Silvestri L, Atorino L, Rampoldi L, Morgante L, Ballabio A, Aridon P, Casari G. Haploinsufﬁciency of ATP1A2 encoding the Naþ/Kþ pump alpha2 subunit associated with familial hemiplegic migraine type 2. Nat Genet 2003; 33:192–196. 122. Vanmolkot KR, Kors EE, Hottenga JJ, Terwindt GM, Haan J, Hoefnagels WA, Black DF, Sandkuijl LA, Frants RR, Ferrari MD, van den Maagdenberg AM. Novel mutations in the Naþ, Kþ-ATPase pump gene ATP1A2 associated with familial hemiplegic migraine and benign familial infantile convulsions. Ann Neurol 2003; 54:360–366. 123. Fahn S. Paroxysmal Dyskinsias. In: Marsden CD, Fahn S, eds. Movement Disorders 3. Oxford: Butterworth-Heineman, 1994:310–345. 124. Bhatia KP. The paroxysmal dyskinesias. J Neurol 1999; 246:149–155. 125. Phillips HA, Scheffer LE, Berkovic SF, Hollway GE, Sutherland GR, Mulley JC. Localization of a gene for autosomal dominant nocturnal frontal lobe epilepsy to chromosome 20q 13.2. Nat Genet 1995; 10:117–118. 126. Mount L, Reback S. Familial paroxysmal choreoathetosis: preliminary report on an hitherto undescribed clinical syndrome. Archives of Neurology and Psychiatry 1940; 44:841–847. 127. Demirkiran M, Jankovic J. Paroxysmal dyskinesias: clinical features and classiﬁcation. Ann Neurol 1995; 38:571–579. 128. Fouad GT, Servidei S, Durcan S, Bertini E, Ptacek LJ. A gene for familial paroxysmal dyskinesia (FPD1) maps to chromosome 2q. Am J Hum Genet 1996; 59:135–139. 129. Fink JK, Rainer S, Wilkowski J, Jones SM, Kume A, Hedera P, Albin R, Mathay J, Girbach L, Varvil T, Otterud B, Leppert M. Paroxysmal dystonic choreoathetosis: tight linkage to chromosome 2q. Am J Hum Genet 1996; 59:140–145. 130. Jarman PR, Davis MB, Hodgson SV, Marsden CD, Wood NW. Paroxysmal dystonic choreoathetosis. Genetic linkage studies in a British family. Brain 1997; 120:2125–2130. 131. Raskind WH, Bolin T, Wolff J, Fink J, Matsushita M, Lift M, Lipe H, Bird TD. Further localization of a gene for paroxysmal dystonic choreoathetosis to a 5-cM region on chromosome 2q34. Hum Genet 1998; 102:93–97. 132. Grunder S, Geisler HS, Rainier S, Fink JK. Acid-sensing ion channel (ASIC) 4 gene: physical mapping, genomic organisation, and evaluation as a candidate for paroxysmal dystonia. Eur J Hum Genet 2001; 9:672–676. 133. Auburger G, Ratzlaff T, et al. A gene for autosomal dominant paroxysmal choreoathetosis/spasticity (CSE) maps to the vicinity of a potassium channel gene cluster on chromosome 1p, probably within 2 cM between D1S443 and D1S197. Genomics 1996; 31:90–94. 134. Kertesz A. Paroxysmal kinesigenic choreoathetosis. An entity within the paroxysmal choreoathetosis syndrome. Description of 10 cases, including 1 autopsied. Neurology 1967; 17:680–690.

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135. Swoboda KJ, Soong BW, McKenna C, Brunt ER, Litt M, Bale JF, Jr., Ashizawa T, Bennett LB, Bowcock AM, Roach ES, Gerson D, Matsuura T, Heydemann PT, Nespeca MP, Jankovic J, Leppert M, Ptacek LJ. Paroxysmal kinesigenic dyskinesia and infantile convulsions. Clinical and linkage studies. 2000. Neurology 2001; 57:S42–48. 136. Guerrini R, Bonanni P, Nardocci N, Parmeggiani L, Piccirilli M, De Fusco M, Aridon P, Ballabio A, Carrozzo R, Casari G. Autosomal recessive rolandic epilepsy with paroxysmal exercise-induced dystonia and writer’s cramp: delineation of the syndrome and gene mapping to chromosome 16p12–11.2. Ann Neurol 1999; 45:344–352. 137. Caraballo R, Pavek S, Lemainque A, Gastaldi M, Echenne B, Motte J, Genton P, Cersosimo R, Humbertclaude V, Fejerman N, Monaco AP, Lathrop MG, Rochette J, Szepetowski P. Linkage of benign familial infantile convulsions to chromosome 16p12-q12 suggests allelism to the infantile convulsions and choreoathetosis syndrome. Am J Hum Genet 2001; 68:788–794. 138. Nemeth AH. The genetics of primary dystonias and related disorders. Brain 2002; 125:695–721. 139. Lotze T, Jankovic J. Paroxysmal kinesigenic dyskinesias. Semin Pediatr Neurol 2003; 10:68–79. 140. Defazio G, Berardelli A, Abbruzzese G, Lepore V, Coviello V, Acquistapace D, Capus L, Carella F, De Berardinis MT, Galardi G, Girlanda P, Maurri S, Albanese A, Bertolasi L, Liguori R, Rossi A, Santoro L, Tognoni G, Livrea P. Possible risk factors for primary adult onset dystonia: a case-control investigation by the Italian Movement Disorders Study Group. J Neurol Neurosurg Psychiatry 1998; 64:25–32. 141. Fletcher NA, Harding AE, Marsden CD. A case-control study of idiopathic torsion dystonia. Mov Disord 1991; 6:304–309. 142. Factor SA. Cervical dystonia in twins. Mov Disord 2002; 17:846–847. 143. Wunderlich S, Reiners K, Gasser T, Naumann M. Cervical dystonia in monozygotic twins: case report and review of the literature. Mov Disord 2001; 16:714–718. 144. Misbahuddin A, Placzek MR, Warner TT. Focal dystonia is associated with a polymorphism of the dopamine D5 receptor gene. Adv Neurol 2004; 94: 143–146. 145. Placzek MR, Misbahuddin A, Chaudhuri KR, Wood NW, Bhatia KP, Warner TT. Cervical dystonia is associated with a polymorphism in the dopamine (D5) receptor gene. J Neurol Neurosurg Psychiatry 2001; 71:262–264. 146. Matsumoto S, Nishimura M, Sakamoto T, Asanuma K, Izumi Y, Shibasaki H, Kamatani N, Nakamura T, Kaji R. Modulation of the onset age in primary dystonia by APOE genotype. Neurology 2003; 60:2003–2005.

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19 Inherited Epilepsies Yr Sigurdardottir Department of Pediatrics, University Hospital Iceland, Reykjavik, Iceland

Annapurna Poduri Division of Epilepsy and Neurophysiology, Children’s Hospital Boston, Boston, Massachusetts, U.S.A.

INTRODUCTION Epilepsy, deﬁned by recurrent, unprovoked seizures, is a relatively common neurologic disorder with a prevalence of 4–8 per 1000 in the United States (1). The hereditary transmission of seizure disorders has long been suspected in humans, and carefully designed epidemiologic studies have identiﬁed a substantial genetic contribution to epilepsy (2,3). These studies have shown that close relatives of index cases have an increased risk of epilepsy compared to the general population, and twin studies have shown higher concordance in monozygotic twins when compared with dizygotic pairs (4). There are over one hundred single gene (Mendelian) disorders whose phenotype includes recurrent seizures (OMIM). These conditions are usually so rare, however, that collectively they only shed light on the etiology of epilepsy in a mere fraction of patients with epilepsy. Nonetheless, it is through the study of these disorders and their genetic underpinnings that we may better understand cortical excitability, seizure threshold, and other aspects of human epilepsy. More often, we face the more complex situation of genetic susceptibility to epilepsy that does not follow a clear Mendelian inheritance pattern. When the genetics of epilepsy was comprehensively reviewed in 1980 by Newmark and Penry (5), the evidence for the genetic basis of epilepsy rested

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largely on animal models and the examination of pedigrees of families with epilepsy. In the past 25 years, emerging discoveries in molecular genetics have conﬁrmed the genetic basis for many epilepsy syndromes, and in some cases the putative mechanisms for epileptogenesis have been established. The mapping of single gene disorders starts with the identiﬁcation of families with multiple affected members, preferably with a single seizure phenotype. The clinician’s task is paramount in identifying appropriate index cases. The next step is to perform linkage analysis to map a disorder to a chromosomal region. If this can be accomplished, positional cloning techniques can be employed to identify the speciﬁc gene involved. Subsequently, functional studies can be performed to assess the function of the associated gene product and its role in epileptogenesis. In the future, such advances should lead to a more precise and targeted pharmaceutical approaches to epilepsy than we have now. There are, of course, numerous conditions such as structural brain malformations and inborn errors of metabolism that include epilepsy in their phenotype and have known or presumed genetic etiologies. We will focus this chapter, however, on diseases that are deﬁned chieﬂy by epilepsy. Table 1 outlines inherited epilepsies for which there is evidence for an involved chromosomal region or locus. Note that there are multiple genes or loci listed for some conditions and that multiple, allelic disorders are found at some loci. Other epilepsy syndromes with a suspected but as yet unconﬁrmed genetic component are listed in Table 2. The important concepts of single gene disorders can be illustrated by focusing on the channelopathies that are associated with epilepsy and some of the myoclonic epilepsy syndromes that follow clear patterns of inheritance. Some of these disorders demonstrate genetic heterogeneity, with several separate mutations at different loci producing the same clinical phenotype. The so-called ‘‘idiopathic’’ generalized and localization-related epilepsies of childhood and adolescence are examples of epilepsies with both single gene and complex, presumed polygenetic, inheritance. CHANNELOPATHIES The channelopathies represent the prototype of epilepsies with single gene abnormalities that give rise to abnormalities in proteins plausibly involved in mechanisms of epileptogenesis. It is not surprising that epilepsy, a condition deﬁned by abnormally synchronous neuronal events giving rise to repeated seizures, can be associated with, if not caused by, abnormalities of ion channel structure or function. Animal models of ion channel defects have established the connection between disease-producing mutations and their associated clinical phenotypes. Several human epilepsy syndromes illustrate the importance of ion channels in the development of seizures and epilepsy. Interestingly, while most of the mouse models involve homozygous mutations

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Chromosal locus

– CLN1 CLN1 ATP1A2 – – CACNB4 FEB3 SCN2A SCN2A – SCN1A SCN1A CLCN2 CLCN2 CLCN2 FEB4 GABRG2 GABRG2 GABRA1 EJM1 – EFHC1 NEU1 EPM2B

Epilepsy syndrome Childhood absence evolving to juvenile myoclonic epilepsy Infantile NCL (Santavuori–Haltia) Some adult NCL (Kufs) Benign familial infantile convulsions (BFIC)a (199) Benign adult familial myoclonic epilepsy (BAFME)a Autosomal dominant cortical myoclonus and epilepsy (ADCME) Juvenile myoclonic epilepsy (JME)a Familial febrile convulsions (FFC)a Benign familial neonatal/infantile convulsions (BFNIC) Generalized epilepsy with febrile seizures plus (GEFSþ)a Benign familial infantile convulsions (BFIC)a Generalized epilepsy with febrile seizures plus (GEFSþ)a Severe myoclonic epilepsy of infancy (SMEI)a Childhood absence epilepsy (CAE) (ECA3) Juvenile absence epilepsy (JAE) Juvenile myoclonic epilepsy (JME)a Familial febrile convulsions (FFC)a Childhood absence epilepsy (CAE) (ECA2) Generalized epilepsy with febrile seizures plus (GEFSþ)a Juvenile myoclonic epilepsy (JME)a Generalized tonic–clonic seizures of awakening Juvenile myoclonic epilepsy (JME)a Juvenile myoclonic epilepsy (JME)a Sialidosis types I and II Lafora body diseasea

Inheritance AD AR AR AD AD AD Complex AD AD AD AD AD De novo Complex Complex Complex AD Complex AD Complex AD Complex AD AR AR (Continued)

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1p 1p32 1p32 1q23 2p11.1-2q12.2 2p11.1-2q12.2 2q22-q23 2q23-q24 2q23-q24.3 2q23-q24.3 2q24 2q24 2q24 3q26-qter 3q26 3q26 5q14-q15 5q31.1 5q34 5q34 6p 6p11 6p12-p11 6p21.3 6p22

Gene

Inherited Epilepsies

Table 1 Epilepsy Syndromes with Established Genetic Basis by Chromosomal Locus

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Table 1

Epilepsy Syndromes with Established Genetic Basis by Chromosomal Locus (Continued )

Chromosal locus

Gene EPM2A CLN7 CLN8 – FEB1 – KCNQ3 – LGI

11p15.5 12p13.31 13q21.1-q32 15q14 15q21-q23 16p12.1 16p12-q12 16p12-q12

CLN2 DRPLA CLN5 CHRNA7 CLN6 CLN3 – –

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Lafora body diseasea Late infantile NCL (Turkish) Childhood NCL Northern epilepsy variant Childhood absence epilepsy (CAE) (ECA1) Familial febrile convulsions (FFC)a Benign adult familial myoclonic epilepsy (BAFME)a Benign familial neonatal convulsions (BFNC)a Partial epilepsy with auditory features Autosomal dominant lateral temporal lobe epilepsy (ADLTE) Late infantile NCL (Jansky–Bielschowsky) Dentatorubral pallidoluysian atrophy (DRPLA) Late infantile NCL (Finnish) Juvenile myoclonic epilepsy (JME)a Late infantile NCL (variant) Juvenile NCL (Spielmeyer–Vogt) Benign familial infantile convulsions (BFIC)a Infantile convulsions and choreoathetosis (ICCA)

Inheritance AR AR AR Complex AD AD AD AD AD AR AD (CAGn) AR Complexa AR AR AD AD

Sigurdardottir and Poduri

6q23-q25 8pter-p22 8p23 8q24 8q13-q21 8q23.3-q24.11 8q24.22 10q 10q22-q24

Epilepsy syndrome

–

16p13 19q12-q13.1 19p13 19q13.1 19p13.3 20q13.2 20q13.32-13.33 21q22.3 22q12 Xp21.3-p22.1 Xp22.3 Mitochondrial loci

– – CACNA1A SCN1B FEB2 CHRNA4 KCNQ2 CSTB – ARX STK9 MTTK MTTLI MTTH MTTS1

Rolandic epilepsy with paroxysmal exercise-induced dystonia and writer’s cramp Familial idiopathic myoclonic epilepsy of infancy (200) Benign familial infantile convulsions (BFIC)a Episodic ataxia type 2 with seizures Generalized epilepsy with febrile seizures plus (GEFSþ)a Familial febrile convulsions (FFC)a Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) Benign familial neonatal convulsions (BFNC)a Unverricht–Lundborg disease Familial partial epilepsy with variable foci (FPEVF) (201) X-linked infantile spasmsa (202) X-linked infantile spasmsa (203) Myoclonic epilepsy with ragged red ﬁbers (MERRF)a

AR AR AD AD, sporadic AD AD AD AD AR AD X-linked X-Iinked Mitochondrial Mitochondrial Mitochondrial Mitochondrial

Inherited Epilepsies

16p12-11.2

a denotes diseases that have been associated with multiple loci. A dash (–) indicates that the information is not established. Abbreviations: CAGn, CAG repeat expansion; NCL, neuronal ceroid lipofuscinosis.

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Table 2 Epilepsy Syndromes with Presumed Genetic Basis Benign myoclonic epilepsy of infancy Familial temporal lobe epilepsy Febrile seizures Common generalized epilepsies of childhood (most cases) Childhood absence epilepsy Juvenile absence epilepsy Juvenile myoclonic epilepsy Benign Epilepsy with Centro-Temporal Spikes (BECTS) Myoclonic-astatic epilepsy (Doose’s syndrome) Reading epilepsy

of channel-associated proteins, the majority of the channelopathies associated with human disease display an autosomal dominant (AD) pattern of inheritance. An exception is childhood absence epilepsy (CAE), in which the T-type calcium channel appears to be the site of abnormality but inheritance appears to be multifactorial in most cases; this condition is discussed below in the ‘‘Generalized Epilepsy Syndromes’’ section. Calcium Channelopathies: Mouse Models and Human Disease Several autosomal recessive (AR) mouse mutants with epilepsy have been identiﬁed. The best known is the tottering (tg) mutant, which has ataxia and absence epilepsy with generalized spike-wave (SW) discharges on electroencephalogram (EEG) (6). In 1996, Fletcher et al. identiﬁed a mutation in a gene that encodes the a1A subunit of the voltage-gated calcium channel (VGCC) in the tottering mouse mutant (7). This remains a prototype of a single gene mutation causing epilepsy. Three additional allelic mouse mutants—leaner (tgla), rolling Nagoya (tgrol), and rocker (rkr)—all demonstrate ataxia with or without seizures (7–9). VGCCs are composed of four primary subunits (a1, b, g, and a2d) that are believed to be arranged in a 1:1:1:1 stoichiometry. Several mouse mutants have been identiﬁed with mutations in the b, g, and a2d subunits, all demonstrating seizure phenotypes (Table 3). Since generalized epileptiform discharges and absence epilepsy dominate the clinical manifestations in these animal models, one might expect that calcium channel subunit mutations would be associated with epilepsy in humans. Until recently, however, the three allelic human disorders with mutations in the CACNA1A gene on chromosome 19—familial hemiplegic migraine (FHM), spinocerebellar ataxia type 6 (SCA6), and episodic ataxia type 2 (EA2)—were not associated with epilepsy, although EEG abnormalities had been described (10). There is mounting evidence that mutations in VGCC subunits cause human epilepsy. CACNA1A mutations have been identiﬁed in a family with

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Table 3 P/Q Calcium Channel Mouse Mutant Models

Subunit alA a2d2 b4 g2 g3

Mouse chromosomal locus

Mouse phenotype

Human chromosomal locus

Human phenotype

8 9 2 15 –

tg, tgla, tgrol rkr du lh stg, stg-wag

19p13.1 3p21.3-21.2 2q22-23 22cen-qter 16p13.3-12

FHM, EA2, SCA6, sz – ataxia, sz, JME – Chorea, infantile sz

A dash (–) indicates that the information is not established. Abbreviations: tg, tottering; tgla, leaner; tgrol, Nagoya rolling; rkr, rocker; FHM, familial hemiplegic migraine; du, ducky (30); lh, lethargic (24); EA2, episodic ataxia type 2; SCA6, spinocerebellar ataxia type 6; sz, seizures; JME, juvenile myoclonic epilepsy; stg, stargazer (31); stg-wag, waggler.

EA2, generalized epileptiform discharges on EEG, and childhood seizures (11). Another report of a patient with developmental delay, ataxia, and epilepsy identiﬁed a de novo CACNA1A mutation (12). Human epilepsy has also been found in patients with mutations in the b and g subunits of the calcium channel as well. The fact that most patients with EA2 do not have epilepsy supports the concept that a critical level of channel dysfunction may be required before SW activity is generated and clinical seizures appear (11–14). The mechanisms by which mutated calcium channels lead to the development of epilepsy have been extensively studied in the above mentioned spontaneous mouse mutants harboring homozygous mutations in genes encoding various subunits of the calcium channel complex (15–18). Some mutants have increased expression of tyrosine hydroxylase, a key enzyme in noradrenergic biosynthesis (19), while in other mutants increased numbers of GABA-B receptors seem to result in thalamo-cortical hypersynchronization that leads to generalized seizures (17). Other Clinical Epilepsy Syndromes Associated with Channelopathies In addition to EA2 with seizures, there are several clinical syndromes characterized chieﬂy by epilepsy that have been associated with mutations in channel-encoding genes. The epilepsies associated with channelopathies are summarized in Table 4. Benign Familial Neonatal Convulsions The syndrome of benign familial neonatal convulsions (BFNC) was ﬁrst described in the mid-1960s by Rett and Teubel (20) and Bjerre and Corelius (21). This AD syndrome is characterized by unprovoked seizures in the neonatal period, which remit within a few months of life and are followed

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Table 4 Inherited Channelopathies Associated with Epilepsy Epilepsy syndrome Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) Benign familial neonatal convulsions (BFNC) Benign familial infantile convulsions (BFIC)

Familial febrile convulsions (FFC)

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Gene

Protein

Chromosomal locus

AD

CHRNA4

nAch-R

20q13.2

AD

KCNQ2

VGPC

20q13.32-13.33 (EBNl)

AD

KCNQ3 ATP1A2

VGPC Na/K-ATPase

8q24.22 (EBN2) 1q23

AD

– – – SCN2A

– – – VGSC

2q24 16p12-q12 19q12-q13.1 2q23-q24.3

CACNA1A

VGCC

19p13

FEB1 FEB2 FEB3 FEB4

– – – –

8q13-q21 19p13.3 2q23-q24 5q14-q15

AD, some de novo AD

Sigurdardottir and Poduri

Benign familial neonatal/infantile convulsions (BFNIS) Episodic ataxia type 2 (EA2) with seizures

Inheritance

Type 3 Infantile convulsions and choreoathetosis (ICCA) Severe myoclonic epilepsy of infancy Severe myoclonic epilepsy of infancy atypical variant

AD

AD

SCN1B SCN1A SCN2A GABRG2 –

VGSC VGSC VGSC GABA-A-R –

19q13.1 2q24 2q23-q24.3 5q34 16p12-q12

De novo

SCN1A

VGCC

2q24

GABRG2 SCN2A

GABA-A-R VGSC

5q31.1-q33.1 2q23-q24.3

Inherited Epilepsies

Generalized epilepsy with febrile seizures plus (GEFSþ) Type l Type 2

A dash (–) indicates that the information is not established. Abbreviations: nAch-R, nicotinic acetylcholine receptor; GABA-A-R, GABA-A receptor; VGCC, voltage-gated calcium channel; VGPC, voltage-gated potassium channel; VGSC, voltage-gated sodium channel.

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by normal psychological development. The seizures are clinically heterogeneous and consist of eye twitching, occasional tonic posturing, and often clonic movements of extremities. Ictal EEG recordings typically reveal a period of voltage attenuation followed by bilateral rhythmic spiking during the tonic–clonic phase (22–24). BFNC is usually categorized as a generalized epilepsy although some reports of focal EEG features have challenged that view (23,24). Each seizure is brief, typically less than two minutes in duration, but up to 40 seizures per day can occur. Patients with BFNC usually respond well to treatment and experience a lasting remission, although the prevalence of subsequent epilepsy is higher than in the general population (15% vs. 1%). In 1989, this disease was mapped to chromosome 20, and in 1992 further analysis narrowed the region to 20q13.32-13.33 (25,26). Evidence for genetic heterogeneity surfaced quickly when in 1991 a family with clinical BFNC mapped to chromosome 8q24.22 (27). In the late 1990s, both involved loci were found to contain genes encoding voltagegated potassium channel subunits, the chromosome 20 gene encoding KCNQ2 and the chromosome 8 gene KCNQ3 (28,29). Numerous different mutations within these genes have been found, all leading to a similar disease phenotype (25–30), and no clear difference has been found between the clinical presentations of individuals with mutations in KCNQ2 and KCNQ3. Functional studies of the role of voltage-gated potassium channels in cortical excitability have shown that wild-type KCNQ2 and KCNQ3 channels are associated with a current comparable to the native M-current, a slowly activating–deactivating potassium current that plays a critical role in determining the subthreshold electrical excitability of neurons (31,32). Mutated channels have smaller associated potassium currents, and recent studies have shown that a 25% reduction of the M-current can lead to epilepsy (33). The age dependency of this syndrome remains fascinating, and most researchers hypothesize that the KCQN2 and KCNQ3 genes are most actively transcribed in the ﬁrst weeks of life. A recently described family with a clinical BFNC phenotype has been found to have a pericentric inversion of chromosome 5 (34), and one family with BFNC phenotype and AR inheritance was described in 1991 (35). This further points to considerable genetic heterogeneity, and it is likely that more loci will be identiﬁed with time. Benign Familial Infantile Convulsions and Infantile Convulsions and Choreoathetosis Benign familial infantile convulsion (BFIC) is another benign AD epilepsy syndrome of early infancy. It is characterized by an age of seizure onset around six months of age (range 3–9 months) with resolution by 12 months of age and no subsequent neurologic abnormalities. The seizures are typically partial or secondarily generalized and respond well to antiepileptic medication. BFIC was ﬁrst described by Vigevano et al. (36) in 1992. He

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reported ﬁve Italian infants with the typical phenotype, each with a family history of infantile seizures. Seventeen additional families were reported soon afterwards (37,38). Linkage analysis in eight affected Italian families ruled out involvement of the BFNC locus on chromosome 20 (39), and the BFIC gene was later linked to chromosome 19 in ﬁve of these eight families (40). The report of a second locus at 16p12-q12 demonstrates the genetic heterogeneity of BFIC (41–43). Three overlapping clinical entities are seen with mutations at this second locus. The earliest described was the BFIC phenotype. Since then, families with infantile convulsions and choreoathetosis (ICCA), a kinesigenic movement disorder, have been reported (43,44), as well as others with paroxysmal kinesogenic choreoathetosis (PKC), a movement disorder without seizures (45). These three syndromes are thought to be allelic disorders (41). Speciﬁc mutations at this locus have not been identiﬁed. A databank gene scan of this region has not identiﬁed a sequence corresponding to an ion channel gene, but several sodium-glucose co-transporters have been identiﬁed as potential candidates. Benign Familial Neonatal/Infantile Seizures Kaplan and Lacey (46) reported a family in which 12 members had idiopathic neonatal or early infantile seizures inherited in an AD pattern. The age of onset ranged from 2 days to 3.5 months, representing an intermediate between BFNC and BFIC. Seizures occurred in clusters, over a period of hours to days. None of the patients developed a subsequent seizure disorder, and all had normal psychological development. A few years later, one of two families initially reported as BFNC was found to have an intermediate age of onset, more typical of Benign familial neonatal/infantile seizures (BFNIS) than BFNC (47,48). This family has been extensively studied and has not shown linkage to the BFNC loci on chromosomes 8 and 20. Linkage to 2q23-q24.3 was established (49), and a mutation in a voltage-gated sodium channel alpha subunit gene (SCN2A) was veriﬁed in 2002 (50). Berkovic et al. (51) recently reported missense mutations in the SCN2A gene in eight families with a BFNIS phenotype. In these families, ictal EEG showed a posterior temporal onset, and the clinical features were that of a secondarily generalized partial seizure. In a few cases, interictal EEG showed generalized epileptiform discharges. All affected members responded well to antiepileptic drugs and remained seizure free after 12 months of age. Voltage-gated sodium channels have one pore-forming a subunit and two b subunits that modulate channel-gating kinetics (52). Whole-cell patch clamp recordings of wild-type and mutated channels have shown the mutant channel to inactivate more slowly, whereas the sodium channel conductance was normal. Prolonged residence in the open state of the mutant channel may augment sodium inﬂux and thereby underlie the neuronal hyperexcitability that induces or facilitates seizure activity (52).

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Familial Febrile Convulsions and Generalized Epilepsy with Febrile Seizures Plus Febrile seizures affect approximately 2–4% of children under six years of age and are by far the most common seizure disorder (53). Up to 6% of children with febrile seizures later develop ongoing epilepsy with afebrile seizures (54). Twin studies have conﬁrmed a major impact of genetic factors in the etiology of febrile seizures (55), and rare families exhibit AD inheritance (56–59). Several loci (19p13.3, 8q13-q21, 2q23-q24, 5q14-q15) have been related to familial febrile convulsions (FFC) (56–59). In one family with febrile and afebrile seizures, a nonsense mutation in the MASS1 (monogenic audiogenic seizure susceptibility 1) gene was discovered (60). Although this loss-of-function mutation may be responsible for the seizure phenotype in this particular family, it is not likely that MASS1 mutations contribute to the cause of febrile seizures in the majority of cases. In the same report, 47 other families with febrile seizures were found not to harbor mutations at this site (60). In 1997, a subset of patients with familial febrile seizures was identiﬁed and their condition termed generalized epilepsy with febrile seizures plus (GEFSþ) by Scheffer and Berkovic (61). In the family they described, the most common phenotype was denoted as febrile seizures plus (FSþ), comprised of childhood-onset febrile seizures. Unlike members of kindreds with a typical FFC phenotype, patients with FSþ had seizures with fever continuing beyond six years of age as well as afebrile seizures. Other phenotypes included FSþ and absence seizures, FSþ and myoclonic seizures, FSþ and atonic seizures, and the most severely affected individual had myoclonicastatic epilepsy (MAE). Seizures usually ceased by mid-childhood (median 11 years). Singh et al. (62) reported the seizure phenotypes of 63 patients in nine families with epilepsy consistent with the GEFSþ syndrome. Approximately 50% had febrile convulsions, 25% had febrile seizures plus (FSþ), and 27% had FSþ mixed with other seizure types (atonic, myoclonic, absence, complex partial, or myoclonic-astatic). Inheritance was AD with approximately 60% penetrance. Wallace et al. (63) established linkage to chromosome 19q13.1 in a large family with GEFSþ and later identiﬁed a mutation in a voltage-gated sodium channel a subunit (SCN1B) gene in that region. This is known as GEFSþ type 1. GEFSþ type 2 has since been described with a mutation in the SCN1A gene on chromosome 2q24 (64–66). An additional mutation in the SCN2A gene on chromosome 2q23-q24.3 was reported in a family with febrile and afebrile seizures (52). These genes code for voltage-gated sodium channel a subunits. In addition to these sodium channel subunit mutations, a mutation in a gene coding for a GABA-A receptor g2 subunit gene (GABRG2) has been found in a family with a third phenotype, GEFSþ type 3 (67). The exact manner by which mutated sodium channels give rise to epilepsy is not known. The fast inactivating sodium current is the principal

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current responsible for the depolarizing phase of the action potential and thus is essential for neuronal excitation in general (68). Pentylenetetrazole, a drug that causes convulsions in experimental models, prolongs sodium-carried action potentials (69), and multiple antiepileptic drugs work by manipulating sodium channel gating (68). It therefore came as no surprise when functional studies of channels with SCN1A, SCN2A, and SCN1B mutations showed prolonged opening of sodium channels, which would hypothetically lead to increased excitability and seizures (52,70). Computer simulation studies have found that despite each individual mutation having a unique effect on the action potential, the end result of all four mutations (SCN1A, SCN2A, SCN1B, GBRG2) is an increased propensity to ﬁre repetitive action potentials (71). More research is needed to understand why a static change in channel properties would lead to a paroxysmal disorder like epilepsy. Severe Myoclonic Epilepsy of Infancy Severe myoclonic epilepsy of infancy (SMEI) is a rare disorder (1:40,000) characterized by generalized tonic, clonic, and tonic–clonic seizures that are frequently initially induced by fever and begin during the ﬁrst year of life (72,73). Later, patients also manifest other seizure types, including absence, myoclonic, and simple and complex partial seizures. Psychomotor development is initially normal but plateaus during the second year of life. SMEI is considered a malignant epilepsy syndrome due to its effect on psychomotor development as well as the tendency for ongoing intractable seizures. SMEI was ﬁrst described by Dravet in 1978 (72), and the ﬁrst suggestion that it might be due to a single gene disorder was in 1992 when monozygotic twins exhibiting the SMEI phenotype were reported (74). Singh et al. (75) reported 12 probands with SMEI and a family history of seizures and proposed that SMEI was the most severe phenotype of GEFSþ. Having observed similarities in the phenotype of SMEI and GEFSþ, Claes et al. (76) screened seven unrelated patients with clinical SMEI for mutations in the neuronal voltagegated sodium channel a-subunit gene SCN1A and identiﬁed a mutation in each patient (four frameshift, one nonsense, one splice-donor, one missense). These patients did not have a family history of seizures, and all of the mutations were de novo mutations. Since then it has become clear that SMEI, although most often sporadic, can be seen within GEFSþ families and depicts the most devastating clinical form of the GEFSþ spectrum (77–80). Multiple SCN1A mutations as well as a GABRG2 mutation (5q31.1-q33.1) have been reported in patients with SMEI (81). An SCN2A mutation has also been described in a patient with an atypical SMEI phenotype (82). Functional studies of the mutated channels have shown various types of dysfunctional channels with some forms leading to complete loss of channel function (82,83). In all types the end result is a state of hyperexcitability and epilepsy.

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Autosomal Dominant Nocturnal Frontal Lobe Epilepsy The last channelopathy-related epilepsy disorder that we will cover in detail is autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). In this syndrome, patients experience brief nocturnal motor seizures with hyperkinetic or tonic manifestations. Seizure onset is in childhood, and psychomotor development is usually normal (84). Milder cases are often undiagnosed or misdiagnosed as nightmares or other sleep disorders, whereas patients with more severely affected family members with frequent nocturnal seizures are more readily identiﬁed. ADNFLE was ﬁrst described in 1994 in ﬁve families, the largest of which mapped to chromosome 20q13.2 (85–87). Further studies of this family led to the identiﬁcation of a mutation in the neuronal nicotinic acetylcholine receptor a4 subunit (CHRNA4) gene in affected family members (88). Genetic heterogeneity has been demonstrated by Phillips et al. (89), who established linkage to chromosome 15q24 in a separate family. This region is close to a cluster of genes coding for acetylcholine receptor subunits (CHRNA3/ CHRNA5/CHRNB4), but the exact mutation has not been identiﬁed. Other families linked to chromosome 1q21 were found to have CHRNB2 (b2) acetylcholine receptor subunit mutations (90–92). Another report describing patients with the classical ADNFLE phenotype excluded linkage to any of the known neuronal acetylcholine receptor subunit genes (93). The CHRNA4 (a4) and CHRNB2 (b2) subunits assemble to form the active receptor (a4b2) (94). Therefore, it is not surprising that similar phenotypes are seen with mutations in either one (95). There is some evidence that a gain of function in mutant (a4b2) receptors might be at the root of the neuronal dysfunction causing epileptic seizures (96). A single case study reported a decrease in the seizure frequency of an ADNFLE patient during use of a transdermal nicotine patch (97). In time, the mechanisms of the acetylcholine receptor subunits and their role in the development of epilepsy should become clearer. MYOCLONIC EPILEPSIES Myoclonic epilepsy is a key ﬁnding in several hereditary syndromes. Some of these have been described in one or a few families, and the gene locus or product has not been identiﬁed. Other syndromes have been well characterized clinically, and more recent advances in molecular genetics have conﬁrmed their genetic basis. We will review both the malignant progressive myoclonic epilepsies as well as the benign myoclonic epilepsy syndromes. SMEI is discussed above in the section covering channelopathies. Progressive Myoclonic Epilepsy The constellation of myoclonus, epilepsy, ataxia, and cognitive deterioration forms the typical phenotype of progressive myoclonic epilepsy (PME).

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There are six main conditions that fall into this category; they are listed in Table 5. Not all are associated with dementia, but typically PME represents a progressive encephalopathy with intractable epilepsy and myoclonus. Some of the individual diseases discussed below have typical features that may distinguish them from the others. The importance of reﬁning a diagnosis to speciﬁc clinical entity, and when possible a speciﬁc genetic diagnosis, lies in the need for establishing a prognosis and for genetic counseling for families of affected patients. Most of the PMEs are inherited in an AR fashion. Exceptions include dentatorubral pallidoluysian atrophy (DRPLA), which has AD inheritance, and the mitochondrially inherited myoclonic epilepsy with ragged red ﬁbers (MERRF). Unverricht–Lundborg Disease Familial myoclonus with progressive mental and motor decline was ﬁrst described by Unverricht and Lundborg in the late 1800s (98,99). This AR disorder, also called Baltic myoclonus, presents with convulsions between 6 and 13 years of age. Asynchronous myoclonus, predominantly in the proximal muscles of the extremities, begins 1–5 years later (100). The stimulussensitive myoclonic jerks are mild at ﬁrst but become so violent late in the clinical course that a patient can be thrown to the ﬂoor. Mental deterioration is usually present but is not a prominent ﬁnding, while emotional lability is a typical feature. Psychotic symptoms are rare. Signs of cerebellar ataxia are seen in later stages. Antiepileptic therapy and better supportive therapy in recent years have led to a marked improvement in survival, but there is no cure. Unverricht–Lundborg disease has been diagnosed worldwide, but its prevalence is highest in Finland (1:20,000) where approximately 100 cases had been identiﬁed by 1980, half of all known cases in the world at that time (101). Areas of high incidence also include Northern Africa, Southern France, and in the Eastern Mediterranean region (102–104). EEG ﬁndings include slowing of the posterior background rhythm, episodic 4–6 Hz bilateral sharp waves, and polyspikes with myoclonus activated by photic stimulation (105). Treatment with N-acetylcysteine or levetiracetam decreases seizure frequency but does not have any effect on the ataxia or myoclonus (106,107). Valproic acid has also been found to be beneﬁcial, while phenytoin has a detrimental effect on seizures and myoclonus (108). Lehesjoki et al. (109) mapped Unverricht–Lundborg disease to chromosome 21q22.3. The same group sequenced the cystatin B gene (CSTB) from affected individuals and identiﬁed two different mutations in the gene (108). Two groups reported a 12 base pair unstable repeat expansion of the 50 ﬂanking region CSTB in affected individuals; this has since been found to be the most common mutation in Unverricht–Lundborg disease (110,111). Two to three repeats are considered normal, up to 17 repeats are considered a premutation state, and clinical signs are seen in individuals with 30–80

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Table 5 Myoclonic Epilepsies with Established Genetic Basis

A. Progressive myoclonic epilepsies Unverricht–Lundborg disease Lafora body disease Sialidosis types I and II Neuronal ceroid lipofuscinosis Infantile (Santavuori–Haltia) Late infantile (Jansky–Bielschowsky) Late infantile (Finnish) Late infantile (Portuguese, Costa Rican) Late infantile (Turkish) Childhood Northern epilepsy variant Juvenile (Spielmeyer–Vogt) Adult (Kufs) Dentatorubral pallidoluysian atrophy (DRPLA) Myoclonic epilepsy with ragged red ﬁbers (MERRF)

Inheritance

Gene

Protein

Chromosomal

AR AR AR AR

CSTB EPM2A EPM2B NEU1

Cystatin B Laforin Malin Neuraminidase

21q22.3 6q23-q25 6p22 6p21.3

AR AR AR AR AR AR AR AR, AD AD, CAG repeat Mitochondrial

CLN1 CLN2 CLN5 CLN6 CLN7 CLN8 CLN3 CLN4 DRPLA MTTK

PPT TPP-I – – – – Battenin PPT (minority) Atrophin-1 Mitochondrial proteins

1p32 11p15.5 13q21.1-q32 15q21-q23 8pter-p22 8pter-p22 16p12.1 1p32 (minority) 12p13.31 Mitochondrial loci

B. Benign myoclonic epilepsies Autosomal dominant cortical myoclonus and epilepsy (ADCME) Benign adult familial myoclonic epilepsy (BAFME) Familial idiopathic myoclonic epilepsy of infancy A dash (–) indicates that the information is not established.

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AD

–

–

2p11.1-2q12.2

AD

–

–

AR

–

–

2p11.1-2q12.2 8q23.3-q24.11 16p13

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MTTL1 MTTH MTTS1

442

Epilepsy syndrome

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repeats (112). Among those with overt clinical manifestations, no correlation has been found between number of repeats and disease severity or age of onset. The repeat expansions of most other repeat expansion diseases lie within the coding regions of the involved genes (113). In the case of CSTB, the repeat expansion is outside the coding region but still results in a marked decrease in CSTB expression (112). CSTB repeat expansion has been shown to lead to apoptosis and decreased neuronal survival, but how that relates to epilepsy remains to be established (112,114). Lafora Body Disease Lafora body disease, or myoclonic epilepsy of Lafora, is an AR disorder that usually presents around 15 years of age with generalized tonic–clonic seizures and/or myoclonus. Rapid and severe mental deterioration ensues, often with psychotic features (115,116). No treatment is available, and survival is usually less than 10 years after onset. Histologic studies of multiple tissues, including brain, muscle, liver, and heart, reveal intracellular polyglucosan inclusions, called Lafora bodies, which play a key role in the diagnosis of this devastating disorder (115,117). Lafora bodies are found in myoepithelial cells surrounding axillary apocrine glands; outside the axilla, they are found in the cells composing the ducts of the eccrine glands (118). Andrade et al. (118) found that all patients with Lafora body disease (with EPM2A or NHLRC1 mutations) exhibit Lafora bodies in both eccrine and apocrine glands. A patient later found to have genetically proven Unverricht–Lundborg disease was erroneously diagnosed with Lafora disease due to a ‘‘false’’ positive axillary (apocrine) biopsy while an exocrine biopsy was negative. Andrade et al. favor biopsy of skin outside the axilla for the diagnosis of Lafora body disease. In 1992, Lehesjoki et al. (119) excluded linkage to chromosome 21q22.3, the locus for CSTB, in three families with Lafora body disease. A few years later, nine families with Lafora body disease were mapped to chromosome 6q23-q25 (120). Since then, mutations in a gene encoding the laforin protein, also known as EPM2A, have been found in patients with Lafora body disease (121). Genetic heterogeneity was suspected early on since at least 20% of affected patients did not map to the chromosome 6q23-q25 locus (122). Chan et al. (123,124) linked four families with Lafora body disease to chromosome 6p22 in 2003 and described mutations in the NHLRC1 gene in 26 families with clinical Lafora body disease. This second involved gene is also known as EPM2B and encodes the protein malin. The role of laforin and malin in the pathophysiology of Lafora body disease is not yet understood. Cherry-Red Spot Myoclonus Syndrome Cherry-red spot myoclonus syndrome, or sialidosis type I, is an AR disorder that typically presents in adolescence with severe myoclonus and tonic–clonic seizures. This is followed by progressive visual loss, macular cherry-red spots,

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and ataxia. Dementia does not usually occur. The characteristic macular cherry-red spot is seen on fundoscopic examinations and is useful for diagnostic purposes. Cherry-red spot myoclonus (sialidosis type I) is the milder form, while sialidosis type II has an earlier onset and signiﬁcant dysmorphic features, leading to earlier diagnosis (125). Sialidoses are caused by a deﬁciency of the enzyme neuraminidase and are characterized by the progressive lysosomal storage of sialidated glycopeptides and oligosaccharides (126). Swallow et al. (127) ﬁrst reported a neuraminidase deﬁciency in cultured ﬁbroblasts from two adolescent brothers with progressive visual loss and myoclonus. Mutations in the neuraminidase gene were found in one patient with sialidosis type I and two patients with sialidosis type II, suggesting the two forms are allelic disorders (128). There is a close correlation between the residual activity of the mutant enzymes and the clinical severity of disease (type I vs. type II phenotypes) (129). No known curative therapy is available for these disorders, although bone marrow transplantation might be a future alternative (130). 5-Hydroxytryptophan has been shown to decrease the myoclonus in at least one patient with cherry-red spot myoclonus syndrome (131). Neuronal Ceroid Lipofuscinoses The neuronal ceroid lipofuscinoses (NCLs) are a group of diseases characterized by dementia, seizures, progressive visual failure, and intralysosomal accumulations of lipopigments in granular, curvilinear, or ﬁngerprint patterns (132–136). These disorders include infantile Santavuori–Haltia disease (CLN1), late infantile Jansky–Bielschowsky disease (CLN2), late infantile form (CLN5, CLN6, CLN8), juvenile Spielmeyer–Vogt disease or Batten disease (CLN3), and adult-onset Kufs disease (CLN4). There are also approximately a dozen atypical variants. What follows is an overview of each disease and its genetic abnormality. Infantile NCL The infantile form of NCL is Santavuori–Haltia disease (CLN1), ﬁrst described by Santavuori in 1973 (133). Onset is most often in the ﬁrst year of life, ranging between 8 and 18 months, with rapid psychomotor deterioration, blindness by age two years, ataxia, and muscular hypotonia. Microcephaly and myoclonic jerks are also features while convulsions are rare (137). The unique clinical picture of these patients supported this being a separate entity from other NCLs, and in the early 1980s Baumann and Markesbery (138) found unique inclusions in circulating lymphocytes and the central nervous system in patients with presumed Santavuori–Haltia disease. Jarvela et al. (139) studied 15 Finnish families with Santavuori–Haltia disease and excluded involvement of the CLN3 gene in these families. They went on to map these families to chromosome 1p32. Mutations in the palmitoyl-protein thioesterase (PPT) gene have since been identiﬁed, and PPT activity was found to be undetectable in

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brain tissue from two Finnish individuals with Santavuori–Haltia disease (140). A relatively simple enzyme assay is available for both postnatal and prenatal diagnosis (141). There is a report describing patients with an adult-onset NCL with a PPT gene mutation, but this represents an exception (142). Late infantile NCL Jansky–Bielschowsky disease (CLN2), the classical late infantile form of neuronal ceroid lipofuscinosis typically presents between the ages of two and four years with seizures, myoclonus, mental retardation, and ataxia. The condition was ﬁrst described in the literature in 1926 (132). EEG ﬁndings are characterized by slow posterior background activity and pseudoperiodic high-voltage slow SW complexes, mostly in the posterior regions (143). In one study, magnetic resonance imaging (MRI) abnormalities included cerebral atrophy and hyperintensity of the periventricular white matter on T2-weighted images; magnetic resonance spectroscopy revealed a decrease in NAA, increases in myo-inositol and glutamine/glutamate, and undetectable lactate (144). Postmortem examinations have shown decreased brain weight, evidence of neuronal loss, and intralysosomal inclusions with a curvilinear pattern (145). Sleat et al. (146) identiﬁed a single lysosomal protein absent in patients with late infantile NCL, and the gene encoding this protein was mapped to chromosome 11p15.5 (147). The involved protein is tripeptidyl amino peptidase-I (TPP-I), a lysosomal peptidase (148). Prenatal and postnatal testing is available (149,150). A majority of late infantile NCL patients are homozygous or compound heterozygous for CLN2 mutations, resulting in complete loss of enzymatic activity (151). As with the other NCLs, there is no treatment and the course is steadily progressive. Other late infantile variants of NCL The other late infantile forms of NCL have a clinical picture of late infantile NCL but gene defects other than CLN2 mutations. CLN5 is a Finnish variant characterized by a slightly later onset than Jansky–Bielschowsky disease, with typical onset between four and seven years. The disease progresses more slowly in individuals with CLN5 mutations, and survival is prolonged. This disease has been mapped to chromosome 13q21.1-q32 (152). CLN6 is a subtype of late infantile NCL that has been mapped to 15q21-q23 (153) and observed in Portuguese and Costa Rican kindreds. CLN7 is a late infantile Turkish variant NCL that has a slightly later onset than Jansky–Bielschowsky disease at approximately ﬁve years of age; it has been mapped to chromosome 8pter-p22. It is thought to be allelic to Northern Epilepsy, a childhood onset NCL found in Finland (154). Visual loss is not a prominent feature of Northern Epilepsy, there is no myoclonus, and the clinical progression is slower than that of the allelic Turkish variant NCL (155).

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Juvenile NCL Juvenile NCL (Spielmeyer–Vogt disease or Batten disease) is among the most common neurodegenerative diseases of childhood with an incidence of 1:21,000 (156). The onset of juvenile NCL is around 5–10 years of age with rapid deterioration of vision and a slower but progressive deterioration of intellect. Seizures and psychotic behavior are seen in later stages. The fundi show pigmentary degeneration. The pathologic features are severe widespread neuronal degeneration resulting in retinal atrophy, massive loss of brain substance, vacuolated lymphocytes, and curvilinear bodies on electronic microscopy (157,158). CLN3, the mutated gene in juvenile NCL, was mapped to chromosome 16 in 1990 and to chromosome 16p12.1 in 1994 (159,160). Most patients carry a 1.02 kb deletion removing nucleotides 461–677 from the coding region of the responsible protein, but various other mutations (missense, nonsense, splice site and intronic mutations, as well as small deletions and insertions) have been identiﬁed (161). There is ongoing investigation regarding the function of this protein and the predilection for this disease to affect the central nervous system. No treatment is known, and patients progress often rapidly to a vegetative state and death. Adult-onset NCL Adult-onset NCL (Kufs disease) has clinical onset at approximately 30 years (162). Berkovic et al. (163) have delineated two clinical phenotypes of Kufs disease. Type A begins with PME, which is followed by dementia and ataxia. Type B is characterized by dementia with cerebellar and/or extrapyramidal motor symptoms. Neither type is associated with retinal degeneration or blindness. MRI typically reveals cerebral and cerebellar atrophy within six years of disease onset. All patients showed ﬁngerprint proﬁles in electron microscopy of skin cells. A few patients have a PPT gene mutation like that seen in Santavuori–Haltia disease (164), but a majority of patients lack that mutation. The gene or genes responsible for the majority of Kufs disease are unknown at this time. Dentatorubral Pallidoluysian Atrophy Dentatorubral pallidoluysian atrophy (DRPLA) is an AD disorder due to a CAG trinucleotide repeat expansion in the DRPLA gene (chromosome 12p13.31), which encodes atrophin 1 (165). The disorder can present in a variety of forms at a variety of ages, but one of the more common is an adolescent presentation with myoclonic epilepsy followed by dementia, ataxia, and choreoathetosis (166). The major neuropathologic ﬁnding is degeneration of the dentatorubral and pallidoluysian systems. DRPLA is relatively common in Japan, where three clinical forms have been classiﬁed: an ataxic-choreoathetoid form, a pseudo-Huntington form, and a myoclonic

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epilepsy form (167). The size of the CAG repeat expansion correlates with the age of onset (168). The normal CAG repeat size is 7–23, and it is expanded to more than 49 repeats in clinically affected patients. Expansion is usually (although not invariably) associated with paternal transmission (165). Treatment is supportive, and survival is 20 to 40 years. Myoclonic Epilepsy with Ragged-Red Fibers Myoclonic epilepsy with ragged-red ﬁbers (MERRF) is a well-known mitochondrial disorder, the phenotype of which includes myoclonic seizures, generalized tonic–clonic seizures, and a paroxysmal hearing disturbance (169). Mental deterioration, muscle atrophy, weakness, and truncal ataxia are also seen. Lactate levels in both blood and cerebrospinal ﬂuid are elevated, and neuroimaging shows cerebral atrophy and bilateral calciﬁcation of the basal ganglia. Muscle biopsies show ragged-red ﬁbers. Inheritance follows a mitochondrial pattern, from mother to offspring, with considerable variability in phenotypic severity. MERRF is associated with mutations in one of at least four known mitochondrial genes (MTTK, MTTL1, MTTH, MTTS1). An in-depth overview of mitochondrial disorders can be found in Chapter 13 (Mitochondrial Disorders). Benign Myoclonic Epilepsies There are at least two familial myoclonic epilepsy syndromes with relatively benign course and an established genetic basis, listed in Table 5. Benign Adult Familial Myoclonic Epilepsy Benign adult familial myoclonic epilepsy (BAFME) and familial adult myoclonic epilepsy (FAME) refer to the same condition, characterized by distal myoclonus (also referred to as cortical tremor), generalized myoclonus, infrequent generalized tonic–clonic seizures, and preserved cognition. Characteristic electrophysiological ﬁndings included generalized polyspike-wave discharges and activation with photic stimulation on EEG and abnormally large cortical potentials on somato-sensory evoked potentials (SSEPs) (170). Two groups of Japanese families were studied, with age of onset ranging from 13 to 18 years in one study (171) and 18 to 45 years in another (170). An associated genetic locus was identiﬁed in 1999 at chromosome 8q23.3-q24.11 in these same families (170,171). Genetic heterogeneity was established for this condition when analysis of a European pedigree with a very similar phenotype demonstrated linkage at chromosome 2p11.1-2q12.2 (172). Autosomal Dominant Cortical Myoclonus and Epilepsy Guerrini et al. (173) described a family with a similar but clinically distinct syndrome called autosomal dominant cortical myoclonus and epilepsy (ADCME). The syndrome is deﬁned by distal myoclonus and generalized

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tonic–clonic seizures but may also include complex partial seizures, intractable epilepsy, and mild mental retardation in some individuals. In addition to generalized EEG abnormalities similar to those seen in patients with BAFME, some patients had focal abnormalities in the frontotemporal regions. ADCME maps to chromosome 2p11.1-2q12.2 (173), making the disorder allelic to BAFME in the European kindred.

GENERALIZED EPILEPSY SYNDROMES Most of the age-dependent idiopathic generalized epilepsy syndromes, although known to have a genetic predisposition, do not follow true Mendelian inheritance patterns. We will review the known genetic contribution to three common generalized epilepsy syndromes. Table 6 lists the known chromosomal locations and genes associated with these conditions, but it is important to remember that these abnormalities have been found in only a minority of cases with these clinical conditions and do not fully explain the genetic basis of the generalized epilepsies. Childhood Absence Epilepsy CAE is a well-known age-dependent epilepsy syndrome, presenting with frequent brief absence seizures between 3 and 12 years of age. The EEG

Table 6 Generalized Epilepsy Syndromes with Associated Genetic Abnormalities Gene

Protein

Chromosomal locus

Complex

– GABRG2 CLCN2 CLCN2

– GABA-A-R CIC-2 CIC-2

8q24 5q31.1 3q26-qter 3q26

Complex

CACNB4

VGCC

2q22-q23

CLCN2 GABRAl EFHCl CHRNA7

CIC-2 GABA-A-R – nAch-R

3q26 5q34 6p12-11(EJMl) 15q14

Epilepsy syndrome

Inheritance

Childhood absence epilepsy CAE1 CAE2 CAE3 Juvenile absence epilepsy (JAE) Juvenile myoclonic epilepsy (JME)

Complex

A dash (–) indicates that the information is not established. Abbreviations: CIC-2, voltage-gated chloride channel; GABA-A-R, GABA-A receptor; nAch-R, nicotinic acetylcholine receptor; VGCC, voltage-gated calcium channel.

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shows 3 Hz generalized SW discharges correlating to patients’ clinical events. These children tend to respond well to medication, and seizures typically remit by late adolescence. Generalized tonic-clonic seizures may occur (174). CAE usually occurs sporadically, but familial forms are seen. One form of familial CAE, designated ECA1, has been linked to chromosome 8q24, but the gene has not been identiﬁed (175,176). Another familial form of CAE, with or without febrile seizures, ECA2, is caused by mutations in the GABRG2 gene on 5q31.1 (177). A third form, ECA3, has been described in a family where affected individuals have various seizure phenotypes including absence seizures in childhood. In this family, a mutation in the CLCN2 gene on 3q26-qter has been identiﬁed (178).

Juvenile Absence Epilepsy Juvenile absence epilepsy (JAE) is another well-known age-dependent epilepsy syndrome that manifests as absence seizures around 10–12 years of age. The frequency of absence seizures is lower than in CAE, while the frequency of generalized tonic–clonic seizures and the probability of ongoing epilepsy into adulthood are considerably higher (179). While most cases of JAE are sporadic, families with the classic JAE phenotype as well as a mixed JAE/myoclonic epilepsy picture have been described. Members of one JAE pedigree have been found to have a CLCN2 gene mutation (178,180), while in others the loci involved have not been identiﬁed.

Juvenile Myoclonic Epilepsy Juvenile myoclonic epilepsy (JME) is a genetically heterogeneous disorder. Forty to 50% of patients with JME have a family history of epilepsy (181). JME usually presents in adolescence with myoclonic jerks as well as myoclonic seizures upon awakening. Generalized tonic–clonic seizures are seen in up to 90% of patients with JME. This syndrome is benign with respect to its excellent response to valproic acid, but relapses are common after drug tapering and lifelong antiepileptic medication is usually required. The characteristic interictal EEG shows 4–6 Hz bihemispheric synchronous polyspike and wave discharges (182). The genetics of JME are complex, and most families do not follow a strict Mendelian inheritance pattern. Early reports believed it to be an AR disorder, while recent evidence suggests an AD pattern with low penetrance (183,184). Numerous single case or family studies have led to the identiﬁcation of mutations in various genes, none of which make the major etiologic contribution to JME. These include mutations in an a1 subunit GABA-A receptor gene (GABRA1), a VGCC b4 subunit gene (CACNB4), and a chloride channel-2 gene (CLCN2) (178,185,186).

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LOCALIZATION-RELATED EPILEPSY SYNDROMES Localization-related epilepsy, also called partial epilepsy, occurs in patients whose seizures are thought to arise in a focal region of cortex. ADNFLE is one such condition, and it is discussed in the section on channelopathies. Two other examples of localization-related epilepsy with known genetic associations are discussed below. A brief summary of these conditions is found in Table 7; we expect this list to expand in the coming years. Benign Epilepsy with Centro-Temporal Spikes Linkage to chromosome 15q14 has been established not only for JME (187) and other types of idiopathic generalized epilepsy (188), but also for the characteristic EEG trait of benign epilepsy with centro-temporal spikes (BECTS), also known as benign rolandic epilepsy (BRE) (189). The inheritance of this EEG pattern is reported to be AD with age-related penetrance (190). Classical BECTS presents between 2 and 13 years of age with simple partial seizures, including hemifacial motor movements, oropharyngeal gurgling or hypersalivation with speech arrest, and sensory and motor phenomena in the extremities. These progress occasionally to generalized tonic–clonic seizures (191). Most seizures are nocturnal. Children with BECTS who require medication—for daytime or recurrent generalized seizures—respond well to carbamazepine. The condition resolves by the mid- to late-teens. There is a unique subset of BECTS with speech dyspraxia and a more deﬁnitive AD pattern of inheritance, for which a locus has not yet been established (192). In another distinct syndrome, rolandic epilepsy is seen with paroxysmal exercise-induced dystonia and writer’s cramp; this syndrome maps to the centromeric region of chromosome 16, 16p12-11.2 (193). Overall, however, the inheritance of BECTS—as for the majority of CAE, JAE, and JME—is likely to be multifactorial (194). Autosomal Dominant Lateral Temporal Lobe Epilepsy Familial temporal lobe epilepsy was ﬁrst reported in 1995 (195). In the patients described, disease onset was in childhood or early adolescence, and the condition was characterized by rare seizures, prominent auditory symptoms thought to be simple partial seizures, and normal interictal EEGs. Linkage analysis in this family showed strong evidence for localization to chromosome 10q22-q24 (195). The term autosomal dominant lateral temporal lobe epilepsy (ADLTE) has been applied to this disorder. Additional families were later found to map to the same region, and the putative gene has been identiﬁed as the LGI (leucine-rich glioma inactivated) gene (196–198). The speciﬁc manner by which these mutations lead to localization-related epilepsy is not yet understood.

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Inherited Epilepsies

Table 7 Localization-Related Epilepsy Syndromes with Established Genetic Basis Epilepsy syndrome Autosomal dominant lateral temporal lobe epilepsy (ADLTE) Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) Benign epilepsy with centro-temporal spikes with speech dyspraxia Familial partial epilepsy with variable foci (FPEVF) Rolandic epilepsy with paroxysmal exerciseinduced dystonia and writer’s cramp Partial epilepsy with auditory features

Inheritance

Gene

Protein

Chromosomal locus

AD

LGI

–

10q22-q24

AD

CHRNA4

nAch-R

20q13.2

AD

–

–

–

AD

–

–

22q12

AR

–

–

16p12-11.2

AD

–

–

10q

A dash (–) indicates that the information is not established. Abbreviation: nAch-R, nicotinic acetylcholine receptor.

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CONCLUSION Neurogenetics is shaping our approach to clinical diagnosis in epilepsy at a remarkable pace. The study of pedigrees with well-deﬁned epilepsy syndromes has led to the identiﬁcation of several involved genes. The fact that a number of the known gene products associated with epilepsy are ion channel proteins substantiates theories that abnormalities in channel function give rise to seizures. We can certainly look forward to an increasingly thorough understanding of the fundamental mechanisms that underlie human epilepsy. Further study will enrich and expand upon our current knowledge, and we will be better equipped as clinicians to diagnose and counsel patients with epilepsy. It would not be surprising to see future clinical epilepsy classiﬁcation schemes reﬂect the genetic basis of many of the epilepsies. Ultimately, a more detailed molecular understanding of this complex set of conditions should enhance our ability to develop targeted treatment strategies for individual types of epilepsy. REFERENCES 1. Hauser WA, Hesdorrfer DH. Epilepsy: Frequency, Causes and Consequences. New York: Demos Press, 1990. 2. Lennox WG. J Am Med Assoc 1951; 146:259. 3. Ottman R. Genetic epidemiology of epilepsy. Epidemiol Rev 1997; 19:120–128. 4. Lennox WG. Epilepsy and Related Disorders. Vol. 1 Boston: Little, Brown, 1960:532–574. 5. Newmark ME, Penry JK. Genetics of Epilepsy: A Review. New York: Raven Press, 1980. 6. Noebels JL, Sidman RL. Inherited epilepsy: Spike-wave and focal motor seizures in the mutant mouse tottering. Science 1979; 204:1334–1336. 7. Fletcher CF, Lutz CM, O’Sullivan TN, Shaughnessy JD, Hawkes R, Frankel WN, Copeland NG, Jenkins NA. Absence epilepsy in tottering mutant mice is associated with calcium channel defects. Cell 1996; 87:607–617. 8. Oda S. A new allele of the tottering locus, rolling mouse Nagoya, on chromosome No. 8 in the mouse. Jpn J Genet 1981; 56:295–299. 9. Zwingmann TA, Neumann PE, Noebels JL, Herrup K. Rocker is a new variant of the voltage-dependent calcium channel gene CACNAIA. J Neurosci 2001; 21(4):1169–1178. 10. Neufeld MY, Nisipeanu P, Chistik V, Korczyn AD. The electroencephalogram in acetazolamide-responsive periodic ataxia. Movement Disord 1996; 11:283–288. 11. Sigurdardottir LY, Dlugos DJ, Solomon D, Kim GW, Baloh RW, Jen J. A mutation in CACNA1A causing familial episodic ataxia and epilepsy. (In Progress). 12. Denier C, Ducros A, Vahedi K, Joutel A, Thierry P, Ritz A, Castelnovo G, Deonna T, Gerard P, Devoize JL, Gayou A, Perrouty B, Soisson T, Autret A, Warter JM, Vighetto A, VanBogaert P, Alamowitch S, Roullet E,

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20 Leukodystrophies James Y. Garbern Department of Neurology, Wayne State University School of Medicine, Detroit, Michigan, U.S.A.

INTRODUCTION Hereditary disorders of myelin formation or its maintenance comprise the ‘‘leukodystrophies,’’ a term derived from leuko ‘‘white’’ and dystroph ‘‘defective nutrition’’ (1). These disorders are characterized based on biological and pathologic features rather than a common clinical syndrome. We will apply the term to monogenic disorders primarily affecting myelin formation or its upkeep, although the nosology of leukodystrophies is more complex (2). The clinical syndromes vary extensively, from severe neonatal encephalopathy to virtually asymptomatic conditions. Complicating the diagnosis of leukodystrophies is the fact that normal early neurologic development depends upon myelination, which mainly occurs during infancy and early childhood (3). Thus, what constitutes a normal examination, both by magnetic resonance imaging (MRI) as well as clinically, varies signiﬁcantly by age. In addition, temporal patterns of neurologic signs from hereditary myelin disorders vary as well, from developmental delay neonatally, to progressive deterioration after a period of normal development. These temporal patterns are better understood if their biological bases are appreciated. Disorders that interfere with normal myelination are characterized by early onset developmental delay and neurologic signs, whereas those that primarily affect the maintenance of myelin (typically involving enzymes that

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degrade lipids) permit normal early childhood development that is followed by breakdown of myelin and progressive neurologic decline. In addition, inheritance patterns span the full Mendelian range, in addition to maternal inheritance associated with mitochondrial disorders, with attendant implications for genetic counseling. In the past, without a distinct clinical syndrome, the diagnosis of leukodystrophy could be made only after cerebral biopsy or autopsy, but MRI now commonly establishes the diagnosis. However, not all myelin disorders have readily appreciated changes on MRI. Recognition of white matter abnormalities must also account for the changes in MRI appearance of white matter during the course of normal development. Radiologists deﬁne myelin by its appearance on MRI scans. Normal myelination is characterized by shortening of the T1 and T2 relaxation times in the white matter, meaning that myelin will be light in intensity relative to gray matter on T1-weighted scans, but dark relative to gray matter on T2-weighted images (3–7). These changes may reﬂect the dehydration of white matter during myelination. In normal infants, myelination has occurred at birth in the pons and cerebellum; followed by the posterior limb of the internal capsule, the optic radiations and splenium of the corpus callosum by three months; the frontal, parietal, and occipital white matter by 8–12 months; and the temporal white matter by around 12 months. The adult pattern of myelination is normally acquired by 18 months of age. While obvious discrepancies from normal should be apparent by this age, close scrutiny and detailed understanding of the normal patterns of myelination may enable earlier suspicion or diagnosis of myelination abnormalities. Somewhat arbitrary decisions were made to compile the list of diseases discussed here, and new diseases will be discovered. Many diseases affect myelination or its maintenance (such as neuronal ceroid lipofuscinosis and mucolipidoses), but cause signiﬁcant disruption of other organs or neurons as well. These will not be discussed here. An early scheme for classiﬁcation of leukodystrophies was based on the histochemical staining properties of white matter. While all leukodystrophies have reduced staining with conventional myelin stains, such as Luxol Fast Blue or Sudan Black, the tinctorial properties of the white matter differed and served as one basis for classiﬁcation. Thus, with orthochromatic leukodystrophies the white matter that remains stains similarly to normal white matter. In contrast, in metachromatic leukodystrophy the abnormal white matter stains aberrantly. The following classiﬁcation is based on current understanding of the causes of leukodystrophies (Table 1). DISORDERS OF MYELIN LIPID METABOLISM While the bulk of myelin is lipid, no classical leukodystrophies are known that are caused by mutations in genes encoding enzymes responsible for

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Disorder

Inheritance

Gene

Locus

OMIM

MRI pattern

XR

ALDP

Xq28

300100

Posterior

AR

SLC17A5

6q14-q15

Global

AR

ARSA

22q13.31qter

604369/ 604322 250100

AR

SUMF1

3p26

272200

Global

Krabbe disease AR Megalencephalic AR leukoencephalopathy with subcortical cysts Canavan AR

GALC MLC1

14q31 22qter

245200 604004

Global Anterior temporal cysts/ Global

ASPA

17pter-p13

271900

Global

PCWH

AD

SOX10

22q13

602229

Global

Alexander disease

AD

GFAP

17q21

137780

Frontal

Adrenoleukodystrophy/ Adrenomyeloneuropathy Salla disease Metachromatic leukodystrophy Multiple sulfatase deﬁciency

Frontal/ global

PNS D

D

Analyte VLCFA

Other signs Addison disease

D

C/R

Sialic acid

C/R

Sulfatide

C/R

Sulfatide and mucopolysaccharides

C/R

C/R

D

–

Testing

NAA urine, CSF

Macrocephaly

C/R

Waardenburg/ R Hirschsprung Macrocephaly C/R

471

(Continued) Copyright 2006 by Taylor & Francis Group, LLC

Leukodystrophies

Table 1 Leukodystrophies

Leukodystrophies (Continued )

Disorder

Inheritance

Gene

472

Table 1

Locus

OMIM

MRI pattern

VWM/CACH

AR

EIF2B1-5

2p23.3;1;14 q24;12;3 q27

603896

Globall/ cystic degeneration Global Global Arrested myelination

PMD/SPG2 PMLD Sjo¨gren-Larsson

XR/XD AR AR

PLP1 GJA12 ALDH3A2

Xq22 1q41q42 17p11.2

300401 608804 270200

Cockayne syndrome

AR

CKN1/ ERCC6

5q12.1/ 10q11

216400

Arrested myelination

Cerebrotendinous/xanthomatosis

AR

CYP27A1

2q33-qter

213700

Global/ focal

PNS

Analyte

Other signs

Testing C/R

D Mixed

C/R R C/R

Abnormal DNA repair D

Cholestanol

Short stature/ skin changes/ retinopathy C/R Short stature, progeroid features, photosenstive C/R Cholesterol deposits in skin and tendons/ retinal degeneration

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Garbern

AD: Most cases are spordic and reproductively unﬁt. Abbreviations: OMIM, Online Inheritance in Man Database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db¼OMIM); AD, autosomal dominant; AR, autosomal recessive; XR, X-linked recessive; XD: X-linked dominant; MRI, magnetic resonance imaging; PNS, peripheral neuropathy D, demyelinating; VLCFA, very long chain fatty acids; NAA, N-acetyl aspartate; C, clinical testing available; R, research testing available (contact information listed at http://www.genetests.org).

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myelin lipid synthesis. Mice deﬁcient in fatty acid synthase die in utero, reﬂecting the importance of lipids in critical processes in addition to myelination (8). Lack of the relatively myelin-speciﬁc lipids galactocerebroside and sulfatide results in severe dysmyelination in mice that are deﬁcient in UDP-galactose:ceramide galactosyltransferase (CGT; in humans, uridine diphosphate glycosyltransferase 8; UGT8; MIM 601291), which is required for GalC synthesis (9). Therefore, it is likely that mutations in the human UGT8 gene would cause a recessive leukodystrophy. Disorders of myelin lipid breakdown are among the best known human leukodystrophies. All are recessive, as with most enzyme-encoding gene mutations. The age of onset and rate of progression of these lysosomal storage diseases reﬂect the degree of residual enzyme activity in the CNS and the normal turnover of individual lipids. Since myelin synthesis may not be affected in these disorders, myelin may form normally, but neurologic signs develop later as myelin turnover proceeds. Accumulation of storage products disrupts oligodendrocyte function and myelin integrity with accompanying clinical signs. In addition, storage products or their derivatives may be directly toxic, as proposed for Krabbe disease, which is characterized by psychosine (galactosylsphinosine) accumulation. Psychosine, like other lipids, has detergent properties, and may also have other cytotoxic effects. Lysosomal Storage Diseases Metachromatic Leukodystrophy (MIM 250100) Description and clinical manifestations: Metachromatic leukodystrophy (MLD) is characterized by metachromatic staining (from sulfatide) of the white matter, pituitary gland, liver, kidney, and testes (10–14). Sulfatide, a sulfated glycolipid, accumulates from reduced activity of arylsulfatase A (ARSA), the lysosomal enzyme that releases galactose-3-sulfate from sulfatides (15,16). This reduced activity results from mutations in the ARSA gene (17–19). Over 100 MLD-related ARSA mutations have been found according to the Human Gene Mutation Database (HGMD) (20). Their frequencies differ between different ethnic groups. MLD is an autosomal recessive disease that is usually classiﬁed by the age of onset into late infantile, early and late juvenile, and adult forms. The late infantile and early juvenile forms are the most common. The late infantile form begins typically between the ﬁrst and second year, and the clinical course is subdivided into four stages (21). In the ﬁrst stage, a child who has already learned to walk becomes unsteady and requires support to stand. Flaccid weakness and hypotonia occur in the legs or all four limbs. Muscle stretch reﬂexes may be diminished or absent. Genu recurvatum is often present. This stage lasts from a few months to a year or more. In the second stage, which may last only a few months, the child can sit but no longer stand; cognitive regression begins; speech is impaired due to a combination

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of dysarthria and aphasia; ataxia and nystagmus are noted; and spasticity develops in the legs and possibly the arms, even though deep tendon reﬂexes may remain diminished from peripheral neuropathy. The child may also complain of intermittent pain in the arms and legs. In stage 3, the child is quadriplegic and bedridden, with decorticate, decerebrate, or dystonic posturing. Bulbar and pseudobulbar palsies cause difﬁculty in maintaining normal breathing. While speech is no longer distinct, the child may be able to smile and respond. In stage 4, which lasts from a few months to a decade or more, the child is blind, deaf, without speech or volitional movement. The juvenile form begins clinically between 4 and 12 years, and is subdivided based on whether the onset is before or after six years of age. The disease is characterized by impaired school performance and behavioral disturbances, such as confusion or bizarre behavior, incontinence, gait disturbance, dysarthria, and extrapyramidal dysfunction. The disease progresses to states that correspond to stages 3 and 4 of the late infantile form (22). The late juvenile form progresses more slowly than does the late infantile and early juvenile variants, and the duration of the illness may be longer than 20 years. Onset in the adult form can range from 12 to over 70 years. The initial symptoms typically affect behavior, manifesting as personality change or in poor school or work performance. There is anxiety, emotional lability, poor memory, and disorganized thinking. Symptoms of depression, schizophrenialike psychosis, feelings of depersonalization, or paranoia often lead to a diagnosis of psychiatric disorder. A survey of 129 patients revealed an overall incidence of psychosis in 53%, auditory hallucinations in 18%, delusions in 27%, and a psychosis was diagnosed in 35% (23). Peripheral neuropathy may also be the initial manifestation (24). Many, but not all, patients develop demyelinating peripheral neuropathy. Dystonic posturing may occur. In the later and ﬁnal stages, bulbar and spastic quadriparesis, decorticate posturing, and optic atrophy occur. Most commonly the duration of the illness is between 5 and 10 years, but in some patients it may last decades. Most MLD patients develop focal or generalized seizures that may be subclinical electrographic seizures (25,26). Some patients have a nonfunctioning gall bladder, although symptoms related to gall bladder involvement are overshadowed by the neurological manifestations. Gall bladder complications can include polypoid masses or papillomatous fronds, cholecystitis, and hyperbilirubinemia (27). Kim et al. (28) performed abdominal ultrasonograms in seven consecutive patients with MLD and found abnormal ﬁndings in four. Etiology: MLD is caused by deﬁciency of ARSA activity. The enzyme is encoded by the ARSA gene at chromosomal locus 22q13.31-qter. The coding region spans eight exons and predicts a primary protein sequence of 507 amino acids with an 18 amino acid leader sequence. ARSA is a protein of

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62 kDa. The protein contains three N-glycosylation sites. More than 100 MLD-related ARSA mutations have been entered in the HGMD or described (20). Among MLD - causing ARSA mutations, about 85% are nucleotide substitutions and about 13% are small deletions. One small insertion and one small combined insertion - deletion have been reported thus far. As with most recessive diseases, the prevalence of MLD and of speciﬁc diseasecausing alleles differs among different ethnic groups. The biological and clinical effects of the mutations vary. MLD mutations can be functionally divided into two broad groups: null (0 or I) alleles associated with no enzymatic activity and R (or A) alleles that are associated with some residual enzyme activity (19). The most common null allele is a mutation at the ﬁrst base in intron 2 (459þ1G > A) that leads to the loss of a splice donor site. This is one of the most frequent mutations (25% of 140 MLD alleles), and leads to a deﬁciency of ARSA messenger RNA (mRNA). No ARSA protein can be detected. Patients homozygous for this mutation have the most severe form of the disease. The P426L R allele is the second most frequent mutation and is present in about 15% of MLD patients. Patients with this mutation have normal production of enzymatically active ARSA, but, within the lysosome, the mutant enzyme has a reduced half-life. In most cases, persons homozygous for this mutation have the adult and sometimes the juvenile form of MLD. About 12% of MLD patients have the I179S mutation. Patients who are compound heterozygous for a null allele and an R allele show intermediate grades of clinical severity, such as the juvenile form of the disease. However, the variation in severity among patients with identical genotypes can be remarkable, and prediction of the clinical course for an individual based on genotype alone is not reliable (19,29–31). As with other metabolic diseases, the best screening test is measurement of enzyme activity or of the storage product in body ﬂuids. Complicating the laboratory diagnosis is the discovery of so-called pseudodeﬁciency (Pd) alleles (32). Pd does, in fact, cause partial deﬁciency of ARSA but since only 5–10% of ARSA activity is sufﬁcient to prevent sulfatide accumulation, it does not cause neurologic signs (33). Pd alleles complicate diagnosis and identiﬁcation of MLD carriers based on the biochemical assay. The Pd condition is associated with two mutations in the ARSA gene: the A2723 ! G, which removes the polyadenylation signal site of the mRNA, and the A1788 ! G, which results in the loss of an N-glycosylated asparagine in the protein (34). This latter mutation causes a severe deﬁciency of a 2.1 kb mRNA, with resultant diminished synthesis of ARSA. The Pd allele has a population frequency of 7–15% (35). The MLD mutations can occur also within this allele (36,37). Numerous reports suggest a relationship between the condition of MLD/ Pd compound heterozygotes and the development of nonprogressive neurological symptoms (38–41). That only a fraction of MLD/Pd compound heterozygotes develops symptoms suggests that other genetic or environmental factors contribute to the development of the neurological phenotype.

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Molecular and cellular pathogenesis: The main pathological feature of MLD is demyelination and the deposition of metachromatic granules in both the central nervous system (CNS) and peripheral nervous system (PNS). The demyelination in the CNS is widespread but spares the U-ﬁbers, while the peripheral nerves show segmental demyelination (42). The metachromatic granules, which under the electron microscope consist of lamellae and vacuoles and are referred to as ‘‘tuffstone bodies’’ (43), are present not only in the nervous system, where they are prominent in microglial cells, oligodendrocytes, and Schwann cells, but also in the kidney, gallbladder, retina, rectal tissue, and other tissues. The accumulation of sulfatide, the pathognomonic feature of MLD (13,14), is caused by deﬁciency of ARSA, or more rarely by a deﬁciency of the sulfatide activator, SAP (PSAP; see below). The mechanisms responsible for the demyelination are not clearly deﬁned. Sulfatide accumulation may induce alterations within myelinating cells. Accumulation of sulfatide in oligodendrocytes has been noted in a ﬁve-month-old fetus, at a time when myelin structure was still intact (44). Another potential mechanism is that abnormally high sulfatide content may cause myelin instability (45). Sulfogalactosylsphingosine, which may be cytotoxic, is present in abnormally high concentrations in the tissues of MLD patients and may have a role in demyelination (46). Data obtained in mouse models suggest that microglial activation in MLD and other lysosomal storage disorders contributes to demyelination (47,48). A ‘‘knockout’’ mouse model of MLD has been developed (47). The mice have the expected deﬁciency of ARSA activity and, starting at the age of 9–10 months, accumulate sulfatide in neuronal and non-neuronal tissues. During the second year of life they develop demyelinating lesions, and neurological examination reveals impairment of motor coordination and learning. Availability of these animals provides important new opportunities for the clariﬁcation of pathogenesis and for testing therapeutic interventions. Epidemiology: MLD is inherited in an autosomal recessive manner, with an incidence of 1 in 40,000 in Northern Sweden (49) but about 1 in 100,000 in the United States (50). A Habbanite community in Israel, where consanguineous marriages are relatively common, has a reported an incidence of 1.3% associated with a 17% carrier frequency (51). Diagnostic evaluation: The ﬁrst description of the use of MRI, the most useful screening test for leukodystrophies, in MLD appeared in 1991 (52). Kim et al. (53) have reviewed the MR ﬁndings in seven patients with late infantile MLD. Only one serial neuroradiological study has been performed on a patient with late infantile MLD (54). As described in the previous studies the brain MRI initially shows a diffuse symmetric hyperintense, but nonenhancing, signal in the periventricular and subcortical supratentorial white matter on T2-weighted images, usually beginning in the

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anterior white matter, but sparing the arcuate ﬁbers. As the disease progresses, other structures are involved such as the corpus callosum, cerebellar white matter, corticospinal tracts, internal capsules, and thalami. In the late stages, the U-ﬁbers are involved and atrophy appears. Another common manifestation is a tigroid pattern of demyelination, as observed in Pelizaeus– Merzbacher disease (53,55). Recently, diffusion weighted imaging performed in a case of lateinfantile MLD revealed a cytotoxic edematous pattern in the deep white matter, suggesting a restriction of mobility of water molecules within the abnormal portions of myelin sheaths, consequent to the impaired myelin turnover (56). Magnetic resonance spectroscopy (MRS) of patients with MLD reveals reduced choline, a marker of myelin turnover (57), but further studies on additional patients will be needed to ascertain the usefulness of this observation. Single positron emission computed tomography (SPECT) imaging in one patient with adult-onset MLD showed reduced cerebral perfusion in the frontal lobes, a nonspeciﬁc ﬁnding (58). CT scans remain very useful for the identiﬁcation of calciﬁcations, which suggests other acquired or hereditary diseases (cytomegalovirus infection, Aicardi–Goutierres syndrome, and Cockayne disease). Peripheral neuropathy is usually present in MLD patients with the late infantile or juvenile forms, but may be absent in adult-onset patients. When present, it manifests as a demyelinating neuropathy with progressive and severe reduction of nerve conduction velocities; later in the disease, nerve action potentials are diminished in amplitude with prolonged latencies, suggesting late axonal injury, as is common in other primary demyelination peripheral neuropathies (59). Neurophysiological signs of peripheral neuropathy may be present while patients are still asymptomatic. The combined presence of PNS and CNS white matter abnormalities helps to differentiate MLD and Krabbe leukodystrophy from Pelizaeus–Merzbacher disease, multiple sclerosis and other disorders that affect the CNS only. Metachromatic deposits can be demonstrated in sural nerve biopsies. This procedure was used frequently in the past, but is now replaced by less invasive biochemical and molecular studies. Biochemical tests: The speciﬁc diagnosis depends on biochemical procedures and mutation analysis. Since the reduction in ARSA activity can be caused by a wide variety of mutations, and genetic analysis may miss some mutations such as those in regulatory regions or introns, direct biochemical analysis of ARSA activity to detect ARSA deﬁciency is generally the ﬁrst step since its assay can be combined with enzymatic assay for other lysosomal enzymes. Certain factors have to be taken into careful consideration. There is considerable variability in enzyme activities among individuals (60), and ARSA activity in cultured ﬁbroblasts varies with culture conditions (61). The presence of the Pd allele, which accounts for 5–15% of residual activity,

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Garbern

may lead to the incorrect diagnosis, incorrect assignment of carrier status, and incorrect assessment of risk of having an affected child. Differentiation of these possible conditions requires the analysis of sulfatides in a 24-hour urine collection and molecular analysis. Large quantities of sulfatides can be detected in the urinary sediment of affected individuals and its measurement on the 24-hour collection may aid in diagnosis in all cases in which ARSA activity values are not clearly diagnostic for MLD (62). Normal ARSA activity is observed in MLD patients with SAP-B deﬁciency, because the colorimetric assay used for determining ARSA activity does not depend on this activator. In these patients sulfatiduria is positive. The most commonly used ARSA assay uses p-nitrocatechol sulfate as the substrate and employs conditions that minimize the contribution of arylsulfatase B (63). The use of labeled cerebroside sulfate (64) or a ﬂuorescent compound as substrates (65) may help in distinguishing MLD from Pd patients. Additional means of distinction between the two disorders are the sulfatide loading test (66) and the measurement of urinary sulfatide levels (67,68). The sulfatide loading test measures the ability of cultured ﬁbroblasts to convert 35S labeled sulfatide to inorganic sulfate. This capacity is reduced more severely in MLD patients than in people with the Pd allele. Urinary sulfatide excretion is increased in patients with MLD, but is normal or only minimally increased in patients with Pd. Mutation analysis: Mutation analysis provides an increasingly important diagnostic tool. While more than 100 MLD-related ARSA mutations have been described, roughly 50% of the MLD alleles remain unidentiﬁed. The three most common mutations (459þ1G > A, P426L, and I179S) are present in less than 50% of the patients. The other mutations described are usually private mutations. Therefore, most of the patients remain undiagnosed at a molecular level if only common mutation screening is performed. In cases where the more common point mutations have been excluded, but prenatal or preimplantation genetic diagnosis is under consideration, then gene scanning or sequencing can be pursued. The Pd allele has a population frequency of 7–15% (35). It can be identiﬁed with DNA-based assays, but its detection cannot rule out the concomitant presence of an MLD allele. In fact, the coexistence of MLD and Pd mutation in the same allele has been estimated at 2.5–13% in MLD patients; in view of the frequent occurrence of the Pd allele in the general population, this ﬁnding is not surprising. SAP deﬁciency also has to be considered in the presence of ARSA activity within normal range and elevated urinary sulfatides. Thus, judicious use of mutation analysis combined with biochemical assays is needed for correct diagnosis, management and counseling of patients and families affected by MLD and related disorders. Prevention: Reproductive planning in conjunction with genetic counseling is currently the main method for the prevention of MLD. An affected

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fetus can be identiﬁed prenatally (69) and prenatal diagnosis can be offered to all couples with a previously affected child. Carrier identiﬁcation may be achieved by assays of ARSA deﬁciency (51), but there is overlap with normal individuals (38). It is of critical importance for prenatal diagnosis and carrier identiﬁcation to distinguish MLD from the benign Pd condition. This distinction is discussed in the section on diagnostic workup. In families where the molecular defects have been identiﬁed in affected members, mutation analysis provides the most accurate method for prenatal diagnosis and carrier identiﬁcation. When mutation analysis is available, preimplantation genetic diagnosis is possible (70). Management: Dietary regimens, pharmacological agents, and attempts at enzyme replacement have not had a favorable effect on the clinical course or biochemical abnormalities (50). At present, no effective therapy is available for MLD except for bone marrow transplantation (BMT) or stem cell transplantation (SCT) in selected cases (71–74). Therefore, the main management remains symptomatic and includes: 1. Physical therapy and antispasticity drugs to improve the ability to walk independently or with aids as long as possible, and to minimize contractures and complications related to relative immobility. Periodic rehabilitation medicine and/or orthopedic evaluations may be helpful in preventing or treating orthopedic complications such as scoliosis and contractures. 2. Respiratory therapy and adequate control of pulmonary infections, which are quite frequent as a consequence of dysphagia. 3. Adequate support of calories, vitamins, and minerals, which may require, in the advanced stages, a nasogastric tube or a permanent gastrostomy. 4. Antiepileptic drugs to control seizures, which may not be clinically evident and therefore require electroencephalographic monitoring. 5. Adjustment of school environment and counseling to maintain contact with family and friends and activities. 6. Psychiatric intervention and medications to control psychosis and behavioral symptoms may be required. BMT and SCT can be effective treatments in some lysosomal disorders (74–77). Their therapeutic efﬁcacy relies on the migration of donor-derived cells of the monocyte–macrophage lineage into diseased target organs where they replace the resident enzyme-deﬁcient population. The donor-derived cells that migrate to the tissues can become a local and steady source of the missing enzyme. In selected MLD patients, such as in the very early stage of the juvenile and adult forms, BMT and SCT may delay the onset and slow the progression of the disease. Furthermore, BMT or SCT represents a valid therapeutic option in patients with late infantile onset if

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performed at a presymptomatic stage with a therapeutic ‘‘window’’ of at least one year, a condition that is generally fulﬁlled in the presence of an affected sibling, where the clinical outcome of an untreated child can be reasonably predicted. BMT has no efﬁcacy if performed in symptomatic patients with early onset or in advanced stages of late-onset patients. Thus, only a minority of MLD patients can beneﬁt from BMT (74). The number of patients eligible for BMT is further reduced by the availability of an HLA-compatible donor. In addition, BMT is associated with fairly high morbidity/mortality due to conditioning regimens, rejection, failure of engraftment, and graft versus host disease. A novel transplantation approach for MLD includes the use of mesenchymal stem cells (MSC) (73). Recently, six MLD patients, who previously underwent successful BMT from HLA-identical siblings, received allogeneic MSC from the same donors. Nerve conduction velocity (NCV) studies of three of these patients showed slight improvement, but without any signiﬁcant clinical beneﬁt. Further studies are required to better understand the potential therapeutic efﬁcacy of MSC transplant for MLD. The absence of robust therapies emphasizes the need for the development of innovative therapeutic approaches, such as gene therapy. Despite previous encouraging results based on the use of retroviral vectors (78–80), retroviral gene transfer into ARSA-deﬁcient hematopoietic stem cells cannot prevent or control the phenotype of transplanted MLD mice (81). Lentiviral vectors, capable of high efﬁciency of infection and long term and sustained expression of transgenes into non dividing cells, represent a unique therapeutic tool for enzyme delivery into the CNS (82). Using this approach, it is possible to correct both central and peripheral neuropathology and clinical impairments in the MLD mouse and is an especially promising potential therapy for MLD. Multiple Sulfatase Deficiency (MIM 272200) and Sulfatide Activator Deficiency (MIM 249900) Accumulation of sulfatides and clinical manifestations that resemble MLD can be caused by at least two other genetic disorders. In multiple sulfatase deﬁciency (MSD; MIM #272200), children are more severely affected than those with late infantile MLD and have clinical features of both infantile MLD and mucopolysaccharidosis (MPS) (50,83). While they may learn to stand and say a few words, affected children usually fail to attain the early normal developmental milestones that most children with late infantile MLD achieve. MPS-like features such as coarse facial features, hepatosplenomegaly, rib ﬂaring, stiff joints, and deformities of the acetabulum and sternum may occur. Ichthyosis (from steroid sulfatase deﬁciency) frequently develops. The disease advances during the second year, when the children lose the ability to stand, sit, or speak. Death often occurs before 10 years of age, but some patients have survived to the second or third decade. A somewhat milder variant, albeit with corneal clouding and severe dysotosis multiplex,

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but with only mild or moderate mental retardation, has been reported in Saudi Arabia (84). In MSD, the activities of at least eight sulfatases are deﬁcient (85), due to a defect in posttranslational modiﬁcation. The gene mutated in MSD, sulfatase modifying factor 1 (SUMF1) (86,87), encodes a factor that is both an essential and a limiting factor for multiple sulfatases. This factor is required for a posttranslational modiﬁcation at the catalytic sites of sulfatases, where Ca-formylglycine is generated by a cysteine modiﬁcation. With sulfatide activator protein deﬁciency (MIM #249900), the clinical manifestations are similar to those of MLD, and range in severity equivalent to late infantile to adult MLD (88,89). This syndrome is caused by mutations affecting the sphingolipid activator saposin (SAP; SAP-B) (90); ARSA activity is normal. In addition to saposin B, other products of the prosaposin or PSAP gene, which also encodes saposins A, C, and D, can be perturbed, and cause an MLD- or Gaucher-like clinical syndrome. The molecular biology of the SAP deﬁciency has been clariﬁed. The SAP, also referred to as SAP-1 or SAP-B, is derived from a larger precursor molecule, prosaposin. The prosaposin (PSAP) gene occupies about 20 kb on the long arm of chromosome 10 (90). Epidemiology and management of MSD and SAP deﬁciency: Approximately, 50 cases of MSD have been reported (50,84,91). Few cases of SAP deﬁciency are described, but this variant may be underdiagnosed. Management of MSD and SAP deﬁciency is similar to that of MLD. Krabbe Disease (Globoid Cell Leukodystrophy; GLD; MIM 245200) Clinical description: Krabbe disease was ﬁrst described in children with a syndrome that began during infancy and was characterized by tonic spasms, nystagmus, periodic fever, muscular rigidity, progressive quadriplegia, and early death (92–94). Postmortem examination revealed loss of myelin staining especially in the cerebellum, degeneration of long spinal tracts, and gliosis. Blood vessel sheaths were inﬁltrated by granule-containing cells that were described as globoid cells. These deposits are enriched in cerebrosides that accumulate due to lack of galactosylceramidase (95,96). The initial clinical reports of Krabbe disease described the early infantile presentation (92). This presentation, which accounts for over 90% of patients, begins before six months of age in most cases, and as early as in the ﬁrst three months of age in 25% of cases (97). It has four stages (94): Presymptomatic stage: Infants generally are able to ﬁx and follow visual targets, smile interactively and reach for objects. Stage 1 (3 to 4 months): Photic and auditory sensitivity, extreme irritability, intermittent neurogenic fever, developmental arrest followed by regression, and tonic spasms with normal to reduced muscle stretch reﬂexes.

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Stage 2: Permanent opisthotonus, tonic ﬂexion of arms and extension of legs, rapid loss of nearly all previously acquired skills, myoclonic jerks, seizures, optic atrophy, blindness and more frequent febrile episodes. Stage 3 (usually before 12 months): Immobile, decerebrate but no longer rigid, and unable to feed. Children can uncommonly present in the neonatal period; prenatal onset was ﬁrst suggested when a ﬁve-month-old fetus was noted to have the characteristic neuropathological ﬁndings (98). The cerebrospinal ﬂuid protein is usually elevated (99). The late infantile variant is much less common and begins between 19 months and four years of age (100). These children have normal intelligence or only moderate retardation during the ﬁrst years, but gradually develop ataxia, weakness, spasticity, and dysarthria. Visual loss accompanied by optic atrophy, mental regression, occasional seizures, deafness, and normal peripheral nerves are the characteristic ﬁndings. Spinocerebellar degeneration associated with peripheral neuropathy, but without visual loss or dementia, has been reported (101). As in other forms of Krabbe disease, cerebrospinal ﬂuid protein is usually, but not always elevated (100). Late-onset forms of Krabbe disease have been deﬁned as juvenile onset (4 years to 19 years), and adult onset (>20 years of age) (102–104). These patients usually have optic nerve pallor, pes cavus, slowly progressive spastic tetraplegia, sensory-motor demyelinating neuropathy, symmetric T2 hyperintense parieto-occipital white matter, and preserved mental function (102,105). Cerebrospinal ﬂuid protein is frequently normal. Survival into the third to fourth decades is possible with juvenile-onset disease. Adult-onset patients can present in the fourth to ﬁfth decades with signs of motor neuron disease, mild and patchy demyelinating peripheral neuropathy, mild elevation of cerebrospinal ﬂuid protein, and mild T2 hyperintensity of deep cerebral white matter (106,107). Survival into the seventh decades is possible. Pathogenesis and pathophysiology: Krabbe disease, an autosomal recessive disorder, is caused by deﬁciency of the enzyme galactosylceramidase (GalC; EC 3.2.1.46; galactosylceramide b-galactosidase; galactocerebrosidase) that catalyzes the catabolism of galactosylceramide. The GALC gene lies at 14q31 (108,109) and spans 17 exons over a 60 kb interval (110). The GALC cDNA encodes a 669 residue protein of about 80 kDa with a 26 amino acid leader sequence (111). The protein is further cleaved to 50 kDa amino terminal and 30 kDa carboxy terminal subunits. Molecular and cellular pathogenesis: The typical neuropathological ﬁndings in Krabbe disease include generalized brain atrophy with replacement of the central white matter by gliotic tissue. There is severe loss of oligodendroglia, myelin, and axons with dense astrocytic proliferation, and the presence of ‘‘globoid cells,’’ phagocytic cells of 30–50 m in diameter ﬁlled with

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periodic acid-Schiff (PAS) stained material ringed by a chain of multiple nuclei. The cells are likely of macrophage–microglial origin. In the murine model of Krabbe disease (the twitcher mouse) (112), oligodendrocytes undergo apoptosis (113). Myelin loss is seen in peripheral nerves; however, typical globoid cells are usually not observed (114). Biochemical analysis of Krabbe disease brains demonstrated the accumulation of the glycolipid, galactosylceramide, within a fraction of brain that was enriched in globoid cells (115). Experimentally, galactosylceramide injected into the cerebral cortex produces globoid cells (116). However, total glycolipid and sulfatide levels in white matter from these brains were reduced (14). Malone and Suzuki conﬁrmed that galactosylceramidase was deﬁcient in the leukocytes, brain, liver, and spleen of Krabbe patients (117,118). Galactosylceramidase hydrolyzes other glycolipids containing a terminal beta-galactose group, such as lactosylceramide, monogalactosyl diglyceride, and galactosylsphingosine (psychosine) in addition to galactosylceramide (119–121). Analogous to ARSA, galactosylceramidase requires a sphingolipid activator protein (saposin A or C) that helps solubilize the protein–lipid complex (122). Saposin A and saposin C also activate the degradation of galactosylsphingosine (122). Lack of the lysosomal enzyme, GM1 ganglioside bgalactosidase, which hydrolyzes GM1 ganglioside, causes GM1 gangliosidosis. Under certain conditions GM1 ganglioside beta-galactosidase can hydrolyze galactosylceramide, but not psychosine (galactosylsphingosine) (123). Though galactosylceramide does not accumulate in the white matter of Krabbe patients except in the globoid cells (115), psychosine is increased in the brains of patients with Krabbe disease (124). Psychosine, normally present at only trace levels in the CNS, causes oligodendrocyte apoptosis in culture that can be reduced by protein kinase C activators, by galactosylceramidase, or by sequestering psychosine (125). Thus, psychosine, but not galactosylceramide, may accumulate in the brains of Krabbe disease patients and may be responsible for a signiﬁcant amount of the observed cellular pathology and paradoxical relative lack of galactocerebroside accumulation (124). The psychosine-induced apoptotic depletion of oligodendroglial cells could cause abnormal myelination and reduce the amount of galactocerebroside that would otherwise accumulate. Over 60 mutations affecting GALC are associated with Krabbe disease including base transitions, polymorphisms, and deletions. Approximately 40–50% of cases of infantile Krabbe disease affecting children of Northern European ancestry have an allele that has a 30-kb deletion beginning in intron 10 and extending past the 30 end of the gene (126,127). This deletion allele also has a C to T transition at position 502, a polymorphism seen in only about 4% of the population. Most of the mutations causing infantile-onset disease are located in the region encoding the 30-kDa subunit of the enzyme, suggesting that this subunit is critical for the normal functioning of the enzyme (97). Adult-onset cases may also have the 502/del mutation on one allele, but many

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other mutations (occurring predominantly in the region coding for the 50 kDa subunit) have been found (103,105–107,128–130). Epidemiology: Krabbe disease is an autosomal recessive disorder that has been reported worldwide. Most reports probably underestimate the incidence of Krabbe disease; an estimate from Sweden suggested an incidence of 1.9 in 100,000 (131), whereas in Japan the estimate was 1 in 100,000 to 1 in 200,000 (132). In Germany, the incidence was estimated as 0.6 per 100,000 live births (133). In a highly inbred Israeli community the incidence was 1 per 6000 live births (134). Diagnostic evaluation: An infant suspected of having Krabbe disease should have an MRI scan to evaluate the white matter. Understanding the progression of normal myelination aids the interpretation of MRI abnormalities seen in infantile-onset Krabbe patients. The posterior limb of the internal capsule is normally myelinated by birth, but infants with early infantile Krabbe disease have abnormal T1 and T2 intensities in this area. As the disease progresses, these areas and gray matter atrophy, even as normal patterns of myelination are developing in the frontal areas (135–137). In infantile Krabbe disease, cerebellar white matter and the pyramidal tracts are usually abnormal. These white matter abnormalities, seen as abnormal T1 and T2 contrast signal, most often affect the pyramidal tracts, the posterior corpus callosum, and parieto-occipital white matter (137). As the disease progresses all of the white matter structures become abnormal. Anomalously low signal in the thalamus on T2-weighted images and high signal on T1-weighted images can be found, suggesting a paramagnetic effect due to hydrophilic material (138). These regions at postmortem have abundant globoid cells and macrophages, some with calcium deposits. Adult-onset Krabbe disease can present with relatively mild changes in the posterior corpus callosum and parieto-occipital white matter (103,106,107,139). Electrophysiological studies are helpful in evaluation of Krabbe disease. Husain studied 26 patients and reported slow nerve conductions in all children with early and in 20% of children with childhood onset disease, whereas 88% of early infantile Krabbe children and 40% of late onset Krabbe children had abnormal brainstem auditory evoked potentials (BAEPs) (140). The severity of abnormalities on electrophysiological testing correlated with severity of abnormalities on MRI scans. Electroencephalograms (EEGs) are usually normal early in the course of disease, but generalized slowing and multifocal epileptic spikes, which may not always be associated with motor activity, are typical in the later stages (140,141). Cerebrospinal ﬂuid in patients with early and late infantile forms of Krabbe disease shows elevated protein (frequently 75–500 mg/dL) with normal cell counts (97). Ultrastructural studies of skin biopsies may identify electron dense inclusions in eccrine sweat glands similar to those seen in Krabbe disease Schwann cells (142,143).

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Since galactocerebroside does not accumulate to signiﬁcant and consistent levels in body ﬂuids of Krabbe patients, measurement of galactosylceramidase activity in serum, leukocytes, or cultured ﬁbroblasts is the only speciﬁc test for conﬁrming the diagnosis of Krabbe disease. Genetic testing is the deﬁnitive test that when positive will permit prenatal or preimplantation genetic diagnosis. Defects in saposin A, similar to those of sulfatide activator protein deﬁciency, may cause Krabbe disease in the setting of normal galactocerebrosidase enzyme levels (144). Prognosis and complications: Children with the early infantile Krabbe disease have a poor prognosis, and rarely live beyond two years of age (99). Children with late infantile Krabbe disease may survive for one to ﬁve years or more after neurologic signs arise. Adult onset patients have been reported who have survived until the seventh decade (106). Management: Therapy for Krabbe disease is limited. BMT has shown limited beneﬁts in small series of patients. Five children with Krabbe disease (four juvenile, one infantile) who received hematopoietic SCT had signiﬁcant improvements in their neurologic symptoms (145). Children transplanted well into the course of the illness have not improved following the procedure. While not as extensively studied as with other lysosomal disorders, BMT and SCT remain among the more promising future therapies for this devastating condition (77,146). Supporting the application of BMT and SCT for treatment of Krabbe are demonstrations of signiﬁcant beneﬁt of BMT in treating the twitcher mouse, a bona ﬁde model of Krabbe disease (147–149). Once neurologic signs begin, therapies such as BMT are less effective and associated with signiﬁcant morbidity and mortality. At these later stages, treatment is limited to symptomatic and supportive measures. The severe irritability can be treated with either benzodiazepines or low doses of morphine (150). Other supportive measures are as described for treatment of complications of MLD (above). Matsuda et al. (151) observed that saposin A deﬁcient female mice that were kept continuously pregnant had improved neurologic outcome compared to affected nonpregnant females. The beneﬁt of pregnancy could be replicated with pharmacologic administration of estrogen. While the mechanism of this beneﬁcial effect was not proven, it may be mediated by down-regulation of some immunological mediators such as Tumor necrosis factor-alpha. Pharmacologic therapies to reduce galactocerebrosidase substrate buildup have shown promise, again using twitcher mice. L-cycloserine, which reduces sphingosine synthesis by inhibiting 3-ketodyhydrosphingosine synthase, prolonged the lives of twitcher mice (152). Combined with BMT, L-cycloserine therapy was even more effective (153,154). In principal, gene replacement therapy using viral vectors is a viable strategy. Transgenic ‘‘cure’’ of the twitcher mouse has been accomplished

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using genomic DNA (155). Ex vivo transduction of hematopoietic stem cells, as with MLD mice, is a potential therapy. Lipid Metabolism Disorders Salla Disease (Sialic aciduria, Finnish variant: MIM 604369) Aula et al. described children with an autosomal recessive disorder characterized by normal infantile development followed at 6 to 24 months of age by progressive cognitive decline, seizures, dystonia, ataxia and hypotonia, followed by spastic paraparesis or quadriparesis (155a). Most cases have been reported from Scandinavia, where the carrier prevalence can be as high as 1 per 100. Coarsening of facial features may appear during adulthood, but is not present during childhood, although short stature is common. Although children typically learn to walk and speak, by 20 years of age they have typically become wheelchair conﬁned and have cognitive capabilities of about a 2 year old (155b). Survival is probably shortened, but survival into the eighth decade has been reported. MR imaging demonstrates abnormal signal throughout the cerebral white matter, although with relative sparing of the internal capsule (155c). The cerebellar white matter almost always has a normal appearance. Tissues, such as skin, show storage deposits typical of lysosomal storage disease, but biochemical assays for lysosomal enzymes are normal. Urine sialic acid (N-acetyl neuraminic acid; NANA) is elevated (155d). Brain MR spectroscopy reveals elevation in a resonance most likely due to NANA rather than to NAA (155e). There is no speciﬁc therapy for Salla disease, so treatments are limited to symptomatic management. Salla disease is caused by mutations in the SLC17A5 gene, which encodes sialin (155f) and is allelic to the more severe infantile acid storage disorder (ISSD: MIM 269920). Sialin is an intrinsic lysosomal membrane protein required for export of sialic acid released by catabolism of lysosomal enzymes and glycolipids, to the cytoplasm. How the defect in sialic acid transport leads to the biochemical and clinical syndrome are not well understood. Null mutations of SLC17A5 lead to ISSD, while those with some residual activity lead to Salla disease. At least one mutation causes misfolded sialin, which is retained in the endoplasmic reticulum (155g). Sjo¨gren–Larsson Syndrome (SLS; MIM 270200) Sjo¨gren and Larsson exhaustively described an autosomal recessive syndrome that is prevalent in Sweden and characterized by short stature, macular degeneration, severe pruritic ichthyosis, spastic quadriparesis, developmental delay, seizures, and mental retardation (156). Sjo¨gren–Larsson syndrome (SLS) patients subsequently were found to have a characteristic leukodystrophy characterized by arrested myelination (157–163). SLS ﬁbroblasts have

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a defect in oxidation of fatty alcohols (164,165), and mutations in the aldehyde dehydrogenase, family 3, subfamily A, member 2, or ALDH3A2 (also fatty alcohol: NAD þ oxidoreductase and fatty aldehyde dehydrogenase) gene cause SLS (166). Clinical features: The triad of ichthyosis, spasticity, and mental retardation constitutes the major clinical signs of SLS. The dermatologic features are usually characterized by brown-yellow discoloration and wrinkled hyperkeratosis that usually develops in the latter half of the ﬁrst year of life. The ichythyosis generally spares the face and is associated with severe pruritis. Spastic paraparesis develops gradually during the ﬁrst two years of life. While SLS children may learn to walk, they become wheelchair dependent during later childhood or adolescence. Cognition typically is mild to moderately impaired. Macular degeneration characterized by the presence of retinal crystals, sometimes referred to as ‘‘glistening white dots,’’ is present in all patients (167,168). Photophobia and poor visual acuity are common symptoms. Seizures may occur, but are generally easily managed. Some patients can have a milder syndrome, with less severe dermatologic and neurologic signs. The cerebral MRI shows delayed myelination during the ﬁrst years of life. The cerebellar white matter is normal, and the internal capsule, splenium, and genu of the corpus callosum are also spared. T2-weighted scans show a zone of high signal in the periventricular white matter with either frontal or parieto-occipital predominance (163). MRS shows peaks at 1.2–1.3 and 1.3 ppm that may correspond to elevated levels of free lipids and their derivatives (161,163). In addition, elevated levels of creatine, choline, and inositol are found, suggesting demyelination and gliosis. The diagnosis is usually established by assay of ALDH3A2 activity in cultured skin ﬁbroblasts, but the diagnosis can also be made by measurement of leukotriene B4 (LTB4), 20-hydroxy-LTB4 and 20-COOH-LTB4 in urine. Levels of LTB4 and 20-OH-LTB4 are elevated, while that of 20-COOH-LTB4 is absent (168) due to inability of ALDH3A2 to oxidize LTB4 to 20-COOH-LTB4 (169). Direct genetic diagnosis is now available and enables prenatal and preimplantation genetic diagnosis. Epidemiology: The prevalence in Vasterbotten county in Sweden was 8.3 per 100,000, suggesting a founder effect and inbreeding. For Sweden overall the prevalence was 0.4 per 100,000, which is roughly the global prevalence (156,168). Molecular pathogenesis: The defect in ALDH3A2 impairs oxidation of aldehydes to their corresponding carboxylic acids. The precise role of this in myelin, in the eye and in skin is not well understood, but may relate in part to turnover of lipids. In the skin, the accumulation of LTB4, a pro-inﬂammatory compound, likely accounts for the ichthyosis and pruritis (168).

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Management: Deﬁnitive therapy for SLS is not available at this time. There are no published reports on the use of growth hormone to correct the short stature in SLS patients. There are some promising disease-speciﬁc treatments. LTB4 synthesis inhibitors would be beneﬁcial at least with respect to the pruritis (169–172). Whether this strategy beneﬁts the ocular and CNS components of the disease is not known. Cerebrotendinous Xanthomatosis (CTX; MIM 213700) CTX is a slowly progressive syndrome of ataxia, spastic paraparesis, palatal myoclonus, dementia, cataracts, and tendinous xanthomas. Pathologic examination shows lipid deposits in the deep cerebellar and cerebral ganglia, partial myelin loss and gliosis. Menkes et al. (173) found elevated levels of cholestanol in CTX brain specimens. Setoguchi et al. (174) found that bile acid production in CTX is reduced, even though activity of cholesterol7a-hydroxylase, the rate-limiting enzyme in bile acid synthesis, is increased. Oftebro et al. (175) found that CTX patient mitochondria were unable to 27-hydroxylate 5b-cholestane-3a, 7a12a-triol. Cali et al. (176,177) found that CTX patients are deﬁcient in sterol 27 hydroxylase, now called CYP27A1 (cytochrome P450, subfamily 27A, polypeptide 1). Clinical syndrome: Chronic infantile diarrhea is the earliest possible sign in CTX and probably is caused by the defect in bile acid metabolism (178–181). Juvenile cataracts are the presenting sign in about 75% of individuals with CTX and usually appear in the ﬁrst decade (182). Tendon xanthomas are a typical ﬁnding that usually appears in the second or third decades, and affects the Achilles, elbow and wrist extensor, patellar and neck tendons. Xanthomas also can occur in internal organs, including lung, bones, and brain. A variety of neurologic signs can occur, including mental retardation, and dementia, psychiatric symptoms such as paranoid delusions, hallucinations, depression, agitation, spastic paraparesis or quadriparesis, ataxia, extrapyramidal abnormalities, and seizures. Palatal myoclonus appears to be an infrequent manifestation of CTX. Peripheral neuropathy with features of both demyelinating and axonal neuropathy has been described (183–189). Premature cardiovascular disease has been reported in CTX patients (190,191), however, others argue that factors other than CYP27A1 deﬁciency account for the vasculopathy, and that CYP27A1 deﬁciency is not a direct cause of lipoprotein abnormalities seen in some CTX patients (192). Diagnostic evaluation: The brain MRI shows cerebellar atrophy, diffuse and/or focal increase of T2 intensity in cerebral or cerebellar white matter that does not involve the U-ﬁbers (193–201). Symmetric elevated T2 or FLAIR signal in the cerebellar white matter adjacent to the dentate nuclei is characteristic (198,200,202). MR spectroscopy is helpful and shows reduced

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N-acetylaspartate, suggesting axonal dysfunction and increased lactate, suggesting mitochondrial dysfunction (200). Electrophysiological studies have shown slowing of nerve conduction in some patients (183,185,203), and neuropathy in others (189). It is possible that neurogenic changes are secondary to primary demyelination, as has been seen with CMT1 (204–209). Delay in central conduction velocities has also been reported (187,188,210). CTX can be diagnosed clinically when the classic ﬁndings are present. It is distinguished from other xanthomatous conditions by the ﬁndings of high blood and tissue cholestanol levels, which are 5–10 times above normal, low to normal blood cholesterol levels, marked reduction of chenodeoxycholic acid, and especially increased bile alcohol levels in blood or urine, which can be 500–1000 fold or more than the normal levels. Identiﬁcation of homozygous or compound heterozygous mutations in CYP27A1 conﬁrms the diagnosis of CTX and enables straightforward presymptomatic and preimplantation genetic diagnosis. Although all conﬁrmed cases of CTX have mutations in CYP27A1, mutations affecting genes encoding cofactors required for CYP27A1 activity, adrenodoxin and adrenodoxin reductase, potentially could also cause CTX-like illness, analogous to sulfatide activator protein deﬁciency and MLD-like disease. Molecular pathogenesis: CYP27A1 deﬁciency precludes bile acid synthesis, leading to accumulation of aberrantly hydroxylated bile alcohols. Reduced levels of bile acids lead to high activity of 7a-hydroxylase, the ratelimiting enzyme in bile acid synthesis. This in turn results in high levels of bile alcohols, such as cholestanol, in body ﬂuids and tissues. Management: CTX is a treatable leukodystrophy. In addition to supportive and symptomatic care as outlined for other leukodystrophies, speciﬁc therapies are indicated. Chenodeoxycholic acid (CDCA), a drug used to medically treat cholelithiasis, has been effective in reversing the biochemical abnormalities and in stabilizing and even reversing the clinical condition in CTX patients by bypassing the metabolic block (211,212). Long-term CDCA treatment (750 mg/day in adults) normalizes the bile acid metabolic derangement and corrects the biochemical abnormalities and neurophysiologic abnormalities (213). CDCA treatment combined with HMG-CoA reductase inhibitors has been proposed as a therapy as well (214), but lovastatin and cholestyramine were found ineffective in reducing abnormal bile alcohol synthesis (215). Canavan Disease (Canavan–van Bogaert–Bertrand Disease MIM 271900) Clinical manifestations and intial description: Canavan disease is an autosomal recessive disorder of macrocephaly, severe mental and motor retardation and spongy white matter degeneration of the brain (218) with

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a high prevalence of this disorder in the Ashkenazim, in which the carrier frequency has been reported to be as high as 1 in 37 (216–221). This disorder results from mutations in the ASPA gene (coding for aspartoacylase) and gives rise to N-acetylaspartic aciduria (222–224). Patients with classic Canavan disease have developmental delay, beginning as early as three to six months of age (225). Around six months of age, the head enlarges disproportionately to somatic growth, and may reach the 90th percentile or more by one year of age. Extremity and truncal hypotonia is an early and generally persistent sign. Seizures and blindness (with optic atrophy) typically occur during the second year. Irritability and sleep abnormalities are common. Feeding difﬁculties and dysphagia ensue, and most children do not survive beyond the ﬁrst decade. Some may live beyond that with attentive care. In these cases, hypotonia evolves into spasticity with persistent quadriparesis and poor cognitive functioning (266). Milder clinical variants with onset at four to ﬁve years of age and more gradual neurologic decline have also been described (227,228). Adult variants have not been reported. Pathogenesis and pathophysiology: While aspartoacylase deﬁciency is the cause of Canavan disease (223,224), how inability to hydrolyze Nacetylaspartate (NAA) causes disease remains uncertain. NAA reaches very high concentrations in the mammalian brain (8–10 mM), making it the second most abundant organic acid (next to glutamate) in brain (229). Numerous attempts to deduce NAA function have been inconclusive (230–232). The striking spongiform change in white matter observed in Canavan disease suggests that NAA is important in the formation and/ or maintenance of myelin, although roles as a neurotransmitter, osmoregulator, or protein synthesis regulator have been suggested (230,232–235). NAA is synthesized in the gray matter in neuronal mitochondria by the enzyme aspartate-N-acetyltransferase (ANAT) (236–238). In contrast, aspartoacylase (ASPA) is speciﬁc to oligodendrocytes, the myelinating cells of the CNS (239–243). NAA can serve as a precursor for myelin lipid synthesis (244–246). While key elements remain to be veriﬁed, a compelling hypothesis has emerged that NAA, synthesized in neurons, is axonally transported, released, and taken up by oligodendrocytes that hydrolyze it with ASPA to acetate and aspartate. These enter multiple metabolic pathways, including lipid synthesis. Chakraborty et al. (247) have recently provided direct support for this concept in the optic nerve and Madavarao et al. have further demonstrated myelin synthesis is severely impaired in Aspa deﬁcient mice and in Canavan disease (187a). While the primary function of NAA may be to serve as an osmolyte, the proposed mechanism would explain the spongiform change as the result of the high extracellular or high glial levels of NAA, which when injected directly into the brain is neurotoxic (248).

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Canavan disease is autosomal recessive. The ASPA gene lies at 17pter-p13 (249). Over 50 ASPA mutations have been catalogued, but two mutations, Glu285Ala and Tyr231Stop, account for over 95% of Canavan mutations among Ashkenazim (219–221,250,251). While ﬁrst recognized in Ashkenazi patients, Canavan disease is found in other ethnic groups as well, where other mutations are found (219,252–260). Epidemiology: Canavan disease occurs in all ethnic groups but is best recognized in the Ashkenazi Jewish population where the carrier frequency is very high (219,220). With carrier testing for Canavan disease and other recessive diseases common among this ethnic group, the number of affected children has declined. Diagnostic evaluation: With an appropriate clinical suspicion of leukodystrophy, MR imaging of the brain should be performed. Early white matter abnormalities are seen in the cerebral white matter including U-ﬁbers, with less noticeable changes in cerebellar and brainstem white matter (261–263). Serial examinations should be obtained if clinical suspicion persists but early MR studies are normal or inconclusive. An important tool for screening for Canavan disease is MRS. NAA is particularly well suited for MRS and can be reliably measured in vivo (264). It is best to request measurement of NAA and other metabolites in an area of white matter, where NAA is normally metabolized and where it accumulates in Canavan disease (265,266). Deﬁnitive diagnosis of Canavan disease begins with biochemical testing for NAA in urine, where levels are usually over 100-fold greater than normal. NAA is also elevated in blood and cerebrospinal ﬂuid (CSF). Measurement of aspartoacylase activity in cultured ﬁbroblasts is possible, but usually not needed if NAA levels in blood or urine are diagnostic. Once the biochemical diagnosis of Canavan is established, genetic conﬁrmation should be obtained, especially if other family members require testing or prenatal or preimplantation diagnosis is being considered. It is important to understand the type of testing being performed at the laboratory, since some laboratories restrict testing to speciﬁc point mutations, such as the common Ashkenazi mutations, and would miss other mutations. Management: There is no speciﬁc treatment for Canavan disease, with symptomatic management as described for other leukodystrophies. Bone marrow or hematopoietic stem cell transplantation is not likely to be effective since microglia do not normally express aspartoacylase (personal observations). Therapy of Canavan disease with recombinant viruses expressing aspartoacylase is the most promising therapy being developed. There is an ongoing trial to test adeno-associated virus 2 (AAV2) vectors to provide aspartoacylase in Canavan patients (267). While results of this trial are awaited, there has been some success in treating a rat model of Canavan disease, the tremor rat (268,269) and a mouse aspartoacylase ‘‘knock-out’’ (270).

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Peroxisomal disorder Adrenoleukodystrophy and adrenomyeloneuropathy(MIM 300100) Description and clinical manifestations: ALD is an X-linked recessive disorder affecting the adrenal glands and white matter (271–273). It is associated with elevation in very long chain fatty acids (VLCFA) and defective b-oxidation of VLCFA in ALD patients resulting from mutations in the ABCD1 (formerly the ALDP) gene that encodes the adrenoleukodystrophy (ALDP) protein (274–278). The term adrenoleukodystrophy should be reserved for the X-linked disorder caused by mutations of the ABCD1 gene that involves the brain clinically or radiographically. The designation adrenomyeloneuropathy is reserved for the allelic syndrome that primarily involves the spinal cord and peripheral nerves, without cerebral involvement. The term neonatal ALD has been used to describe an autosomal recessive disorder that results from defects in peroxisomal biogenesis and should be considered a subtype of Zellweger syndrome. ALD classically begins during childhood, usually between 5 and 15 years of age, with (1) behavioral changes, including withdrawn, pseudo-autistic and/or hyperactive behavior, (2) visual impairment, (3) auditory impairment, and (4) signs of parietal lobe dysfunction, such as apraxia. As the disease progresses, gait disturbance and ataxia appear, often with asymmetric involvement initially. About one-third of patients develop seizures, which may herald more rapid progression that leaves the child bedridden, spastic, quadriplegic, and totally dependent for daily functions (279–281). Adrenal insufﬁciency occurs in over 90% of childhood onset disease but the temporal course does not mirror that of the neurologic signs. Hyperpigmentation due to increased ACTH levels often occurs. Isolated Addison disease may be caused by mutations in the ABCD1 gene. Milder disease variants are recognized and can begin during adulthood. First described by Grifﬁn et al. (282) adrenomyeloneuropathy (AMN) usually presents around 20–30 years of age with leg stiffness and clumsiness. Clinical signs of spastic paraparesis, dorsal column deﬁcits, neurogenic bladder, erectile dysfunction, and mild peripheral neuropathy develop relatively slowly. Cognitive dysfunction occurs in about 40% of patients, and Addison’s disease in 85%. Gait disturbance progresses gradually over about 10–15 years, and may necessitate use of ambulatory aids or conﬁning the patient to a wheelchair. In one series, 35% of over 1400 patients had childhood onset (281), similar to the proportion of childhood ALD in Auborg’s group of 378 patients (283). AMN is the most common adult form, and occurred in about 30% of patients in the Kennedy Krieger Institute series (281) and in 37% of patients in France (283). Five to 20% of patients with ABCD1 mutations do not have neurologic disability. Half of these patients have adrenal insufﬁciency and are referred to as the ‘‘Addison only’’ phenotype. A signiﬁcant proportion of male

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patients with Addison disease have elevated VLCFA (284–287). These patients and those who are neurologically intact without adrenal insufﬁciency are at risk of developing childhood cerebral ALD or AMN, but onset of neurologic signs may be delayed until the fourth decade or later. A Japanese patient with ALD has presented as a spinocerebellar syndrome (288). While ALD is generally regarded as an X-linked recessive disorder, about 40% of heterozygotes develop neurologic signs and symptoms, typically consistent with AMN (289). Rarely, presumably due to unfavorably skewed X inactivation, girls can present with childhood ALD. Pathogenesis and pathophysiology: ALD is caused by mutations in the ABCD1 gene, which is homologous to peroxisomal membrane protein 70. It is a member of the adenosine 50 -triphosphate-binding cassette (ABC) superfamily, a group of proteins that is involved in membrane transport. ABCD1 encodes a peroxisomal membrane ‘‘half transporter,’’ that probably is responsible for transport of VLCFA into peroxisomes for further metabolism. Over 670 ABCD1 mutations have been identiﬁed thus far (catalogued at www.x-ald.nl). Most families have unique or ‘‘private’’ mutations and there is no clear-cut genotype–phenotype correlation. ABCD1 knockout mice have mild neurologic signs compared to ALD patients, and do not develop the striking MRI changes or inﬂammatory changes characteristic of the human disease (although they do accumulate VLCFA) (290–292). The mechanism whereby VLCFAs accumulate in ALD patients is still unclear (293). The accumulation of VLCFAs logically could be explained by the observation that degradation of VLCFA by peroxisomal oxidation was impaired in cultured ﬁbroblasts of ALD patients (276). The gene encoding VLCFA CoA synthetase (VLCS), which initiates the catabolism of VLCFA was a candidate gene for ALD. McGuinness et al. (294) recently found, however, that VLCFA oxidation was not altered in tissues of ABCD1 knockout mice, an animal model for ALD. This group provided evidence that the primary biochemical defect was interference with interactions between mitochondria, where fatty acid synthesis occurs, and peroxisomes, the site of fatty acid degradation by beta oxidation. Increased levels of long chain fatty acids, such as C16:0, impaired peroxisomal VLCFA oxidation. Thus, increased long chain fatty acid levels are the primary abnormality in ALD, and elevation of VLCFA is a secondary effect in this new model. The role of ABCD1 in this model is unclear but this explanation will likely force reexamination of long held predictions. In the earlier model, it was thought to be involved in either transport of VLCFA or of VLCS into peroxisomes. However, localization of VLCS to peroxisomal membranes is not perturbed in ALD ﬁbroblasts. Further study will be necessary to clarify the key functions of ABCD1. How VLCFA accumulation leads to neurologic and adrenocortical damage is unclear. There is not a clear correlation between severity of the

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biochemical defect and neurological impairment or disease progression (295). Rather, the extent of perivascular lymphocytic inﬁltration in the white matter is the determining factor in childhood cerebral disease (296,297). There is true demyelination, mediated by mononuclear cells, of white matter. The accumulation of VLCFA in myelin may lead to vulnerability to an inﬂammatory ‘‘second hit’’ mediated by cytokines such as interleukin-1 and oligodendrocyte-toxic tumor necrosis factor-alpha (298,299). Inﬂammation leads to demyelination and oligodendrocyte loss. The inﬂammatory phase of the illness generally correlates with rapid clinical deterioration. The inﬂammatory component is mild or absent in patients with AMN, where more subtle mechanisms, such as appearance of gangliosides that contain VLCFA and interfere with axo-glial interactions, may operate, causing a ‘‘dying back’’ axonal degeneration (300). How the inﬂammatory phase is initiated is not understood. The adrenocortical pathology is characterized by accumulation of lamellar lipid striations that contain cholesterol esteriﬁed with VLCFA, which is thought to be cytotoxic (273). Similar cellular inclusions occur in the testis in Leydig cells (301). Epidemiology: The most recent estimate of ALD in the United States is that 1 per 21,000 males is affected, and that 1 per 14,000 women is heterozygous for ALD. In France, the most recent estimate is that about 1 per 15,000 males is affected by ALD (302). Differential diagnosis: Early in the disease, the main differential diagnoses for the childhood onset cerebral form of ALD are attention deﬁcit hyperactivity disorder (ADHD) or mild autism (Asperger syndrome). In ALD the loss of cognitive abilities, handwriting skills, vision, and/or auditory discrimination are important clues that differentiate this disease from ADHD or Asperger. The differential diagnosis of more advanced cerebral ALD adds seizure disorders, neoplasms, meningoencephalitis, acute disseminated encephalomyelitis, and other leukodystrophies. The differential diagnosis of AMN should include spastic paraparesis, both acquired (e.g., transverse myelitis) and familial forms, spinal cord compression, spinal vascular malformations, multiple sclerosis, spinal neoplasm, and Chiari malformation. Differentiation between X-linked ALD and autosomal recessive neonatal ALD is very important, since the two disorders have very different inheritance. The signiﬁcantly more severe clinical syndrome caused by so-called neonatal ALD should permit diagnosis of this from the X-linked syndrome, which does not have signs of cranial dysmorphism: peculiar facies, prominent forehead, dolichocephaly, low set ears, anteverted nares and wide nasal bridge, cataracts, esotropia, and epicanthal folds. Biochemical testing reveals elevations of pipecolic acid and phytanic acid in addition to VLCFA, reﬂective of a more extensive peroxisomal disorder.

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Heterozygous females can develop neurologic signs, typically similar to AMN. If such a female has no family history of ALD the diagnosis can be elusive without a high index of clinical suspicion. The syndrome is frequently mistaken for multiple sclerosis. Symmetric parietal–occipital white matter abnormalities on MRI can suggest the disease, although this ﬁnding is present in only a small fraction of these females. VLCFA levels in the blood are elevated (303). Diagnostic evaluation: The initial diagnosis is often suggested on the basis of the brain MRI scan, which in about 85% of cases shows symmetric parieto-occipital periventricular white matter changes best appreciated on T2-weighted or ﬂuid attenuated inversion recovery (FLAIR) scans (304). About 15% of ALD patients curiously have initial MRI changes in the frontal white matter (305). The MRI is also valuable in predicting the clinical course of disease and subtyping the syndrome (305,306). Children whose MRI scans remain normal at seven years of age are not likely to develop the cerebral form of ALD. Gadolinium contrast enhancement of lesions is a poor prognostic ﬁnding, however (307). Less than 5% of heterozygous women tested have brain MRI abnormalities suggestive of ALD (308). While standard MRI imaging studies are normal in these women, newer MR techniques such as MRS may be more sensitive to differences from normal. Elevated levels of VLCFA are the essential ﬁnding in ALD. All male patients and about 80% of female heterozygotes have high VLFCA levels (309). Blood VLCFA levels are also increased in other peroxisomal disorders, such as the Zellweger syndrome and neonatal ALD. The latter two disorders are clinically distinguishable from ALD, and biochemically also have elevations in phytanic acid and pipecolic acid, both of which are normal in ALD. Conﬁrmation of ALD by mutation analysis is now possible, and of value not only for deﬁnitively establishing the diagnosis, but also enabling reliable prenatal and preimplantation genetic testing. Prognosis: The cerebral form of ALD carries the most severe prognosis. Once clinical signs of cerebral involvement are apparent, clinical decline proceeds at an alarming rate, and leads to a quadriplegic and totally dependent state within two years. Patients may remain in a severely disabled state for many years with attentive care. Loes et al. (306) have developed a quantitative scoring system for MRI scans of ALD patients that is helpful for estimating the likelihood of clinical progression. This has particular importance in selecting patients for BMT or SCT therapy. However, up to 30–40% of patients with ABCD1 mutations have a relatively mild AMN syndrome. While the Loes score is helpful in predicting which patients are likely to worsen, there are no reliable tests for predicting whether an asymptomatic child will have a poor or mild outcome. Mutation analysis and biochemical testing results correlate poorly with clinical outcome, and even null mutations due to large deletions can have a mild phenotype (278).

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The prognosis with AMN and ‘‘Addison-only’’ syndrome is considerably more favorable than that of ALD and is often compatible with normal lifespan. About 40% of these patients ultimately develop some evidence of cerebral involvement (310). Some AMN patients progress rapidly after developing cerebral abnormalities. Other complications are decubitus ulcers, bowel perforation secondary to constipation, or urinary infections. Adrenal insufﬁciency occurs in most patients, and can manifest as weakness, vomiting, and hypotension that can be provoked by minor infections. This potentially life-threatening complication readily responds to hormone replacement. Management: The treatment of adrenal insufﬁciency is important, and is managed as with primary adrenal insufﬁciency. Annual measurements of ACTH levels and of serum electrolytes should be done to monitor for adrenal insufﬁciency in patients who have not yet developed the complication and to evaluate therapy in those who are on replacement therapy, such as oral hydrocortisone 20–25 mg in the morning, and 10–15 mg in the afternoon. While corticosteroids correct the adrenal insufﬁciency, they do not alter the neurologic disease. The treatment of the increased VLCFA levels has received much attention because of the remarkable story of Lorenzo’s oil. Lorenzo’s oil is a mixture of glyceryl trioleate and glyceryl trierucate oils with erucic acid, the key ingredient that lowers VLCFA in cultured ALD ﬁbroblasts and in ALD patients as well (311–313). These monounsaturated fatty acids reduce synthesis of saturated VLCFAs, presumably by competitive inhibition. Recent clinical trials have suggested that Lorenzo’s oil, which does not help children with active cerebral disease (312), may help slow the disease in presymptomatic individuals (293,313,313a). Lorenzo’s oil is generally well tolerated, but can cause symptomatic thrombocytopenia (314,315). BMT can arrest or even improve neurologic function in patients at the early stages of cerebral ALD (293,316–319), but is not effective once signiﬁcant neurologic signs have developed. Related therapy using umbilical cord blood transplants is being explored, but requires further study. Imprecision in predicting clinical outcome in presymptomatic children complicates the assessment of these aggressive therapies that have signiﬁcant morbidity and mortality. Pharmacologic therapy based on known aspects of disease biology and biochemistry has been tested or proposed. Beta interferon, which reduces the rate of multiple sclerosis exacerbations, failed to beneﬁt patients with demyelinating lesions due to ALD (320). Other treatments focused more directly on the biochemical defect are promising. 4-phenylbutyrate (4PB) improves VLCFA catabolism in cultured ALD ﬁbroblasts and in the ABCD1 knockout mouse, in which it also normalizes VLCFA blood levels (321). 4PB probably acts by different mechanisms, including altering expression of several genes including ABCD2 (formerly ALDR), which encodes a

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peroxisomal protein homologous to ABCD1 that partially complement ABCD1-deﬁcient ﬁbroblasts (321). 4PB may also directly augment VLCFA metabolism in both mitochondria and peroxisomes, separate from its effects on ABCD2 (322). 4PB was well tolerated in a brief clinical trial in AMN patients and moreover did lower levels of VLCFA (323). Further studies are needed to determine the safety and efﬁcacy of this promising agent. Statins, the pharmacological inhibitors of 3-hydroxy-3-methylglutaric acid coenzyme A reductase (HMG), promote oxidation of VLCFA and lower their levels in ﬁbroblasts from ALD patients (324). In a small clinical trial, Lovastatin lowered VLCFA blood levels in a group of 12 ALD and AMN patients, although no deﬁnite clinical improvement was observed in this short-term study (325). Symptomatic and supportive care is also important. In addition, intrathecal baclofen has been reported to be quite effective; however, patients and their caretakers should be alert to signs of intrathecal baclofen withdrawal, which can cause severe spasticity, delirium, seizures, and life-threatening hyperthermia which respond to prompt reinstallation of intrathecal baclofen (326,327). Astrocytic Leukodystrophy Alexander Disease (MIM 203450) Description and clinical manifestations: Alexander is a progressive disorder of megalencephaly, hydrocephalus, developmental delay, quadriparesis, and seizures (328). Pathologic examination of the brain noted the widespread occurrence of astrocytic inclusions called Rosenthal ﬁbers (RFs), and symmetric white matter abnormalities that are especially prominent in the frontal lobes. Although Alexander disease is probably an autosomal dominant disorder, virtually all cases are sporadic, since affected individuals do not reach reproductive age. The lack of familial cases impeded identiﬁcation of the involved gene since standard linkage analysis was not possible. The fortuitous recognition of RFs in transgenic mice that overexpressed glial ﬁbrillary acidic protein (GFAP), the major intermediate ﬁlament speciﬁc to astrocytes, prompted analysis of the GFAP gene in Alexander disease patients. Heterozygous mutations in the GFAP gene were identiﬁed in 11 of 12 infantile onset AD patients and in one juvenile onset patient (329). The most common presentation of Alexander disease is the infantile variant, which becomes apparent during the ﬁrst or second year with developmental delay, followed by loss of acquired abilities, quadriparesis, cognitive impairment, seizures, and megalencephaly. The head circumference is usually greater than the 98th percentile between 6 and 18 months of age. Head enlargement may be due to obstructive hydrocephalus secondary to aqueductal stenosis caused by subependymal RFs, and can cause more rapid clinical decline. These patients usually do not live beyond the ﬁrst decade.

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Juvenile Alexander disease may not become apparent until adolescence, when bulbar signs are prominent and associated with ataxia and spastic quadriparesis. Cognitive function may only be mildly, if at all, affected, but brainstem dysfunction can be life threatening (330–333). Juvenile onset patients can have focal lesions (334). Adult onset Alexander disease is the least common form of the disease, and resembles the juvenile onset disease, although with later onset and slower progression (332,335–339). Presentation with palatal myoclonus and abnormal eye movements can occur (340). Cases with patchy white matter lesions that may resemble those of multiple sclerosis have been described (338). Etiology: Most instances of infantile and juvenile Alexander disease are new mutations that are likely to be autosomal dominant, but are not transmitted since patients usually are not reproductively ﬁt. The affected gene encodes GFAP, the main intermediate ﬁlament in, and speciﬁc to, astrocytes (329). In the adult variant, the autosomal dominant nature of GFAP mutations has been demonstrated (337,341,342). Pathogenesis: While there may also be locus heterogeneity, since there are cases of pathologically demonstrated Alexander disease in which no mutations have been identiﬁed (329,343), the cause of Alexander disease in the majority of cases is mutations in the GFAP gene (329). To date, 23 point mutations, all missense, have been catalogued (http://www.waisman.wisc.edu/alexander/index.html). GFAP is a rod-shaped protein, like other members of the intermediate ﬁlament family of proteins. As with many other dominant disorders, mutations may exert a dominant gain-of-function effect that leads to the mutated GFAP associating both with itself and also with its normal counterpart, forming aggregates that accumulate as RFs. RFs are round or elongated astrocytic hyaline bodies that stain with eosin, Luxol Fast blue, and hematoxylin. At the electron microscopic level, RFs are osmiophilic, granular, nonmembrane-bound bodies up to 50 mm or more in size. RFs occur at astrocytic processes and end-feet and are prominent at subpial, subependymal, and perivascular locations in Alexander disease, as well as diffusely in the white matter. They contain GFAP, aB-crystallin, and heat shock protein 27 (hsp27). Another mechanism, suggested by the observation that GFAP transgenic mice develop RFs, is that the mutated GFAP increases expression of GFAP, which in some way is toxic to astrocytes. Whether RFs are cytotoxic, per se, or represent a protective cellular response to the mutated protein has not been resolved. Study of ‘‘knock-in’’ mice with human disease-associated mutations in a GFAP-null background may help clarify key pathogenic mechanisms (344,345). Interestingly, GFAP-null mice have very minimal neurologic abnormalities further strengthening the hypothesis that disease-causing mutations act in a gain-of-function manner (346).

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One of the most interesting facets of Alexander disease is the dramatic white matter change, suggesting a disturbance of oligodendrocyte dysfunction, even though the mutation targets astrocytes. This observation points to an important role for astrocytes in myelin function, as inferred experimentally in GFAP-null mice (347). Astrocytic function may be more important in the frontal lobe white matter since the changes are more prominent there. Alternatively, in the well-established role of astrocytes in forming the blood–brain barrier, GFAP mutations may disrupt the barrier, which would be consistent with observations that lesions enhance with contrast on MR imaging (348). Epidemiology: The true incidence is not known in the United States. A study of leukodystrophies in Germany indicated that Alexander disease constituted about 1.6% of all leukodystrophies. Differential diagnosis: Infantile Alexander disease should be distinguished from other leukodystrophies associated with megalencephaly, including Canavan disease, glutaric aciduria type I, and megalencephalic leukoencephalopathy with subcortical cysts (MLC), which each can be clinically similar to Alexander disease, but are usually more slowly progressive. The CACH syndrome should also be distinguished from Alexander disease, but this disorder is more slowly progressive and MRI scans do not show contrast enhancement. Adult cases need to be distinguished from multiple sclerosis and tumors. Diagnostic evaluation: An infant presenting with megalencephaly, developmental delay, and hypotonic quadriparesis should have MRI imaging and biochemical testing to evaluate conditions outlined in the previous section and other leukodystrophies as well. Symmetric frontal white matter abnormalities, with relative sparing of the occipital and cerebellar white matter should raise suspicion for Alexander disease. Contrast enhancement, especially around the frontal horns and basal ganglia, is characteristic of Alexander disease. MRS is helpful to exclude Canavan disease. Deﬁnitive testing can now be accomplished by GFAP gene analysis, obviating the need to perform brain biopsy. In the event brain biopsy shows RFs, GFAP mutation analysis should be performed. Prognosis and complications: The infantile form of disease has a poor prognosis and children typically do not survive beyond 5–10 years. With some of these children rapid decline may be due to obstructive hydrocephalus due to aqueductal stenosis, which can be temporized with shunting. Clinical decline as well as severity of neurologic signs is less in the juvenile and adult onset forms of disease. Management: No speciﬁc therapy is available for Alexander disease. Supportive and symptomatic management as described for other

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leukodystrophies is necessary. In addition, the added complication of obstructive hydrocephalus may necessitate ventriculo-peritoneal shunting.

Leukodystrophies Caused by Myelin Structural Proteins Pelizaeus–Merzbacher Disease and Spastic Paraplegia 2 (PMD/SPG2;MIM 312080/312920) Friedrich Pelizaeus described a family with an unusual disease for which he also noted ‘‘that the disease is passed on by the mother but does not hurt her’’ (349), consistent with what we now know is consistent with an X-linked mode of inheritance (350,351). Ludwig Merzbacher re-investigated 12 affected individuals from the same family and performed neuropathological analysis on the brain of one member. He identiﬁed the widespread loss of myelin in the cortical white matter (352). Merzbacher noted that the disease began in early neonatal life with aimless, wandering eye movements, followed by nystagmus. Infants failed to develop normal head control and displayed tremors or shaking movements of the head. The disease slowly progressed, with additional signs including bradylalia, scanning speech, ataxia and intention tremor of the upper limbs, spastic contractions of the lower limbs, athetosis, and cognitive impairment (349,352). Seitelberger subsequently described a disorder with similar pathology (353). There was nearly complete absence of myelin sheaths, and a profound loss of oligodendrocytes. Seitelberger suggested that this disease, designated the connatal form of PMD, was similar to that of Pelizaeus and Merzbacher, designated the classical form of PMD. In addition, he noted that in both disorders the absence of myelin was the primary biochemical defect, suggesting that both were leukodystrophies. Zeman et al. (354) emphasized the X-linked recessive inheritance of the disorder and speculated that the defect in PMD affected a proteolipid. Hudson et al. and Trofatter et al. (355–357) demonstrated mutations affecting the major protein component of myelin, proteolipid protein 1 (PLP1; also known as lipophilin or Folch-Lees protein, formerly PLP), cause PMD. Boespﬂug-Tanguy and collaborators identiﬁed several patients with PLP1 mutations who presented with spastic paraplegia without the other signs of PMD [(358), also reviewed in Ref. (359)]. Evaluation of additional patients with X-linked spastic paraplegia and mutations in the PLP1 gene showed that this syndrome could exist as either a ‘‘complicated’’ or a milder ‘‘pure’’ form in which the clinical phenotype is conﬁned to lower limb spasticity. Clinical manifestations of PMD/SPG2: PMD usually presents in one of three typical patterns. The most severe form of disease, connatal PMD, begins during the ﬁrst weeks of life, and is associated with hypotonia,

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respiratory distress, stridor, nystagmus, and sometimes seizures. Because of the prominence of hypotonia and respiratory symptoms, connatal PMD can be confused with motor neuron disease or spinal muscular atrophy (300). Individuals with connatal PMD develop severe spasticity with little voluntary movement, and never ambulate. They have very poor head control and cannot sit unsupported. Growth is poor, and expressive language usually does not develop, although understanding of language is acquired to a limited degree. These individuals usually die before the second or third decade of life. The most common form of disease, classic PMD, begins during the neonatal period, usually within the ﬁrst two to four months, and is associated with nystagmus, hypotonia with lower extremity weakness, and head titubation. Respiration is normal. Muscle tone progresses to spasticity later during childhood. Motor milestones are also delayed in classic PMD, and most individuals never walk independently. Patients with this disease develop spastic quadriparesis. Ataxia of trunk and limb movements is also a prominent feature of classic PMD, and dystonic posturing and movements may occur. Most individuals acquire some expressive and receptive language, which may even approach normal levels, but patients are usually dysarthric and have some degree of cognitive disability. These patients can survive until the sixth decade of life. The mildest form of PMD merges clinically with X-linked spastic paraparesis (SPG2). This disease usually begins/presents within the ﬁrst ﬁve years of life, and is associated with mild to severe spastic paraplegia. There may also be limb and gait ataxia. Patients with SPG2 may have nystagmus as an early sign (358,361) or it may manifest later sign as end-gaze rather than primary position nystagmus (362,363). Motor milestones are usually delayed, but most individuals learn to walk independently during childhood; this ability may be lost later in life. Language skills and intelligence can be normal. Individuals with this form of PMD usually have a normal life span and may reproduce (358). Females heterozygous for a PLP1 gene mutation may have neurological signs and symptoms but are rarely the propositus in a PMD family. Usually symptomatic females do not have neurologic signs until adulthood, when they develop an SPG2 syndrome. Occasionally girls can have signs of PMD during infancy and childhood, but they may resolve with maturation (364). A proposed mechanism to explain this interesting phenomenon is discussed later. Some patients with PMD, typically with a complicated spastic paraparesis phenotype, have a demyelinating peripheral neuropathy (365). The neuropathy in these patients is mild, however, and not usually clinically signiﬁcant (365). Etiology of PMD/SPG2: PMD and SPG2 are caused by mutations of the PLP1 gene. The most frequent cause of PMD (60–70% of cases) is duplication of the segment of the long arm of the X chromosome containing

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the PLP1 gene (366–369). PLP1 duplications are typically tandem in nature, involving a large genomic segment that includes neighboring genes (366, 370,371). In addition to duplications, triplications and quintuplication of the PLP1 locus also cause PMD (370). Striking variation in the position of the breakpoints occurs in different PMD families (370,371), unlike the duplication in Charcot–Marie-Tooth disease type 1A (CMT1A) (372). The duplicated segment can be as large as ﬁve megabases, which is over 150 times the size of the PLP1 locus (371). Therefore, not only might PLP1 be overexpressed in these patients, but also so might be a number of other X-linked genes. Only a fraction of genes are sensitive to dosage effects, and in the segments of the X chromosome duplicated in the PMD patients, PLP1 is apparently the sole gene in this region of the X chromosome for which changes in copy number cause phenotypic aberrations. Most families with duplications have a classical PMD phenotype (368,370,371). Unequal sister chromatid exchange in male meiosis is the major mechanism leading to duplication of the PLP1 gene (369,371). Additional mechanisms of genomic rearrangements operate at the PLP1 locus, as indicated by several families in which the duplicated copy invades another spot on the X chromosome (373). Despite the large number of duplications arising from sister chromatid exchange, the reciprocal recombination event, namely deletion of the PLP1 locus, rarely occurs (372,374). Deletion of PLP1 encompasses a much smaller segment of the X chromosome, with only two neighboring genes (372). The deletion of larger sections of the X chromosome may cause lethality or infertility. Based on the deletion breakpoints in the three identiﬁed families, a mechanism of DNA breakage followed by nonhomologous end-joining (NHEJ) similar to that proposed in some cancers (375–380) causes deletions spanning the PLP1 locus (381). Such a mechanism may also generate PLP1 duplications. In addition to the loss-of-function mutations arising from deletion events, point mutations in the PLP1 coding region at the initiation codon (382) or the second codon (363) are null for PLP1 expression. Unlike the PLP1 deletions characterized to date, these null point mutations allow for a direct examination of PLP1 loss without complicating considerations from deletion of those genes neighboring PLP1, namely the RAS superfamily member RAB9L and the thymosin b family member TMSNB (381). About 20% of PMD patients have point mutations (single base changes or small deletions or insertions) at the PLP1 locus that alter the amino acid sequence of the PLP/DM20 proteins. These include missense, nonsense, frame shift, and splicing mutations, all of which produce abnormal PLP/DM20 proteins. Of the abnormal PLP/DM20 proteins, most result in severely affected patients through a toxic gain of function (see below), while the remainder create milder disease that may be categorized as loss of function. Approximately 100 distinct mutations have been discovered to date, most of which result in missense substitutions (http:// www.geneclinics.org/proﬁles/pmd/pmd-table3.html).

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PLP1 splice site mutations have been uncovered in PMD patients. Some of these are not located at the strictly conserved positions in the donor and acceptor splice sites, including a deletion of 19 bp within intron 3 and 26 bp in intron 5 (383,384). Although the spliced products have not been characterized in these families, splicing mutations most likely result in skipping of an exon, an event that would create an internally deleted and possibly a frame-shifted abnormal PLP1 protein. However, mutations within intron 3 that eliminate the donor splice site may leave the DM20 transcript and protein unscathed. The atypical splicing mutations at the PLP1 locus (383,384) suggest that even more splicing mutations may be found in PMD/SPG2 patients, as sequencing efforts usually concentrate on coding regions and intron/exon junctions. Point mutations in the regulatory regions of the PLP1 gene potentially could alter the expression of PLP1 without affecting the protein sequence. A putative promoter mutation has been reported in a PMD family at -34 of the PLP1 gene (385). Whether this alters PLP/DM20 expression in the reported family is not known. Additional undeﬁned changes may occur within PLP1 regulatory elements or splice sites that affect PLP1 expression. Nonetheless, the C to T transition at -34 is of interest as it is within the area bound by the RNA polymerase complex prior to the initiation of transcription at the upstream initiation site. Clinical testing: All patients with typical signs of PMD eventually develop MRI ﬁndings consistent with a leukodystrophy, with diffusely increased signal intensity within the central white matter of the cerebral hemispheres, cerebellum, and brainstem, best seen on either T2-weighted or ﬂuid attenuated inversion recovery (FLAIR) sequences. Indeed, the MRI abnormalities are often the ﬁrst observations that lead to consideration of PMD or other leukodystrophies. Patients with the spastic paraplegia phenotype have similar MRI changes, but these may be patchy in nature (386,387) or very subtly abnormal in comparison to those of patients with the more severe forms of PMD (351,384,388). Deﬁnitive diagnosis of PMD or SPG2 requires demonstration of a mutation in PLP1. Approximately 80% of patients with clinical, genetic, and MRI features consistent with PMD have PLP1 mutations. Some of the 20% of PMD patients without PLP1 mutations have mutations in the GJA12 gene (389) (see below). PLP1 mutations can arise spontaneously with fairly high frequency, and the distribution of mutations in sporadic cases is similar to that of familial ones (369). The genetic etiology for the remaining patients is not known, but may be due either to mutations in areas of the PLP1 gene not routinely analyzed (introns and regulatory regions) or the presence of an additional autosomal or X-linked mutation causing the same phenotype (390). Because duplication of the PLP1 region is the most common cause of PMD, identiﬁcation of a PLP1 gene duplication is the most efﬁcient initial

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genetic screening test for diagnosing PMD. The duplications are of variable size, but are usually found within an 800-kb region of the X chromosome including the PLP1 gene (366,368,370,371). The duplicated region can also be found, however, at some distance from Xq22. One PLP1 duplication has been identiﬁed at Xp22 and a second at Xq28 (370,373). Both interphase ﬂuorescent in situ hybridization (FISH) and quantitative polymerase chain reaction (QPCR) can detect PLP1 duplications (366,376,383,391). Duplications smaller than 50 kb, however, may not be resolved by FISH, while QPCR does not provide cytogenetic information on the location of the duplication, which could have genetic counseling implications (391,392). For these reasons, both interphase FISH and QPCR should be employed for the molecular genetic diagnosis of PMD. If neither interphase FISH nor QPCR demonstrates a PLP1 duplication, direct sequence analysis of the PLP1 gene should be performed. The PLP1 gene encodes a relatively small protein of 277 amino acids (831 bp of DNA) and the coding sequences are contained within only seven exons. Using automated sequencing methods, therefore, it is cost effective and technically straightforward to obtain the DNA sequence of the PLP1 exons and portions of their surrounding introns. When a small mutation is found, it is sometimes possible to design an allele-speciﬁc oligonucleotide hybridization test or a simple PCR/restriction digestion assay to detect the mutation, which can be particularly helpful for assessment of carrier status in females (383). Prenatal DNA diagnosis of PMD in affected males at risk for the disease has been accomplished by several groups of investigators and is now currently available at several centers (393). A PLP1 duplication in an at-risk male fetus has been identiﬁed using both interphase FISH (394) and QPCR (395). A PLP1 point mutation in an at-risk male fetus, however, has not been reported, although prenatal testing has successfully excluded such mutations (396,397). Preimplantation genetic diagnosis (PGD) for PMD is possible. Direct identiﬁcation of a PLP1 duplication in single cells is technically difﬁcult and might not be reliably distinguished from DNA replication, but indirect identiﬁcation of the duplication-bearing X chromosome is possible by multiplex PCR ampliﬁcation of polymorphic repeats that ﬂank the PLP1 gene, and could potentially be used for PGD. Differential diagnosis: Other diagnostic considerations for individuals with the clinical features of PMD include MLD, ALD, Krabbe disease, Salla disease, Cockayne disease, Pelizaeus–Merzbacher-like disease, and Canavan disease. Most of these diseases, however, are not associated with nystagmus, which is common in PMD. Their diagnosis can usually be made by analysis of the appropriate enzyme or analyte. PMLD is established by analysis of the GJA12 gene. In addition, the white matter abnormalities in these conditions are often regional rather than diffuse: the occipital white

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matter is most affected in ALD, while the frontal white matter is most affected in MLD. Infants with merosin deﬁciency can also have dramatically increased T2 signal in the cerebral white matter, but the presence of severe weakness and hypotonia and the absence of nystagmus should direct the clinician toward consideration of myopathy. A fatal X-linked syndrome of ataxia, blindness, deafness, and mental retardation has been described and is linked to Xq21-24, but the MRI does not show a pattern of leukodystrophy, and mutations in the PLP1 coding regions have been excluded (398). Finally, mutations in the cell adhesion molecule gene L1CAM cause X-linked spastic paraplegia type 1 (SPG1), a disorder associated with mental retardation and adducted thumbs which is allelic to the MASA syndrome (mental retardation, aphasia, shufﬂing gait, adducted thumbs) and X-linked hydrocephalus (399). MRIs of these disorders may show enlarged ventricles or agenesis of the corpus callosum, but not the diffuse abnormalities of white matter consistent with a leukodystrophy. Epidemiology: The incidence of PMD is not known precisely, but was estimated in Germany to account for about 6.5% of leukodystrophies in that country, or about 1 per 750,000 births (or 1/375,000 male births) (133). The true incidence may well be higher since the diagnosis is not infrequently missed. In a survey in the Czech Republic, the incidence was estimated at 1 per 70,000 births (399a). Molecular pathogenesis of PMD: PMD and SPG2 are caused by mutations affecting the most abundant protein in CNS myelin, proteolipid protein 1 (PLP1). PLP1 is unusually hydrophobic, and its name derives from the observations that it is soluble in organic solvents rather than aqueous buffers (400). In addition to a high composition of hydrophobic amino acids, six fatty acid chains are covalently linked to the PLP1 molecule (401). These fatty acids may mediate the association of PLP1 with the adjacent lipid leaﬂet in compact myelin (402). Acylation occurs autocatalytically at a stage following translation of PLP1 mRNA, a temporal pattern consistent with a role for this posttranslational modiﬁcation in the stabilization and/or compaction of myelin (403,404). PLP1 is synthesized in the rough endoplasmic reticulum as a tetraspan intrinsic membrane protein with both termini on the cytoplasmic face (405,406) and subsequently transported through the Golgi complex, where other myelin lipid constituents such as cholesterol and galactocerebroside associate with PLP1 in ‘‘rafts’’ (407). Raft formation is one of the initial stages of myelin assembly, followed by the vesicular transport of PLP1 to the myelin membrane. PLP1 is extremely well conserved. PLP1 gene structure is preserved among tetrapods and readily discernible in the primordial gene of the lipophilin family present in invertebrates (408). Mammals share a nearly identical coding capacity for PLP1. Moreover, no amino acid polymorphisms have been detected in the thousands of coding regions sequenced in the human

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PLP1 gene. The PLP1 gene has seven exons (409). The third exon contains an internal donor splice site which is used to generate transcripts encoding the smaller (20 kDa) DM20 isoform. While identical to PLP1 in topology, DM20 is missing part of the intracellular loop that contains two acylation sites, an absence accounting for the altered conformation and physical properties of DM20 (405,410,411). DM20 is expressed during embryonic development before PLP1, but is present at about 20% the level of PLP1 in mature CNS white matter (412). The two proteins can form heteromers (413). PLP1 and DM20 perform distinct roles in the maintenance of myelin structure, since DM20 cannot fully compensate for deﬁciency of PLP1 in myelin (402,408). Other functions have been proposed for PLP1/DM20 based on features of these lipophilin family members resembling channel proteins, the detection of secreted fragments of PLP1/DM20, and the expression in other glial cells and outside of the nervous system (414,415). PLP1 may act as a sensor in transmitting information across the lipid bilayer (416) as PLP1, but not DM20, interacts with av-integrin as part of a signaling complex (417). Apart from oligodendrocytes, the PLP1 gene is transcriptionally active in the nervous system in olfactory ensheathing cells (418), satellite cells (419), and Schwann cells (363,420,421), where the predominant isoform expressed is DM20 (422). Schwann cell expression of PLP1/DM20 is an order of magnitude lower than that observed in oligodendrocytes, and most of the proteins produced are not normally incorporated into the myelin sheath (363,423). A low level of PLP1/DM20 expression also occurs outside of the nervous system, in the heart (424), fetal thymus, spleen (425), thyroid, testes and skin (Skoff, unpublished). In general, cells other than myelinating oligodendrocytes favor the synthesis of DM20 over PLP1. Even in oligodendrocytes, the DM20 expression proﬁle does not always coincide with myelination, as immature oligodendrocytes selectively express DM20 (426–430). Gow and Lazzarini proposed that differences in clinical severity in patients with PLP1 coding region mutations reﬂect differential effect of the speciﬁc mutation on the folding and intracellular trafﬁcking of the protein [reviewed in Ref. (431)]. Mutations that affect the folding and transport to the cell surface of both PLP1 and DM20 cause the most severe PMD phenotypes and oligodendrocyte cell death, while mutations that impair transport of PLP1 but not DM20 produce a less severe PMD phenotype that is not associated with oligodendrocyte cell death (416,432). Since mutations in which no mutant protein is synthesized cause a relatively mild syndrome, the predominant effect of PLP1 missense mutations is probably due to misfolded PLP1 gene products. The cellular and molecular effects of the accumulation of misfolded PLP1 and DM20 in the rough endoplasmic reticulum (RER) of oligodendrocytes, rather than the absence of these proteins in the myelin sheath, cause the clinical signs and symptoms of PMD. The protein misfolding also explains the effect of these mutations on female carriers. Female dogs that are heterozygous for a severe mutation

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in the canine Plp1 have neurological abnormalities early in life, but by adulthood are clinically normal, have normal numbers of oligodendrocytes, and express very little mutant PLP1 messenger RNA (433). Female PMD carriers are also usually clinically unaffected, although some may have transient neurological abnormalities as children (364). In some PMD families, however, female heterozygotes are clinically affected (349). Because of random inactivation of the X chromosome on which the PLP1 gene is located, females who are heterozygous for PLP1 mutations should express the abnormal protein in approximately 50% of their oligodendrocytes. Oligodendrocytes expressing a more severe PLP1 mutation, however, in which both PLP1 and DM20 are affected, undergo increased cell death and are eliminated during myelination and replaced by normal oligodendrocytes. In contrast, oligodendrocytes expressing a less severe PLP1 mutation, which does not cause cell death, are not eliminated, thus producing abnormal myelin and neurological dysfunction. Paradoxically then, females who are heterozygous for the less severe PLP1 mutations are more likely to experience neurological difﬁculties as adults than are females who are heterozygous for the more severe PLP1 alleles (364,434,435). Similar observations have been noted with experimental and naturally occurring murine PLP1 mutations (Ian Grifﬁths, DVM, PhD, personal communication). How does accumulation of misfolded DM-20/PLP1 in the ER of oligodendrocytes affect their function? Several lines of evidence point to the involvement of the unfolded protein response (UPR), a network of genes that are induced in response to unfolded proteins and regulate expression of molecular chaperones, transcription factors, caspases, and other genes [reviewed in Ref. (436)]. Two bZip transcription factors, CHOP (CEBPbhomologous protein) and ATF3, are induced during the UPR and cause apoptosis when overexpressed in transfected cells. Expression of CHOP, ATF3, and several other RER-resident molecular chaperones are induced in oligodendrocytes in response to the synthesis of mutant PLP1 gene products, implicating the UPR in the pathogenesis of oligodendrocyte cell death in PMD (437). These investigators also discovered that rumpshaker (rsh) mice without a functional Chop gene (rsh/chop-null double mutants) have more severe disease than rsh mice, directly implicating CHOP expression in the pathogenesis of PMD. It is thus likely that the set of genes induced during the UPR plays a role in PMD pathogenesis by protecting oligodendrocytes from the toxic effects of misfolded DM20 and PLP1. Protein misfolding has been implicated in several other neurodegenerative diseases, including Alzheimer, Parkinson, and Huntington disease (438–440). The morphological features associated with protein accumulation in these diseases include amorphous aggregates in the RER, cytoplasm, or nucleus, and intermediate ﬁlament-containing aggresomes in the cytoplasm (441). Perinuclear inclusions are also observed in a variety of cell types, particularly in cultured cells treated with proteasome complex

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inhibitors, and are thought to form when the RER-to-cytoplasm delivery of unfolded proteins exceeds degradation by the proteasome complex. Aggresome-like inclusions are rarely found in PMD, however, because myelinating oligodendrocytes do not normally synthesize intermediate ﬁlaments. Proliferating oligodendrocyte precursor cells express vimentin and nestin in culture, but the expression of these genes stops as the cells differentiate (442). Although protein misfolding has been implicated in all of these diseases, the molecular mechanisms of oligodendrocyte cell death in PMD may thus be different than those in the more classic neurodegenerative diseases. A second pathogenic mechanism in PMD is the overexpression of PLP1 in patients with duplications or even greater dosage increases of the PLP1 gene. Excessive amounts of normal PLP1 proteins accumulate in the late endosome and lysosomal compartments of rodent cells overexpressing PLP1 (443). Since PLP1 typically associates with cholesterol and other lipids to form myelin ‘‘rafts’’ as it trafﬁcs through the Golgi compartment (407), the shunting of excess PLP1 into the endosomal/lysosomal compartment effectively drains myelin lipids from the Golgi (443). Presumably the transport and assembly of myelin constituents is altered in cells overexpressing PLP1. Thus, while abnormal PLP1 proteins trigger a protein misfolding response in the rough endoplasmic reticulum, excessive PLP1 proteins create an imbalance in myelin constituents that adversely affects the subsequent stage of nascent myelin assembly in the Golgi network. A third mechanism of molecular pathogenesis in PMD/SPG2 occurs by loss of function in patients with a deletion of the PLP1 gene (374,381,444) or with point mutations at the beginning of the coding region that preclude translation (363,382). These patients have less severe forms of the disease, with the PLP1 deletions giving rise to either a complicated form of SPG2 or a mild form of PMD (381). In mice lacking Plp1, oligodendrocytes develop normally and assemble a myelin sheath, yet defects in the intraperiod line of these sheaths translate into reduced conduction velocities and impaired motor coordination (445–449). In addition, null mutations also produce axonal pathology (see below). These pathological changes suggest there is an absolute requirement for PLP1 both to maintain the structure of compact myelin, and to maintain axonal integrity and function. Thus, the absence of PLP1 would neither trigger the unfolded protein response nor derail myelin assembly, but would instead negatively affect maintenance of the myelin sheath. Axonal damage has been found in both PMD and its animal models, which is important for understanding the pathogenesis of demyelinating disease and its treatment (450–453). Garbern et al. (453) have found evidence for axonal damage in both mice and patients with a PLP1 null mutation by a combination of direct pathological examination of brain tissue and MRS. The axonal injury is not due to demyelination, since myelin is intact in both patients and experimental animals, or oligodendrocyte cell

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death, since these cells appear healthy and ensheath axons. The extent of axonal injury increases with age, and probably accounts for the progression of neurological signs and symptoms. In addition, the axonal degeneration is length dependent, suggesting that impaired axonal transport is a cause. Edgar et al. (454) have directly demonstrated that Plp1-deﬁcient optic nerves have reduced axonal transport. Interestingly, retrograde transport was disturbed earlier and to a greater extent than was anterograde transport. These data suggest that progressive axonal damage is not only a common feature of the pathogenesis of PMD, but it may also be clinically relevant. Because axonal degeneration occurs without signiﬁcant demyelination, it probably arises from the absence or perturbation of PLP1-mediated oligodendrocyte–axonal interactions (455). Consistent with this notion, Scherer and collaborators have shown that axoglial junctions at the paranodal region are disrupted in md rats, an animal model with a Plp1 point mutation, and that these changes are probably involved in disease pathogenesis (456). No simple correlation has been found between a particular PLP1 mutation or genotype, and the clinical manifestation of the disease or phenotype. Although most patients with duplications have the classic form of PMD (371), some have the more severe connatal form (457), while others have a milder spastic paraparesis. Inoue and co-workers recently analyzed the duplication size and structure in 20 families with PMD and suggested that the size of the PLP1 duplication correlated with the clinical phenotype, so that patients with larger duplications had more severe disease (371). In a similar study of 16 families with PLP1 duplications, however, Hobson and co-workers did not conﬁrm this ﬁnding (Hobson et al., in preparation), suggesting that other structural features of the duplication, such as the location, breakpoint, or orientation may also play a role. Callioux et al. (384) recently compared the clinical phenotype and genotype in 33 families with PLP1 point mutations. They found that single amino acid changes within evolutionarily conserved regions of the protein produced the most severe disease, while substitutions of less conserved amino acids, protein truncations, null mutations, and mutations within the PLP1-speciﬁc region (amino acids 116–150) produced a milder form of disease. Although exceptions to this rule occur, more severe forms of disease are likely to be associated with missense mutations within highly conserved regions of the protein. Garbern et al. (363,453) analyzed several families with PLP1 mutations in which no protein product was produced, so-called null mutations. These patients all had a relatively mild spastic paraparesis which progressed during adolescence and an associated demyelinating peripheral neuropathy identiﬁed during electrophysiological testing. The neuropathy was not correlated with disease severity, and was not found in patients with either duplications or point mutations in which protein was produced. Taken together, these

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data suggest that individuals with a relatively mild form of disease and peripheral neuropathy are likely to have a null mutation. Management and future prospects: Currently, there is no therapy for patients with PMD apart from supportive and symptomatic care. Developing disease-speciﬁc therapies will require understanding of the genetic bases of disease. The observation that most patients with PMD have a gene duplication and thus overexpress PLP1, or have a point mutation causing a gain of function precludes simple replacement gene therapy, even if appropriate delivery vehicles become available. For most patients, the more appropriate goal might be the reduction of PLP1 expression, such as through siRNA or antisense RNA therapy, since lack of PLP1 results in a less severe syndrome than connatal or classic PMD. The ﬁnding that axonal degeneration is clinically relevant in the pathogenesis of PMD also raises the possibility that therapy directed at maintaining the integrity of axons might be effective in this disorder. Pharmacologic modulation of the UPR potentially could improve myelination by preventing oligodendrocyte apoptosis. Cellular therapy, such as transplantation of oligodendrocyte precursors into the CNS, has shown potential in animal models of PMD (458–464) and might therefore be effective in patients. Cellular therapy has not yet reversed the clinical deﬁcits in animal models, however, and for maximum effectiveness, this therapy may need to be initiated either in utero or shortly after birth. Pelizaeus–Merzbacher-Like Disease (PMLD; MIM 608804) Uhlenberg et al. (389) identiﬁed a syndrome caused by mutations in the GJA12 gene, which encodes the GJA12 gap junction protein (connexin 46.6). The GJA12 gene lies at 1q41–q42, and mutations result in the autosomal recessive Pelizaeus–Merzbacher-like disease (PMLD) syndrome. In one consanguineous Turkish family and in two nonconsanguineous families in whom PLP1 mutations had been excluded, the clinical syndrome began with nystagmus between birth and three months of age followed by delayed developmental milestones. Only one of the ﬁve children walked, and she became wheelchair dependent after just 1.5 years of independent ambulation. Four of the ﬁve children developed partial seizures, and most had mild slowing of motor and sensory nerve conduction velocity in the legs. The MRI abnormalities were indistinguishable from those found in PMD. While mice deﬁcient in either Gjb1 (connexin 32) or Gja12 (connexin 47 in the mouse) display no behavioral abnormalities, mice that lacked both Gjb1 (connexin 32) and Gja12 develop a fatal hypomyelinating syndrome, demonstrating an important, but as yet to be characterized, role in CNS myelination (404,405). Mutations in the human GJB1 cause X-linked Charcot-Marie-Tooth disease (CMTX1; MIM 302800), a peripheral neuropathy. Interestingly, some CMTX1 patients develop transient central

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neurologic signs associated with brain white matter abnormalities, particularly in the posterior corpus callosum and parietal white matter (466–472). Uhlenberg and colleagues did not ﬁnd GJB1 mutations in their PMLD families, showing that both GJB1 and GJA12 may have important biological roles in man that are not manifested in mice, which are able to compensate for deﬁciency of either connexin alone, but not both together. Disorder of protein synthesis CACH/Vanishing White Matter (CACH/VWM; MIM 603896) This syndrome has been described as childhood ataxia with central nervous system hypomyelination (CACH) (473) and as vanishing white matter disease (VWM) (474). Subsequently, the Cree leukoencephalopathy (475,476) and leukoencephalopathy with ovarian failure (477–479) were discovered to be allelic to CACH/VWM. The syndrome is characterized by delayed ataxia, quadriparesis, and optic atrophy that may worsen in a stepwise fashion and is associated with leukodystrophy and cystic degeneration of the cerebral white matter. The syndrome is caused by autosomal recessive mutations affecting any of ﬁve subunits of the eIF2B translation initiation factor (480,481). Clinical Syndromes Many phenotypes of CACH/VWM can occur, from a severe infantile form to an adult-onset form. The infantile form rapidly progresses after a period of relatively normal early development, followed by loss of acquired abilities, ataxia, impaired consciousness, and irritability that often follow a febrile illness or relatively minor head trauma (482,483). Among the North American Cree Indians, the syndrome is characterized by seizures, spastic quadriparesis, hyperpnea, vomiting, and loss of abilities that begin at about three to six months of age and is fatal by about two years of age (475). In the early childhood form, children have normal early development, although there may be mild motor or cognitive delay. Ataxia is the ﬁrst signiﬁcant sign and develops between one and ﬁve years of age. Neurologic deterioration may occur with febrile illness or mild head trauma (473,474,484). Later, progressive spastic quadriparesis, vision loss, dysarthria, dysphagia, and seizures ensue, although there may be relative stability at times. Peripheral nerve function remains normal, and cognition is relatively spared. There can be great intra- and inter-familial variability in progression rates. Patients with the juvenile form of CACH/VWM have onset of symptoms between about ﬁve and 15 years of age. The syndrome is similar to that of the early childhood form, but with slower progression, and even periods of relative stability and clinical improvement of motor function (473,485). Rapid deterioration ending in fatality can unfortunately occur (485).

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The adult-onset form of CACH/VWM may present with transient motor or visual symptoms and signs followed by behavioral, cognitive, and/or motor decline. Some patients develop ovarian failure, sometimes without overt neurologic signs (although the brain MRI is abnormal) (479,486). Primary ovarian failure may be the presenting syndrome in some women. Other organs can be involved as well, including the lens, kidneys, and liver (426). Patients who are compound heterozygotes for mutations in a subunit of eIF2B can remain asymptomatic even when another family member has neurologic signs; therefore, there may be genetic modiﬁers or environmental factors that have an important effect on disease pathogenesis (487). Etiology: Recessive mutations in any of the genes encoding subunits of the eIF2B translation initiator factor cause CACH/VWM (480,481). Patients can either be homozygous or compound heterozygous for mutations in a given gene (eIF2B1 through eIF2B5). Diagnostic evaluation: With the above clinical syndromes and MRIs that show diffuse nonenhancing white matter abnormalities (including the U-ﬁbers but sparing the putamen) and evolve into cystic areas with the imaging and MRS characteristics of cerebrospinal ﬂuid, the diagnosis of CACH/ VWM should be entertained (474). Mutations have been found in more than 95% of patients with a characteristic syndrome and MRI ﬁndings. The eIF2B5 gene is the most frequently affected subunit gene, accounting for 18 of the 37 known mutations (http://www.genetests.org/proﬁles/cach/ index.html and at http://www.hgmd.org). Mutations affecting subunits 2, 3, and 5 of eIF2B are associated with ovarian failure and white matter disease. Pathologically, CACH/VWM is characterized by cavitation of the white matter, with relative sparing of axons (483,488–490). Where the white matter has not been destroyed, there are increased numbers of oligodendrocytes with ‘‘foamy’’ cytoplasm and that express oligodendrocyte-speciﬁc markers such as carbonic anhydrase II (490). There is also marked apoptosis of oligodendrocytes, leading to the cavitation of white matter. Molecular pathogenesis: The eIF2B translation initiation factor is a complex of ﬁve subunits that activates initiation factor 2 (eIF2) from an inactive GDP-bound form to an active eIF2–GTP complex, permitting formation of the 43S complex, and initiating protein translation. How mutations in this ubiquitous protein complex cause such a restricted syndrome is not clear (491–494). Presumably oligodendrocytes, among the most biosynthetically active cells in the body during active myelination, that are deﬁcient in one of the eIF2B subunits cannot meet the demands of myelination, triggering apoptosis. While this explanation would be reasonable for the infantile syndrome, it does not explain the adult-onset disease adequately. The frequent clinical worsening with febrile illness suggests that the mutations may cause thermoinstability of the affected protein, but direct examination of this

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possibility failed to conﬁrm the hypothesis (494). All mutations tested thus far impair eIF2B activity. Since mutations alter protein structure, misfolding of eIF2B subunits may activate the UPR, as with some PLP1 mutations (discussed above). So far, all mutations are either missense mutations or result in altered splicing of the affected gene. Null mutations would likely be lethal, as is the case in Saccharomyces. Epidemiology: The precise incidence and prevalence of CACH/ VWM are not known at this time, but it may be as common as MLD (http://www.genetests.org/proﬁles/cach/index.html). Differential diagnosis: MRI can help differentiate CACH/VWM from other leukodystrophies. Most of the leukodystrophies do not have cystic degeneration, and tend to spare the U-ﬁbers and/or have a regional brain predilection. Some mitochondrial disorders such as pyruvate dehydrogenase and pyruvate carboxylase deﬁciencies may cause very similar MRI changes that can be difﬁcult to distinguish from those seen in CACH/ VWM (495,496). MR spectroscopy can be very helpful in these cases, showing elevation of cerebral lactate (496). In some cases, evaluation of tissue mitochondrial function may be needed. In megalencephaly with leukoencephalopathy and cystic degeneration (MLC), subcortical cysts are always present in the anterior temporal area and sometimes elsewhere, but do not show the diffuse changes characteristic of CACH/VWM. In Alexander disease the white matter changes usually occur with frontal predominance and there is a relatively unique pattern of contrast enhancement that is not seen in CACH/VWM (348). Management: Speciﬁc treatment is not available for CACH/VWM. In view of the frequent acceleration of disease with fever, it is logical to recommend aggressive treatment, and prevention of infections. Disorders of uncertain pathophysiology Cockayne Syndrome (MIM 216400) Description and clinical manifestations: Cockayne (497,498) syndrome is a multisystem disorder of dwarﬁsm, cataracts, optic atrophy with pigmentary retinitis without spicules characteristic of retinitis pigmentosa, mental retardation, facial dysmorphism, and deafness (497–500). Clinical features have been interpreted to mimic progeria and ﬁbroblasts show abnormalities of DNA repair (501). CKN1 mutations (also CSA) cause Cockayne (502) syndrome, while a clinically similar but more severe syndrome, Cockayne syndrome II, is caused by mutations in the excision-repair cross-complementing rodent repair deﬁciency, complementation group 6 gene, ERCC6 (CSB) (503).

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Cockayne syndrome is an autosomal recessive syndrome characterized by short stature, microcephaly, mental retardation, dental caries, malformed ears, slender nose, hypertension, hepatosplenomegaly, skeletal abnormalities, characteristic senile appearance, retinopathy, sensorineural hearing loss, and leukodystrophy (504). Growth failure begins postnatally during the ﬁrst two years of life in a child with normal stature at birth. By four to ﬁve years, the child is at least two standard deviations below the mean. The characteristic facial appearance that gives the child ‘‘wizened’’ facies also develops over the ﬁrst few years. Mental retardation of moderate to severe degree, ataxia, and extreme skin photosensitivity to sunlight are common. In contrast to xeroderma pigmentosum, skin malignancies are not common. Dry scaly skin and thin hair are often present. Eye abnormalities are prominent in Cockayne syndrome. Abnormalities include a ﬁne pigmentary retinopathy that worsens over time and is associated with abnormal electroretinography. Other ﬁndings include cataracts, nystagmus, strabismus, and decreased lacrimation, but functional vision is often not noticeably affected. Sensorineural deafness occurs later in childhood and can be mild to severe. Deafness may impair speech, which is usually delayed, but most children learn to speak, and are socially interactive. Endocrine, renal, and hepatic abnormalities may also occur. As a result of both central and peripheral nervous system myelin abnormalities, children develop progressive spastic paraparesis, ataxia, dystonia, and cognitive decline. Joint contractures, including scoliosis, are common late in the course of the disease. The disease is typically fatal by early adulthood. Lowry et al. (505) recognized children with abnormalities present at birth that resembled the major features of Cockayne syndrome, and that carried an even poorer prognosis. This is now designated Cockayne syndrome II. Etiology: Cockayne syndrome I and II are autosomal recessive syndromes that result from abnormalities in the transcription-coupled repair (TCR) subpathway of nucleotide excision repair (NER) (506,507). The syndromes are caused by mutations in the CKN1 (CSA) and ERCC6 (CSB) genes. Pathogenesis and pathophysiology: Defects in the transcriptioncoupled DNA repair subpathway cause Cockayne syndromes I and II. This defect can be detected directly in Cockayne syndrome patient ﬁbroblasts which cannot repair ultraviolet (UV) light-induced DNA damage (506). UV irradiation impairs DNA repair in normal and Cockayne syndrome cells, but the latter do not recover as do healthy cells, which regain the ability to repair DNA from transcriptionally active regions (508). The transcription-coupled DNA repair is a part of the so-called nucleotide excision repair pathway that is important for repair of DNA damage (509–511). Classic

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genetic complementation experiments in cultured patient ﬁbroblasts were instrumental in deﬁning major pathways involved in DNA repair (501). The gene for Cockayne syndrome I, CKN1 (also CSA), was assigned to complementation group A and identiﬁed as a WD repeat protein (named after the occurrence of tryptophan-aspartate repeats) (512). WD repeat proteins are involved with diverse cellular regulatory functions, including gene regulation, signal transduction, and apoptosis (513). The gene for Cockayne syndrome II, ERCC6 (also CSB), was assigned to complementation group B and is an excision repair gene (514,515). ERCC6 belongs to a family of ATPases that are not involved in unwinding DNA (515). The precise function(s) of ERCC6 are not well understood, but it may have a more prominent role in repair of oxidative DNA damage (507,516). How the defects in CKN1 and ERCC6 cause the observed neurologic syndrome is poorly understood, but in addition to their roles in DNA repair, they probably are also involved in transcription (511). Epidemiology: Cockayne syndrome is very rare and precise estimates of its incidence and prevalence are not known. Differential diagnosis: The non-neurologic features of Cockayne syndrome are distinctive enough to enable ready distinction from other leukodystrophies. The photosensitivity is also seen in children with xeroderma pigmentosum, with which it shares biological similarity since both conditions are caused by defects in proteins involved in DNA repair. Xeroderma pigmentosum is generally distinguished from Cockayne syndrome by the increased incidence of skin neoplasms in xeroderma pigmentosum and the lack of leukodystrophy. Sjo¨gren–Larsson syndrome is characterized by short stature, mental retardation, skin abnormalities, photophobia, seizures, macular degeneration, and arrested myelination. The skin rash is pruritic and icthyotic and should be distinguishable clinically. DNA repair in ﬁbroblasts is normal. Diagnostic evaluation: The clinical syndrome of Cockayne syndrome is distinctive and should be considered in a child with acquired short stature, typical facies, mental retardation, photosensitive skin, ataxia, and pigmentary retinopathy. The MRI shows a delayed myelination pattern, with myelin in the basal ganglia, internal capsule, splenium and genu, similar to that in Sjogren–Larsson syndrome. Children with Cockayne syndrome generally have thickened skulls, however, and the skin photosensitivity, facial appearance, and ocular ﬁndings should be distinguishable clinically from Sjogren–Larsson syndrome. Screening for Cockayne syndrome is most often accomplished by testing ﬁbroblasts for ability to repair UV lightinduced DNA damage. Direct genetic testing for CKN1 and ERCC6 is available in a few laboratories.

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Prognosis: Cockayne syndrome patients may survive into the third or fourth decades, and some exceptional patients have lived even longer (507). Management: There is currently no speciﬁc therapy for Cockayne syndrome, but attention needs to be paid to the unique complications of the disorder. Protection from UV light is important to minimize dermatologic complications. Unfortunately, growth hormone does not improve stature (504). Low vision and hearing impaired skills should be taught in anticipation of blindness and deafness occurring. Treatment of hypertension, renal, dental, and orthopedic complications should be anticipated. Megalencephalic Leukoencephalopathy with Cysts (MLC: MIM 604004) Megalencephalic leukoencephalopathy with cysts (MLC) is an autosomal recessive syndrome originally described in 1995 (518). Excessive head growth was apparent during the ﬁrst year of life in patients, after which it stabilized at about the 98th percentile. Early development was usually at most mildly delayed, and most children learned to walk independently. During early childhood, slow progression of spastic quadriparesis, ataxia, and dysarthria occurs. Cognition is generally relatively well preserved until late in the course of the disease. Seizures are common. Children are unusually sensitive to minor head trauma, which can provoke seizures or impairment of consciousness. Occasionally patients may present only with macrocephaly without neurologic abnormalities, even by teenage years, and then have slow progression with retention of independent ambulation until middle adulthood. Survival data are limited, but survival into the third through ﬁfth decades appears to be typical. There can be considerable intra and interfamilial variability in disease onset and progression. MLC is caused in some patients by mutations in the MLC1 gene but there is likely locus heterogeneity (519). The MRI ﬁndings in MLC are diagnostic and characterized by (1) diffusely mildly swollen cerebral white matter, with relative sparing of the cerebellar white matter and central white matter (corpus callosum, internal capsule, and brainstem tracts), (2) subcortical cysts that are always present in the anterior temporal lobes and usually in the fronto-parietal regions as well, and (3) subsequent replacement of white matter swelling by cerebral atrophy. Pathologically, there are numerous vacuoles in the myelin, suggesting lamellar splitting along the intraperiod line (520). Epidemiology: MLC is rare with higher rates in communities where consanguinity is more prevalent, including Turkey (521), the Agarwal community in Eastern India (522,523), and among Libyan Jews (524,525). MLC has characteristic MRI ﬁndings and a relatively mild disease course. Some

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speciﬁc points can differentiate MLC from (1) Alexander disease, which has frontal predominance of MRI abnormalities and contrast enhancement (348) and does not usually undergo cystic degeneration and from (2) CACH/VWM, which has more widespread cystic changes, does not have megalencephaly, and is usually more clinically severe. Etiology: About 65% of MLC patients are homozygous or compound heterozygotes for MLC1 mutations (519,526). Existence of another locus is suspected. Thus far, all patients with mutations in MLC1 have a leukoencephalopathy; no other phenotypes have been observed. Management: There is no speciﬁc therapy for MLC. Genetic conﬁrmation of a MLC1 mutation enables prenatal and preimplantation testing in addition to deﬁnitive diagnosis of this disorder. Molecular pathogenesis: The biological role of MLC1 is not known, but its structure suggests that it is an integral membrane protein of relatively recent evolutionary origin. It is expressed widely throughout the CNS and in extraneuronal tissues as well. Peripheral Demyelinating Neuropathy, Central Demyelinating Leukodystrophy, Waardenburg Syndrome and Hirschsprung Disease (PCWH; MIM 277580) Inoue et al. (527,528) described several patients with severe abnormalities, including long-segment Hirschsprung megacolon, congenital hypomyelinating peripheral neuropathy, sensorineural deafness, white forelock, dysmorphic facies, and impaired brain myelination. They have designated this syndrome PCWH, for the major features of Peripheral demyelinating neuropathy, Central dysmyelinating leukodystrophy, Waardenburg syndrome, and Hirschsprung disease. The disorder is allelic to one form of the Waardenburg–Shah syndrome (MIM 277580) caused by null mutations of the SOX10 gene. Patients with PCWH so far are all heterozygous for spontaneous mutations in the last exon of SOX10 (529). Clinical ﬁndings: The syndrome begins with recognition of longsegment Hirschsprung megacolon, which requires surgical treatment; other early signs include severe hypotonia due to the congenital dysmyelinating peripheral neuropathy, and skin pallor. Nystagmus can be present at birth, or develop later. Developmental delay and sensorineural deafness are present in all patients. The most severely affected patients have cerebral white matter abnormalities that resemble those seen in PMD. White forelock and heterochromia iridis are apparent during infancy or childhood. Most patients have spastic paraplegia, though some patients may learn to walk. Patients described thus far are new mutations, but the mode of inheritance should be autosomal dominant. Most patients though are not reproductively ﬁt.

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Diagnosis: The combination of clinical signs should be highly suggestive of the syndrome. Direct SOX10 mutation detection, which should reveal an exon 5 nonsense mutation, conﬁrms the diagnosis. Molecular pathogenesis: The SOX10 gene encodes a transcription factor of the high mobility group family. SOX10 has been implicated in the regulation of expression of several CNS and PNS myelin genes as well as in regulation of genes important in neural crest development (529–535). Mutations that are responsible for the full PCWH syndrome generate transcripts that escape nonsense-mediated decay (NMD) RNA surveillance and encode products with truncations in the transactivation domain, but do not affect the DNA binding domain of the protein (529). These truncated SOX10 products act in a dominant-negative fashion and bind to target DNA sequences with greater afﬁnity than does the normal SOX10 protein. Transcripts with nonsense mutations in more proximal exons are subject to NMD and in effect act as null alleles and demonstrate haploinsufﬁciency of the SOX10 gene. This results in the Waardenburg–Shah syndrome, which is a more restricted and less severe syndrome than PCWH. CLOSING THOUGHTS The leukodystrophies are diverse genetic disorders illustrating the roles of myelin in nervous system function, and deﬁning the biochemical pathways and molecules important in myelin synthesis and maintenance. Additional genes will be identiﬁed as affected in leukodystrophies that currently remain unclassiﬁed as to cause. To date, no human diseases are linked to mutations in the genes for myelin basic protein (MBP), 20 ,30 -cyclic nucelotide 30 –phophodiesterase (CNP1) or UDP-galactose: ceramide galactosyltransferase (CGT), which are all major proteins in CNS myelin. Viable null mutations in mice for each of these result in dysmyelination and neurologic syndromes of different degrees and suggest that human diseases linked to these genes are likely (9,536,537). With better understanding of the complex biosynthesis, organization, and maintenance of myelin components, rational approaches to diagnose and treat leukodystrophies should appear. RESOURCES Genetests: www.genetests.org United Leukodystrophy Foundation: www.ulf.org Myelin Project: www.myelin.org PMD Foundation: www.pmdfoundation.org NORD: www.rarediseases.org European Leukodystrophy Association: www.ela-asso.com Human Gene Mutation Database (http://uwcmml1s.uwcm.ac.uk/uwcm/ mg/hgmd/search.html)

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21 Lysosomal Storage Disorders David A. Wenger and Stephanie Coppola Department of Neurology, Jefferson Medical College, Philadelphia, Pennsylvania, U.S.A.

INTRODUCTION Lysosomal storage disorders (LSDs) constitute a group of genetic diseases caused by defects in genes that control the lysosomal degradation of naturally occurring proteins, glycoproteins, sphingolipids, glycosaminoglycans (GAGS) and carbohydrates, and the transport of certain smaller molecules from the lysosomes. The birth of an individual affected with an LSD is estimated to occur in about 1 in 5000–8000 births in the United States and Europe (1). Therefore, about 500–800 potential patients are born each year in the United States. While some disorders manifest in utero with features such as hydrops, others do not have recognizable symptoms until the eighth or ninth decade. Over 40 different genes are involved in these diseases, and most are inherited in an autosomal recessive (AR) manner. Many diseasecausing mutations and polymorphic changes have been identiﬁed in these genes, and many patients are compound heterozygotes, having different mutations in the two copies of the same gene. This results in the wide range of clinical features that is characteristic of most LSDs and is responsible for the difﬁculty in predicting the clinical course in newly diagnosed patients, especially those with juvenile and adult onset. While neurological symptoms are found in many patients with a LSD, the age of onset, level of impairment and disease progression are quite variable. The accurate diagnosis of patients, especially those early in the course of their disease, is a challenge for both

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the clinician and laboratory performing diagnostic testing. As the clinical features observed frequently overlap both among the different LSDs and with other genetic and non-genetic disorders, a wide range of testing might be indicated after examination of a patient. Testing for most LSDs is relatively straight-forward and can result in a deﬁnitive diagnosis within a short period of time. This usually entails the measurement of enzymatic activities by using synthetic or natural substrates in easily obtained tissue samples, detection of an abnormal level of a metabolic product in cells or urine, or identiﬁcation of a known mutation(s) in certain families or within certain ethnic groups. With that said, it is clear that many patients with a LSD do not get diagnosed at all or as early as they should. With recent developments in therapy and the absence, at this time, of newborn screening for LSDs, it is obvious that early recognition of clinical features followed by accurate testing in an experienced laboratory is increasingly important. Due to the high costs of some treatments and the signiﬁcant morbidity and mortality of other treatments, predicting the clinical course in newly diagnosed patients is an important issue that must be addressed. In this chapter, we will cover some general concepts regarding the effects of lysosomal storage, outline the clinical features that could suggest a diagnosis of a LSD, highlight those disorders that require special diagnostic attention and then present the treatment options available and under development for future clinical trials.

GENERAL CONCEPTS The great majority of LSDs are caused by mutations in genes coding for lysosomal enzymes that are required for the catabolism of naturally occurring compounds produced in situ or endocytosed into cells. Depending on the speciﬁcity of the enzyme and the nature of the mutation(s) present in the patient, there will be one or more substrates whose catabolism is affected by the deﬁciency. Tissues with no substrate naturally present will have little, if any, pathology. While the accumulation of undegraded substrates within the lysosomes of certain tissues will usually result in cellular pathology, a direct correlation between the clinical symptoms and the pathological changes is less compelling. A good example of the wide range of pathology and clinical symptomology that can be found is demonstrated by defects in acid b-galactosidase (EC3.2.1.23) (2). This enzyme catalyzes the hydrolysis of the terminal beta-linked galactose present in GM1 ganglioside, asiaoloGM1 ganglioside (GA1), glycoproteins, oligosaccharides, and keratan sulfate. Depending on the mutation(s) in an individual, all or only some of these substrates may not be catabolized properly. This leads to the broad spectrum of clinical and pathological ﬁndings observed in patients with b-galactosidase deﬁciency, including fetuses with ascites and fetal demise, infants with dysostosis multiplex

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and hepatosplenomegaly (HSM) with severe developmental deﬁcits, adults with only severe dysostosis multiplex and short stature with completely normal speech and intelligence, and adults with mild vertebral changes and dysarthria with normal intelligence throughout their lives. While the extreme clinical variability found in patients with b-galactosidase deﬁciency is due to different mutations affecting the hydrolysis of the numerous potential substrates, all LSDs occur in a wide range of clinical presentations. This variability is at least partially related to the rate of accumulation and nature of the undegraded storage products and to other genetic and environmental factors that have not yet been identiﬁed. In addition to the pathology caused by engorging the lysosomes with undegraded storage products and possibly disrupting normal lysosomal functions, some undegraded products may be toxic to other organelles leading to the death of certain cell types. For example, the accumulation of psychosine (galactosylsphingosine) in globoid cell leukodystrophy (Krabbe disease) to levels up to 10–20 times normal in infantile human patients and animal models has been shown to kill oligodendrocytes, apparently by an apoptotic mechanism (3,4). This results in the greatly diminished amount of myelin, an astrocytic gliosis, and the production of characteristic globoid cells containing undegraded galactosylceramide and other myelin components. This ability of certain lysosphingolipids (those containing fatty acid-free ceramide) to initiate cell death may play a role in other sphingolipidoses (5). Besides those disorders that are caused by a deﬁciency of a lysosomal enzyme, there are several LSDs that result from defects in non-enzymatic proteins. Some are caused by mutations in genes coding for transport proteins that are required for the export or import of cystine, cholesterol and charged sugars, such as sialic acid and glucuronic acid, in cystinosis, Niemann–Pick disease type C (NPC) and sialic acid storage disease, respectively (6–8). Other LSDs result from defects in the so-called sphingolipid activator proteins (saposins) that are required for the interaction between sphingolipid substrates and the lysosomal enzyme necessary for the hydrolysis of a speciﬁc substrate. While relatively rare, they should be considered when a patient’s clinical features are suggestive of a disorder with a known enzymatic cause, such as Tay–Sachs disease, Gaucher disease or metachromatic leukodystrophy (MLD), and the enzyme usually deﬁcient is normal. In addition to the above disorders, a few others are caused by defects in enzymes that are involved in the post-translational modiﬁcation of speciﬁc lysosomal enzymes. This would include mucolipidoses II and III and multiple sulfatase deﬁciency. It is understandably difﬁcult for the clinician to remember all the possible phenotypes and causes for the LSDs; however, sending samples to an experienced and interested laboratory with clinical, imaging and previous test information would lead to a screen of relevant tests that could result in a deﬁnitive diagnosis.

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CLINICAL FEATURES THAT SUGGEST A LSD The diagnosis of a patient with a LSD initially requires a physician to consider whether the individual’s clinical features could suggest this possibility. Table 1 lists only those LSDs with neurological involvement and some of the presenting signs and symptoms. LSDs should be considered in any individual experiencing developmental delay, loss of learned skills, ataxia, seizures, weakness and dementia in the presence or absence of HSM and other somatic changes. This is especially true if the individual is regressing after a period of relatively normal development and the disease seems progressive. While many features are not speciﬁc and could result from other genetic and/or environmental factors, diagnostic testing can be useful in some situations. The genetic causes of such symptoms could include mitochondrial, peroxisomal and lysosomal disorders as well as defects in amino and organic acid metabolism. The samples required for the study of each group of disorders may be different, and one diagnostic laboratory rarely performs testing for all disorders. As discussed below, testing for most of the LSDs diagnosed in the authors’ laboratory can be performed on leukocytes or plasma isolated from whole, heparinized blood sent at room temperature. In this laboratory, the selection of tests is based on suggestions from the physician and our experience utilizing the patient’s clinical history and the results of other testing and imaging studies. Some disorders that require special consideration because of diagnostic difﬁculties, and clinical variability will be highlighted below. Table 2 outlines for the clinician and laboratory the key features that determine which LSDs should be considered when certain signs and symptoms are observed. The disorders tested for in the authors’ laboratory are clearly indicated. It should, of course, be remembered that most LSDs are progressive, and early diagnosis is of the utmost importance for both genetic counseling purposes and consideration for treatment. Therefore, all physicians, including neurologists, should make the extra effort to examine the whole patient for hints that could aid in test selection. Even noting moderate organ enlargement, slightly coarse facial features, unusual skin changes and mild bone involvement, if present, could be critical to obtaining a diagnosis. Neurological symptoms, in the absence of additional ﬁndings, could signal testing for GM1 and GM2 gangliosidoses, the neuronal ceroid lipofuscinoses, Schindler disease, MLD, and Krabbe disease. NPC, which requires more complicated testing, might also be considered if other features are present. While the ﬁndings from magnetic resonance imaging (MRI), computed tomography and nerve conduction velocities could suggest a particular disorder, a broad range of testing would still be required before a deﬁnitive diagnosis may be made. Based on the authors’ experience, seizures are rarely the only presenting feature of a LSD. Patients with mild to severe neurological symptoms who also have evidence of short stature, dysostosis multiplex, coarse facial features, HSM,

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Name of disease

Defective protein

GM1 gangliosidosisa

Acid b-galactosidase

GM2 gangliosidosis, B variant, Tay-Sachs diseasea

Hexosaminidase A

GM2 gangliosidosis, O variant, Sandhoff diseasea GM2 gangliosidosis, AB varianta Fabry diseasea

Gaucher disease, Types 2 & 3a

Glucocerebrosidase

Niemann–Pick Type A/Ba Niemann–Pick Type C1a

Sphingomyelinase NPC1

Initial signs and symptoms

Samples acceptable for diagnosisb

Treatment optionsc

L, P, F

SC, HSCT, EET

L, P, F

SC, HSCT, SRT

Hexosaminidase A & B

IO: hypotonia, DD, coarse facial features, HM, CRS () LO: DD, ataxia, dysarthria, PR, dystonia IO: hypotonia, hyperacusis, DD, CRS LO: ataxia, dystonia, psychoses, PR Similar to Tay-Sachs disease

L, P, F

SC, HSCT, SRT

GM2 activator protein

Similar to Tay-Sachs disease

F, CSF

SC

a-galactosidase

Acroparesthesia, pain crises, corneal opacities, fatigue, strokes, angiokeratomas HSM, DD, strabismus, Sz, myoclonus, horizontal supranuclear gaze palsy HSM, hypotonia, DD, CRS () Neo-natal onset: jaundice, HSM, hypotonia LO: emotional lability, ataxia, dystonia, HSM (), VSO

L, F

SC, ERT, HSCT, SRT, EET

L, F

SC, ERT, HSCT, SRT

L, F F

SC, ERT SC, HSCT, SRT 559 (Continued)

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Table 1

Presenting Features in LSDs with Neurological Findings (Continued )

Name of disease Niemann–Pick Type C2a

Defective protein NPC2

Metachromatic leukodystrophya Arylsulfatase A

Galactocerebrosidase

a-Mannosidosisa

a-mannosidase

b-Mannosidosisa

b-mannosidase

Sialidosis, Mucolipidosis Ia

Sialidase

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Similar to Niemann–Pick Type C1 Late IO: weakness, hypotonia, DD, genu recurvatum JO: weakness, PR, ataxia, behavior changes AO: pyramidal or cerebellar signs, behavioral changes, psychoses, dementia IO: spasticity, irritability, hypotonia, ﬁsting, DD LO: spastic paraparesis, weakness, burning paresthesia, ataxia, vision loss DD, hearing loss, mildly coarse facial features (large jaw), mild DM DD, MR, hearing loss, mild facial coarsening, angiokeratomas IO: NIFH, DD, coarse facial features, DM, HSM, PR, renal disease LO: myoclonus, CRS, ataxia, visual defects

F

Treatment optionsc SC

L, F, U

SC, HSCT

L, F

SC, HSCT

L, F

SC, HSCT

L, F

SC

L, F

SC

Wenger and Coppola

Krabbe diseasea

Initial signs and symptoms

Samples acceptable for diagnosisb

IO: severe DD, fair hair and skin, HSM, coarse facial features LO: hypotonia, MR, ataxia, DD, speech delay, coarse facial features Neo-natal onset: NIFH, HSM, severe DD IO and late IO: coarse facial features, HSM, kidney and heart defects, DD, DM, MR LO: coarse facial features, DM, corneal clouding, MR, ataxia, Sz, CRS () Spasticity, DD, coarse facial features, DM, MR, angiokeratomas Coarse facial features, DD, DM, MR, hearing loss, corneal clouding, hernias DD, DM, hearing loss, coarse facial features, joint stiffness Aggressive behavior, DD, mildly coarse facial features, hirsute, coarse hair, mild DM Similar to MPS III A Similar to MPS III A

Protective protein, cathepsin A

Fucosidosisa

a-L-fucosidase

MPS I (Hurler and HurlerScheie)a

a-L-iduronidase

MPS II (Hunter)

Iduronate-2-sulfatase

MPS III A (Sanﬁlippo)

Glucosamine-N-sulfatase

MPS III Ba MPS III C

a-N-Ac-glucosaminidase AcCoA:a-glucosaminide-Nacetyltransferase N-acetylglucosamine-6-sulfatase Similar to MPS III A

MPS III D

L, F

SC

L, F

SC

L, F

SC, HSCT

L, F

SC, HSCT, ERT

F, P

SC, HSCT, ERT

F

SC, HSCT

P, F F

SC SC

F

SC

561

Galactosialidosisa

Lysosomal Storage Disorders

Sialic acid storage disease, Salla Transport protein diseasea

(Continued) Copyright 2006 by Taylor & Francis Group, LLC

562

Table 1

Presenting Features in LSDs with Neurological Findings (Continued )

Name of disease MPS VIIa

Mucolipidosis II/IIIa

Mucolipidosis IV Multiple sulfatase deﬁciencya

Saposin defect, generalized typea

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Initial signs and symptoms

IO: NIFH, DM, DD, coarse facial features, HSM, MR LO: DM, DD, mild coarse facial features UDP-N-Ac-glucosaminyl Coarse facial features, DD, phosphotransferase weakness, DM, gingival hyperplasia, macroglossia Mucolipin 1 DD, corneal opacities, retinal degeneration, strabismus Sulfatase modiﬁer protein DD, ichthyosis, coarse facial features, deafness, mild DM, PR Palmitoyl-protein thioesterase 1 Vision loss, motor dysfunction, hypotonia, DD, MR Tripeptidyl peptidase 1 Sz, motor dysfunction, DD, MR, ataxia, dementia Saposin B Similar to JO MLD Saposin C Similar to Gaucher disease, Type 3 Prosaposin HSM, DD, motor abnormalities, exaggerated Moro reﬂex, MR b-glucuronidase

Treatment optionsc

L, F

SC

P, F

SC, HSCT

L, F

SC

L, F

SC

L, F

SC, HSCT

L, F

SC

F, U L, F

SC SC

L, F

SC

Wenger and Coppola

Neuronal Ceroid Lipofuscinosisa Neuronal Ceroid Lipofuscinosisb Saposin defect, MLD typea Saposin defect, Gaucher typea

Defective protein

Samples acceptable for diagnosisb

Aspartylglucosaminidase

Farber diseasea

Acid ceramidase

Wolman diseasea

Acid lipase

Schindler disease

a-N-acetylgalactosaminidase

Pompe disease

a-glucosidase

Delayed speech, motor clumsiness, mildly coarse facial features, behavioral problems Painful and swollen joints, nodules, DD, hoarse cry, hypotonia HSM, vomiting, diarrhea, anemia, PR DD, blindness, Sz, spasticity, PR Hypotonia, DD, cardiac enlargement

L, F, U

SC

L, F

SC, HSCT

L, F

SC, HSCT

L, P, F F

SC

Lysosomal Storage Disorders

Aspartylglucosaminuria

SC, ERT

a

Diseases diagnosed in the authors’ laboratory. These are the samples that have been used to diagnose a given disorder by measurement of a biochemical parameter. Molecular studies can utilize any DNA-containing sample. c Supportive care indicates any procedure performed to alleviate pain, discomfort, and seizures and may include splenectomy and kidney transplantation when indicated. Additional therapies may not be available for all patients with a given disorder because of their clinical status. ERT, SRT and EET may only be at the investigative stage for some disorders. Abbreviations: AO, adult-onset; CSF, cerebrospinal ﬂuid; CRS, cherry-red spots; DD, developmental delay; DM, dysostosis multiplex; EET, enzyme enhancement therapy; ERT, enzyme replacement therapy; F, ﬁbroblasts; HSCT, hematopoietic stem cell transplantation; HM, hepatomegaly; HSM, hepatosplenomegaly; IO, infantile-onset; JO, juvenile-onset; LO, late-onset; L, leukocytes; MR, mental retardation; NIFH, non-immune fetal hydrops; P, plasma; PR, psychomotor regression; Sz, seizures; SRT, substrate reduction therapy; SC, supportive care; U, urine; VSO, vertical supranuclear ophthalmoplegia. Source: Modiﬁed from Arch Neurol 2003; 60:322–328. b

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corneal clouding and other more subtle signs (e.g., unusual skin ﬁndings) require a larger battery of testing to arrive at a deﬁnitive diagnosis (Table 2). Patients with disorders affecting the lysosomal catabolism of GAGS (mucopolysaccharides) and oligosaccharides have a wide range of clinical features. Neurological deﬁcits, beginning at any age from the neonatal period to adulthood, can be mild or severe. Not all features may be present in every patient. For example, patients with mucopolysaccharidosis II (MPS II, Hunter syndrome) have coarse facial features but do not have corneal clouding, and patients with MPS III (Sanﬁlippo syndrome) have less obvious dysostosis multiplex but can have severe neurological impairment and signiﬁcant behavioral problems. There are also a number of subtle features found in patients that should signal speciﬁc testing and could result in a diagnosis (Tables 1 and 2). These include the presence of angiokeratomas, enlarged gums, fairer than expected hair and skin, large jaw, acroparesthesias, macular cherry-red spots, hearing loss, cardiomyopathy and cardiomegaly, tortuosity of conjunctival blood vessels and vertical supranuclear ophthalmoplegia (VSO). In addition, about 10% of infants born with non-immune fetal hydrops (NIFH) have a LSD. These include MPS VII, mucolipidosis II, GM1 gangliosidosis, sialidosis, galactosialidosis, Gaucher disease, Farber disease, and Niemann-Pick type C (NPC) (9). While effective therapy is not currently available for these infants, a deﬁnitive diagnosis allows genetic counseling and prenatal testing to be offered in subsequent pregnancies. DIAGNOSIS OF LSDS Most LSDs can be diagnosed by measuring the activity of selected enzymes with commercially available synthetic or radiolabeled natural substrates by using an appropriate sample sent to an experienced laboratory (10,11). The use of molecular analysis in the diagnosis of LSDs remains limited to a few disorders and for certain speciﬁc ethnic groups. Molecular analysis may be beneﬁcial once a diagnosis has been made to aid in the carrier screening of family members. There may also be a role for molecular studies in the prediction of phenotypes, which might facilitate the identiﬁcation of patients requiring immediate and aggressive therapy. While most laboratories will require the physician to list the tests requested, it would be best to utilize a laboratory that has experience with these disorders and can, with clinical and imaging information, aid in test selection. It is the authors’ opinion that enzymatic studies should be performed without delay if the patient’s clinical features suggest a LSD. Studies of oligosaccharides and GAGS in urine may be requested, but this may only cause a delay in obtaining a diagnosis because of a high rate of false-negative and false-positive results. In most cases, enzymatic or other (e.g., sialic acid content) testing would be required anyway.

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Table 2 Key Features That Determine Which LSDs Should be Considered for Testing 1. Neurological and cognitive abnormalities including developmental delay and/or regression of previously learned skills, mental retardation, ataxia, spasticity, hypoor hypertonia, or behavioral changes without mention of coarse facial features, organomegaly or eye changes GM1 and GM2 gangliosidoses, metachromatic leukodystrophy, Krabbe disease, neuronal ceroid lipofuscinoses, Schindler disease 2. Coarse facial features, dysostosis multiplex, short stature, joint contractures, cardiac problems, or hirsutism with or without apparent neurological and cognitive features, hepatosplenomegaly or eye changes GM1 gangliosidosis, a- and b-mannosidosis, sialidosis, sialic acid storage disease, galactosialidosis, fucosidosis, MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS VI, MPS VII, ML II/III, multiple sulfatase deﬁciency, aspartylglucosaminuria, Pompe disease 3. Hepatosplenomegaly with or without apparent neurological and cognitive abnormalities and no mention of coarse facial features or dysostosis multiplex GM1 and GM2 gangliosidoses, Gaucher disease, Niemann-Pick disease types A/B and C, Wolman disease, Saposin defects, Pompe disease 4. Abnormal eye ﬁndings A. Macular cherry-red spots GM1 and GM2 gangliosidoses, Niemann-Pick disease types A/B and C, Krabbe disease, sialidosis, galactosialidosis B. Corneal Clouding GM1 gangliosidosis, Fabry disease, a- and b-mannosidosis, sialidosis, galactosialidosis, fucosidosis, MPS I, MPS VI, MPS VII, ML II/III, ML IV, multiple sulfatase deﬁciency C. Miscellaneous eye ﬁndings Tortuosity of conjunctival vessels (Fabry disease, fucosidosis); vertical supranuclear ophthalmoplegia (Niemann-Pick disease type C) 5. Miscellaneous features A. Angiokeratomas (Fabry disease, b-mannosidosis, sialidosis, galactosialidosis, fucosidosis) B. Light pigmented skin and hair (sialic acid storage disease) C. Non-immune fetal hydrops (GM1 gangliosidosis, Gaucher disease, NiemannPick disease type C, sialidosis, sialic acid storage disease, galactosialidosis, MPS I, ML II, Farber disease, Wolman disease) D. Subcutaneous nodules (Farber disease) Note: Those disorders listed in bold can be diagnosed in the authors’ laboratory.

Typically, the ﬁnding of low activity for one lysosomal enzyme and normal values for others results in a deﬁnitive diagnosis. There are some very important exceptions. For example, patients with mucolipidoses II and III have normal enzyme values in leukocytes but greatly elevated enzyme values in plasma. In cultured skin ﬁbroblasts all lysosomal enzymes tested, except

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glucocerebrosidase, would be low in these patients. While normal or low normal sphingomyelinase activity in leukocytes and cultured skin ﬁbroblasts does rule out Niemann–Pick disease types A and B, more detailed studies are required to diagnosis NPC (discussed further). Measurement of normal enzymatic activities does not rule out disorders resulting from defects in sphingolipid activator proteins, such as the AB variant of GM2 gangliosidosis, Gaucher disease caused by saposin C deﬁciency and MLD caused by saposin B deﬁciency (reviewed in chapter 12). On the other hand, the ﬁnding of low activity for an enzyme in a patient may not indicate the diagnosis due to the presence of the so-called pseudodeﬁciency (Pd) alleles. Pd alleles are mutations in a gene that either change an amino acid or alter the expression of the encoded protein. When inherited homozygously or together with a disease-causing mutation, the presence of the Pd allele will result in a signiﬁcant reduction of enzymatic activity but not to the levels that cause symptoms. These types of mutations and other enzyme-lowering polymorphisms have been found in the genes coding for the a- and b-chains of hexosaminidases A and B, a-L-iduronidase, a-galactosidase, b-glucuronidase, galactocerebrosidase and arylsulfatase A (ASA) (13). The high frequency of the Pd allele, especially in the ASA gene, creates a very serious problem that can result in the misdiagnosis of MLD both pre- and post-natally (discussed further). The measurement of sialic acid content in leukocytes, urine, or cultured skin ﬁbroblasts is useful in screening for four genetic disorders (8,10). Sialic acid is a nine carbon, negatively charged sugar that is a component of most glycoproteins and gangliosides. Once sialic acid is hydrolyzed from these complex molecules by speciﬁc lysosomal sialidases, it requires sialin, a transport protein, to expedite its exit from the lysosomal compartment (14). Sialic acid content should be measured in patients with NIFH, short stature, coarse facial features, nephrosis, cherry-red spots and myoclonic seizures. If the total content of sialic acid is elevated, the test is repeated without acid hydrolysis to determine if it is free or bound. High levels of bound sialic acid are found in leukocytes and ﬁbroblasts from patients with sialidosis and galactosialidosis who have mutations in the genes coding for sialidase and protective protein/cathepsin A, respectively (15,16). Additional studies to measure b-galactosidase activity in leukocytes and plasma and sialidase activity in cultured skin ﬁbroblasts may be necessary to conﬁrm the diagnosis. High levels of free sialic acid are found in samples from patients with sialic acid storage disease or Salla disease, who have mutations in the gene coding for sialin (14), and sialuria (not a LSD), caused by mutations in the gene coding for UDP-GlcNAc 2-epimerase (17). GM2 gangliosidosis should be considered in any infants or children who are losing interest in their surroundings and have spasticity, macrocephaly, hyperacusis, and macular cherry-red spots. With three genes involved in GM2 ganglioside degradation, deﬁning the genetic type is critical for

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accurate genetic counseling of family members and subsequent prenatal testing. Due to defects in the a-chain, measurement of low hexosaminidase A activity in any tissue sample could result in the diagnosis of Tay–Sachs disease. Since the institution of the carrier testing program in the Ashkenazi Jewish community in the early 1970s, the number of cases of Tay–Sachs disease in this ethnic group has dropped dramatically. However, there has been no decrease in the number of non-Jewish cases of Tay–Sachs disease. The ﬁnding of low total b-hexosaminidase activity, due to defects in the b-chain common to hexosaminidases A and B, results in the diagnosis of Sandhoff disease. It should be noted that additional patients who clinically resemble those with Tay–Sachs and Sandhoff diseases could have the B1 variant of GM2 gangliosidosis. Despite the presence of a mutation in the a-chain of hexosaminidase A, these patients have normal or carrier levels of hexosaminidase A activity when measured using the heat denaturation method (18). However, use of the sulfated derivative of the synthetic substrate for screening will diagnose all patients who have a defect in the a-chain of hexosaminidase A (19). Besides the majority of patients with mutations in the a- and b-chains of hexosaminidases A and B, there are additional infants with similar clinical features who have defects in the GM2 activator protein that is required for hydrolysis of GM2 ganglioside by hexosaminidase A (20). These patients with the AB variant of GM2 gangliosidosis have normal hexosaminidase A and B activities measured with all synthetic substrates. While a brain biopsy could reveal characteristic membranous, cytoplasmic bodies and a great excess of GM2 ganglioside, the easiest way to diagnose these patients is for an experienced laboratory to detect the elevated GM2 ganglioside in 1–2 ml of cerebrospinal ﬂuid (21). Additional studies could identify the mutation(s) in the gene coding for the GM2 activator protein (22). While most patients with defects in the lysosomal catabolism of GM2 ganglioside have the infantile form, adolescents and adults with cerebellar ataxia, symptoms resembling amyotrophic lateral sclerosis, and psychiatric problems should have their hexosaminidase levels checked. Many disease-causing mutations have been found in patients with Tay–Sachs and Sandhoff diseases, and the delineation of speciﬁc mutations in certain ethnic groups has greatly aided carrier identiﬁcation. However, the initial screening of undiagnosed patients is most reliably done by enzymatic testing. MLD is probably the most common LSD involving signiﬁcant neurological features. Late infantile, juvenile and adult onset forms are recognized. MLD is also the LSD causing the most problems with regard to accurate patient identiﬁcation. In specialized laboratories, ASA activity can be measured in leukocytes from individuals of any age with evidence of weakness, ataxia, developmental delay, mental regression, psychiatric problems, or white matter changes on MRI. The ﬁnding of low ASA activity could indicate a diagnosis of MLD. However, due to the high frequency of the Pd allele and the measurement of low ASA activity in patients with

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multiple sulfatase deﬁciency, this diagnosis must be conﬁrmed by additional studies. Pd is a mutation in the polyadenylation signal that results in only about 10% of the normal amount of ASA mRNA (23). About 1 in 7 individuals in the general population is heterozygous for this polymorphism. Therefore, about 1 in 200 individuals, whether completely normal or with neurological problems, is homozygous (ASAPd/ASAPd ) for this mutation and has a low (5–15% of normal) ASA activity. These low ASA values overlap those found in patients conﬁrmed to have MLD (ASAMLD/ASAMLD) and in healthy carriers of MLD who have one MLD-causing mutation and one Pd allele (ASAMLD/ASAPd). The fact that 14 MLD–causing mutations have now been found on the Pd allele causes further complications (24). However, the accurate diagnosis of suspected patients is not difﬁcult when the proper samples are analyzed. ASA activity should be measured in leukocytes, and if low, DNA can be isolated from the remaining sample, and the presence of the Pd allele can be determined by PCR-based testing (24). If the Pd allele is not present and the clinical features suggest a leukodystrophy, the diagnosis of MLD is almost certain. If it is present, MLD must still be considered, and a ﬁrst morning voiding of urine should be analyzed for sulfatides. If excess sulfatides are being excreted, the diagnosis of MLD has been conﬁrmed. It is very important to obtain ASA values on the parents of all patients identiﬁed. One in 14 of the healthy parents of MLD patients will have ASA activity near to that of their affected child due to the high frequency of the Pd allele. This will be critical to know if the couple pursues prenatal testing on subsequent pregnancies or if testing of a newborn is requested. Obtaining ASA values and, if needed, performing molecular analysis for the presence of the Pd allele in the parents is ideal for accurate prenatal diagnosis. The inheritance of the Pd allele (without an additional mutation on that allele) from one parent and an MLD-causing mutation from the an other will result in low ASA activity in any fetal sample (chorionic villi, cultured trophoblasts, and amniotic ﬂuid cells) or cord blood received for testing. The ﬁnding of low ASA activity in a newborn from an at-risk family in which the parents were not tested could result in a misdiagnosis with possible serious implications if life threatening therapy was instituted. Figure 1 shows the excess urinary sulfatide excretion pattern of a four year patient with MLD and the lack of sulfatide excretion in a newborn sibling with an identical, low ASA value who was being prepared for hematopoietic stem cell transplantation (HSCT). Subsequent enzyme and DNA analysis on relevant family members led to correct disease assignment and the determination that the newborn would not be affected with MLD. In addition to testing for the Pd allele, testing can be done routinely for the two most common disease-causing mutations (25). Patients who are homozygous for the G459þ1A mutation have classic late infantile MLD, and those who are homozygous for the P426L mutation have typical adult MLD. Compound

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Figure 1 Thin layer chromatography of urine sulfatides extracted from a 4-year-old child with MLD (ASAMLD/ASAMLD) (1), newborn sibling of (1) with low arylsulfatase A activity but with the ASAMLD/ASAPd genotype (2), standard sulfatide from brain (3).

heterozygotes having these two mutations usually have a juvenile presentation illustrating, in a simple way, the predictive effect of these two alleles. In addition to the above problems related to the diagnosis of MLD, there are some patients who have normal ASA activity but have MLD caused by defects in a sphingolipid activator protein known as saposin B (26). The few individuals who have been identiﬁed clinically resemble those patients with the late infantile or juvenile types of MLD. Such patients

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are most easily diagnosed by detecting excess sulfatides in urine, and subsequent molecular analysis of the saposin gene could identify a mutation(s) in the saposin B region. Finally, patients with multiple sulfatase deﬁciency have low activity for all sulfatases, including arylsulfatase A, and excrete sulfatides plus GAGS in urine (27). However, these patients usually have features suggestive of a MPS disorder, and their parents do not have carrier levels of any sulfatases. NPC is another relatively common disorder with signiﬁcant diagnostic challenges. This is because the neurological and somatic features are variable, NPC cannot be diagnosed by relatively simple tests performed in leukocytes or plasma, and ‘‘variants’’ have biochemical ﬁndings that are difﬁcult to interpret. Testing for NPC is often requested by neurologists who have exhausted other options by more straight-forward testing. Patients range in age from neonates with NIFH to adults with evidence of dystonia and VSO (7). Many patients with the ‘‘classic’’ form have a history of jaundice at birth, but it can be resolved with phototherapy, and they appear normal until the middle of the ﬁrst or second decade when they develop behavioral disturbances and drop in school performance. Some, but not all, have signiﬁcant HSM. Diagnostic testing requires cultured skin ﬁbroblasts for special studies to detect excess free cholesterol using ﬁlipin staining (Fig. 2) and, if positive, to measure cholesterol esteriﬁcation. Mutations in two different genes, NPC1 and NPC2, can cause NPC (28,29). As there have been many disease-causing mutations identiﬁed in the NPC1 gene, DNA analysis is not useful for screening patients, except in suspected cases from the original Hispanic population of northern New Mexico and southern Colorado where one mutation, I1061T, has been identiﬁed (30). However, identiﬁcation of disease-causing mutations, once a biochemical diagnosis has been made, greatly improves carrier testing in relatives and the accuracy of prenatal diagnosis.

Figure 2 Filipin staining of cultured skin ﬁbroblasts from a control (A) and patient with classic NPC (B). Intense peri-nuclear staining is seen in the cells from the patient with NPC.

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Many of the LSDs are very severe, and they can result in early death without the availability of effective therapy. However, prenatal diagnosis is extremely accurate for most LSDs using a direct chorionic villus sample (CVS), cultured trophoblasts, and cultured amniotic ﬂuid cells. Prenatal testing using either enzymatic or molecular analysis usually can be performed within one day from receipt of either a CVS taken at 9.5 to 10 weeks gestation or cultured trophoblasts or amniotic ﬂuid cells. If preliminary studies are performed on a direct CVS, additional testing on cultured trophoblasts from the same original sampling should conﬁrm the prediction of an affected or unaffected fetus. It is critically important that all samples analyzed be free of maternal contamination. Measurement of a very low value for the enzyme in question and normal values for other enzymes completes the diagnosis, and the couple can act on this information. For most LSDs, knowing the mutation(s) in a proband does not necessarily make prenatal testing more accurate or efﬁcient, and mutation analysis is not always available for prenatal diagnosis. It is very useful to have enzyme values from the parents to aid in the interpretation of values obtained from fetal samples. Although most couples are aware of the availability of prenatal diagnosis after conception, some families inquire about pre-implantation diagnosis. Theoretically, pre-implantation diagnosis could be done if the mutation(s) in the proband is known and methods to identify that mutation(s) in the very small number of cells obtained from the developing blastocysts after in vitro fertilization are available (31). However, the successful use of this technology in more pregnancies must be demonstrated before its use can be encouraged. The success of most treatments depends on the accurate diagnosis of patients before symptoms, especially those affecting the nervous system, become overwhelming. There have been recent attempts to develop tests to screen newborn infants who have no family history of a LSD using samples collected at birth. These tests would utilize either blood spots obtained from heal sticks to measure the activity of certain lysosomal enzymes or identify abnormal analytes, or small plasma samples to determine the concentrations of certain proteins, including lysosome-associated membrane proteins (LAMP-1 and -2) and saposins (32–35). The use of dried blood spots for enzymatic screening does seem feasible, but without a family history of one LSD, improved technology will be needed to cover even those LSDs that have treatment options. The ﬁnding of elevated LAMP-1 and -2 and certain saposins in samples from a newborn could indicate a diagnosis of a LSD. Any individual with elevated levels would have to be studied in more detail to determine which, if any, LSD was the cause of the elevation. False-negative and false-positive results remain a problem. If a diagnosis was made, it would be difﬁcult to predict the clinical course and need for treatment in patients diagnosed without a family history. Molecular studies looking for known mutations might be helpful, but genotype–phenotype correlations have not always been useful for predicting clinical course,

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especially in later-onset patients. However, the identiﬁcation of a patient at birth would result in careful clinical observation and serial imaging studies, and treatment could begin when deemed appropriate. TREATMENT OF LSDS When a child or adult is diagnosed with a LSD, one of the ﬁrst questions asked, besides the availability of treatment, is what clinical course lies ahead for the patient. This information would also be helpful in selecting those patients who would beneﬁt most from aggressive therapy. Residual activity, as measured in leukocytes, plasma or cultured skin ﬁbroblasts, using natural or synthetic substrates, generally is not an accurate predictor of phenotype. Molecular studies can be useful for predicting the possibility of neurological involvement in some LSDs; however, the large number of mutations identiﬁed coupled with the fact that many patients are compound heterozygotes makes phenotype prediction difﬁcult from molecular studies alone. For example, ﬁnding even one copy of the N370S mutation in the glucocerebrosidase gene in a newly diagnosed patient with Gaucher disease indicates that there will be no neurological involvement. However, the presence of this mutation does not help to predict the severity of the hematological and bone complications. From the experience with patients who have late-onset forms of Krabbe disease, it is clear that there is tremendous variability even between siblings who have the same genotype for the galactocerebrosidase gene (36). In fact, there is a strong suggestion that the start of symptoms in some late-onset patients may occur after some stressful insult, such as infection or blow to the head. While many disease-causing mutations have been identiﬁed in the genes coding for proteins involved in lysosomal metabolism, most laboratories usually do not perform mutation analysis on newly diagnosed patients. Mutation analysis of all newly diagnosed patients would be useful for academic or research purposes and possibly for helping with carrier testing in relatives; however, mutation identiﬁcation may add little to the management of the patient. Careful clinical evaluation together with sequential imaging studies will usually result in a reasonable prediction of the clinical course a patient will take. Most infants who are diagnosed with a LSD will follow a rather predictable clinical course. The loss of any gained skills and continued neurological deterioration will proceed until the death of the child usually by infection. Predicting the clinical course in patients with chronic and later-onset forms is nearly impossible. This is true for some of the LSDs where enzyme replacement therapy (ERT) is or will soon become available, including Gaucher disease, Fabry disease, Pompe disease, Niemann–Pick disease type B, MPS I, and MPS II. While there is a large body of evidence demonstrating that providing the missing enzyme clears years of undegraded storage products in certain organs, the correlation between the amount of storage and

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the physical health of the patient is less clearly deﬁned. This last point raises the issue of proving effectiveness of any new treatment that slows the expected clinical course but does not reverse previous pathological changes and clinical features. For many patients with a LSD, supportive care is the only available option at this time (Table 1). This is especially true for those patients diagnosed after signiﬁcant neurological involvement has occurred. However, an increasing number of patients with speciﬁc disorders are undergoing HSCT and ERT in an attempt to slow the course of the disease, prevent the onset of clinical features and improve some pathological ﬁndings. There have been recent developments in the treatment of patients with certain LSDs, even those with signiﬁcant neurological involvement. Table 1 denotes the treatments that are in use, that have been tried in a limited number of cases, or are under investigation for each disorder. One treatment that has shown promise in some patients is HSCT in pre-symptomatic or mildly affected individuals. More than 400 patients with a LSD have received HSCT. This includes many patients with Gaucher disease, MLD, Krabbe disease and MPS I plus a limited number of cases with other LSDs (37,38). Some of these patients were identiﬁed in utero or at birth because of family history, and others were mildly affected when transplanted. While most of the donors were initially HLA-identical siblings, most donor HSCs are now isolated from unrelated umbilical cord blood. This increases the number of available donors because of a lower standard of histocompatibility required between donor and recipient. In addition, most donor HSCs would not come from carriers of the disease and thus supply a normal level of the enzyme needed. It has been shown in animal studies that donor-derived macrophages inﬁltrate the brain of the recipient (39,40). These donor-derived cells act as mini-pumps synthesizing and secreting a portion of their lysosomal enzymes that can be taken up by neighboring cells. Those lysosomal enzymes endocytosed by the mannose-6-phosphate receptor-mediated pathway should localize in the lysosomes and hydrolyze the stored substrate. In humans, the replacement of brain microglial cells by donor-derived macrophages can take many months or even years to accomplish. Therefore, HSCT may not be beneﬁcial for those diseases that are rapidly progressing. Although patients with Niemann–Pick disease type A, Tay–Sachs disease and other LSDs resulting in neuronal deterioration have undergone HSCT, the results are not conclusive regarding substantial long-term correction. However, the evidence from HSCT of patients with Krabbe disease shows a positive effect if infantile patients are treated very early in life before the onset of symptoms or when neurological symptoms are minimal in the later-onset forms (41). With successful engraftment of donor HSCs, enzyme levels in leukocytes reach those of the donor and the elevated cerebrospinal ﬂuid protein concentration drops signiﬁcantly. In most cases, the disease progression slows, the MRI pattern improves and the intelligence quotient rises. In general, HSCT has been most effective in

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improving the visceral and hematological symptoms of the treated patients. Severe changes in the bones appear to be difﬁcult to correct; however, with time and the transplantation of mesenchymal cells together with HSCs, signiﬁcant improvements may be noted. ERT is now available or under investigation for a number of LSDs, including Gaucher disease, Fabry disease, Niemann–Pick disease type B, MPS I, MPS II, MPS IVB, MPS VI, MPS VII, and Pompe disease (42). ERT started more than 15 years ago with the large-scale isolation of glucocerebrosidase from human placentas for the treatment of Gaucher disease (43). The treatment of the non-neuronopathic forms of Gaucher disease with this original source of glucocerebrosidase and the later recombinant form has been very successful. The use of ERT to treat subacute neuronopathic Gaucher disease type 3 has not been conclusively demonstrated to prevent the onset and progression of the neurological symptoms (44). It is not used to treat acute neuronopathic Gaucher disease type 2. While there is little evidence that infused enzymes reach the brain due to the so-called blood–brain barrier, the rationale for the use of ERT to treat the milder forms of some neuronopathic LSDs, such as MPS I and II, is to help the somatic manifestations of these patients and make their management easier for care givers. One LSD that could beneﬁt greatly from ERT is Fabry disease. This X-linked disease, caused by a deﬁciency of a-galactosidase activity, is characterized by burning paresthesias, kidney failure, strokes and heart disease in both male hemizygotes and some female heterozygotes. ERT has been shown to greatly improve many aspects of the disease probably by supplying enzyme to the microvasculature (45). There are a number of clinical trials for patients with Pompe disease using a-glucosidase from several sources (46,47). The genes for almost all of the lysosomal enzymes have been cloned, and their encoded proteins theoretically could be produced in large quantities. Despite pressure from concerned families and healthcare providers, signiﬁcant problems still remain before ERT can be tried for most LSDs. The small number of patients with certain LSDs makes the effort to produce and test the protein unproﬁtable. The high cost of these enzymes, the need for life-long treatment and the frequency of injections are signiﬁcant issues for many patients and their families. Involvement of the CNS and the rapid course of many disorders also make it unlikely that intravenously supplied enzyme would reach the brain in signiﬁcant quantities to reverse damage that occurred early in life or possibly during fetal development. This uncertainty does not rule out the use of ERT for patients who have signiﬁcant somatic involvement in addition to neuropathology. In fact, there have been recent efforts to treat patients with MPS I initially with ERT followed by HSCT. While ERT for most LSDs has resulted in the production of antibodies against the injected protein in many patients, these antibodies do not appear to diminish the effectiveness of the treatment or result in a severe immunological reaction.

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Substrate reduction as an adjunctive treatment for certain sphingolipidoses is receiving some attention (48–50). Since symptoms in the patients are thought to result from the storage of undegraded substrate, a slower rate of accumulation accomplished by slowing the rate of synthesis of substrate might be effective, especially in later-onset patients whom we suspect have some residual enzymatic activity. This theoretically could be accomplished by compounds such as L-cycloserine, which inhibits a very early step in the synthesis of sphingolipids, or N-butyldeoxygalactonojirimycin, which inhibits the synthesis of glycosphingolipids derived from glucosylceramide. Mice with Sandhoff disease treated with N-butyldeoxygalactonojirimycin had delayed onset of symptoms and increased life span (50). In addition, the amount of enzyme needed in ERT for Gaucher disease or Fabry disease could possibly be lowered if an inhibitor of glucosylceramide synthesis was also provided. It must be remembered that sphingolipids play very important functions in cellular metabolism, including signal transduction, cell adhesiveness, nerve impulse transmission, etc., and modiﬁcation of these and other functions by these drugs in a developing child must be carefully tested in animal models before human trials are instituted. Also, these drugs have signiﬁcant side effects that may limit their use. A novel approach to the treatment of certain LSDs and some other genetic disorders that result from protein misfolding or aberrant intracellular transport is under investigation (42,51). Under normal conditions, proteins that are signiﬁcantly altered from normal are retained in the endoplasmic reticulum and degraded by the proteasomal protein degradation pathway. This process is monitored by naturally occurring proteins called chaperones. It has been demonstrated that some competitive enzyme inhibitors in sub-inhibitory concentrations can bind to the active sites of certain proteins stabilizing them long enough to permit their localization to the correct organelle, including lysosomes (52). At the low pH of the lysosomes and in the presence of cofactors, these mutant enzymes could be partially stabilized and catalyze the hydrolysis of the stored substrate to a small, but signiﬁcant, degree. This use of the so-called chemical or pharmacological chaperones has been termed enzyme enhancement therapy. An increase in b-galactosidase activity in certain organs, including brain, has been demonstrated in the mouse model of GM1 gangliosidosis following short-term oral administration of N-octyl-4-epi-b-valienamine (NOEV) (53). Alpha-galactosidase activity can be raised by 1-deoxy-galactonojirimycin in lymphoblasts from human patients with Fabry disease and in vivo in the mouse model (54). A human patient with the cardiac variant form of Fabry disease treated with intravenous injections of galactose showed signiﬁcant clinical improvement in cardiac function (55). N-(n-nonyl) deoxynojirimycin can raise glucocerebrosidase activity two-fold in cultured cells from certain patients with Gaucher disease (56). This treatment requires that the mutant enzyme under investigation be synthesized although it normally would be unstable

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and rapidly degraded. As only low levels (1–5% of normal) of most lysosomal enzymes are needed to elicit a positive effect, this approach may have some practical relevance. Chemical chaperones could have additional beneﬁts, including the ability of some to be administered orally, their potential to cross the blood–brain barrier possibly affecting activities in brain, their relative safety, and low cost. While gene therapy to treat LSDs with neurological involvement has been under investigation using the large number of available animal models, no protocols in humans are currently in use. The role for gene therapy in treating neurogenetic disorders is discussed in detail in Chapter 4. Many papers have been published describing ‘‘successful’’ in vivo and in vitro gene therapy for LSDs using different viral vectors in animal models (reviewed in Ref. 57). The mouse model of MPS VII has been used for many gene therapy studies. A large number of viral vectors containing the human b-glucuronidase cDNA have been developed, and upon injection into the ventricles or the brain parenchyma of mice, there is evidence for gene expression and clearing of stored material. Signiﬁcant functional improvement was documented in adult MPS VII mice using feline immunodeﬁciency virus-based vectors (58). While these studies may provide ‘‘proof of principle’’ results, differences in protein defects as well as protein processing and stability, nature of storage products, disease course, cell types affected and other variables make extrapolation to other LSDs difﬁcult. Attempts to transfer the gene of interest into HSCs for autologous HSCT would be very useful since allogeneic HSCT has been successful for some LSDs (59). These HSCs could be engineered to express more of the needed enzyme than normal and, in addition, could supply blood-derived factors that could enhance repair of damaged neural cells. The use of neural stem cells to replace damaged cells and supply needed cofactors or enzymes is under intense investigation. Numerous neural stem cell lines have been isolated, characterized, and injected into the brains of animal models (60–62). When injected into developing brain, they have the potential to migrate and differentiate, thereby providing a source of healthy replacement cells and enzymes that can be taken up by neighboring cells. Some studies have documented improvement in pathology, but clinical improvement has been minimal. Before human trials are proposed, issues of safety and effectiveness must be addressed by studies in larger animals for longer periods of time. In conclusion, the diagnosis of most LSDs is relatively simple using easily obtained tissue samples, such as blood or cultured cells from a skin biopsy. A screen of enzymatic activities and certain analyte concentrations dictated by the information supplied with the sample can result in a deﬁnitive diagnosis within a few days. Molecular analysis to identify the diseasecausing mutations may or may not be subsequently performed depending on the disorder. Carrier testing for interested family members is usually

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available by enzymatic testing. One exception is the Krabbe disease, where polymorphisms in the GALC gene make a very wide carrier and non-carrier range (36). Prenatal diagnosis is available using CVS and cultured amniotic ﬂuid cells for at-risk couples and for those concerned about having an affected child. At this time, treatment of pre-symptomatic individuals and those with mild symptoms is limited to HSCT or ERT for certain disorders. The method of choice depends on the availability of enzyme or a suitable HSC donor and the need to stop or reverse neurological damage. Eventually, it may take more than one treatment to ‘‘cure’’ a patient with a LSD—one acting quickly to prevent storage and arrest degeneration and one providing long-term correction. With effective therapy becoming available for more disorders, it will be increasingly important to recognize the presenting symptoms in a patient and seek diagnostic help from a reliable laboratory. Therefore, there is pressure to obtain a diagnosis as early as possible, especially before neurological symptoms are signiﬁcant. This requires educating physicians to recognize the early signs of these disorders and possibly instituting screening methods that identify patients at or near birth before symptoms are present. While such testing is theoretically possible, it does present some problems related to the need for expensive or life-threatening therapy in individuals identiﬁed without a family history of a disorder. However, early diagnosis would permit careful clinical evaluation of the individual so that appropriate treatment could begin as early as indicated to obtain the most beneﬁcial response.

ACKNOWLEDGMENTS This research was supported in part by a grant from the National Institutes of Health (DK 38795). For information on the Lysosomal Diseases Testing Laboratory please visit our website at www.tju.edu/lysolab.

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22 The Tuberous Sclerosis Complex: Clinical Manifestations and Molecular Genetics John R. Pollard and Peter B. Crino Department of Neurology and Epilepsy Center, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, U.S.A.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that affects one in eight to 10,000 live births. The clinical pathology has been recognized for over a century, and recent research has yielded major breakthroughs not only in the genetics of this disease but also in the molecular pathophysiology. This chapter will address the clinical features and molecular genetics of TSC as well as the impact that research ﬁndings have had on the clinical progression of TSC. CLINICAL FEATURES OF TSC The diagnosis of TSC is based on deﬁned major and minor clinical criteria that include hamartomatous tissue growths (Table 1). Most commonly, TSC affects the brain, skin, heart, kidneys, eyes, and lungs in the form of highly organ speciﬁc hamartomas or benign tissue growths. Hamartomatous growths can cause morbidity by compression of adjacent normal tissues. While true malignancy is rare in TSC (largely limited to renal cell carcinoma), some hamartomas will require procedural or operative intervention to prevent tissue damage. Dermatologic manifestations of TSC including hypopigmented (hypomelanotic) macules, facial angioﬁbromas, ungula ﬁbromas, and the Shagreen

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Table 1 Diagnostic Criteria Deﬁnite TSC: Two major features or one major feature plus two minor features Probable TSC: One major feature plus one minor feature Possible TSC: One major feature or two or more minor features Major Features

Minor Features

Facial angioﬁbromas or forehead plaque Nontraumatic ungual or periungual ﬁbromas Hypomelanotic macules (three or more) Shagreen patch (connective tissue nevus) Multiple retinal nodular hamartomas Cortical tuber 1 Subependymal nodule Subependymal giant cell astrocytoma Cardiac rhabdomyoma, single or multiple Lymphangiomyomatosis 2 Renal angiomyolipoma 2

Multiple randomly-distributed pits in dental enamel Hamartomatous rectal polyps Bone cysts Cerebral white matter radial migration lines 1,3 Gingival ﬁbromas Nonrenal hamartoma Retinal achromic patch ‘‘Confetti’’ skin lesions Multiple renal cysts

1. Cerebral cortical dysplasia and cerebral white matter migration tracts occuring together are counted as one rather than two features of TSC. 2. When both lymphangiomyomatosis and renal angiomyolipomas are present, other features of tuberous sclerosis must be present before TSC is diagnosed. 3. White matter migration lines and focal cortical dysplasia are often seen in individuals with TSC; however, because these lesions can be seen independently and are relatively nonspeciﬁc, they are considered a minor diagnostic criteria for TSC. Source: From Ref. 101.

patch are quite speciﬁc for TSC. Each is considered a major diagnostic feature of TSC. Hypopigmented macules are typically greater that 3 mm in size and can be seen on the limbs and trunk as well as the face. They are best identiﬁed with ultraviolet light (Wood’s lamp). Facial angioﬁbromas are’typically observed over the malar, chin, and forehead regions. Ungual ﬁbromas are ﬂeshy excrescences that can grow to several millimeters in size that can be seen around the edges of the nailbeds. The Shagreen patch is an area of thickened skin located in the ﬂank or lumbosacral region. Other organs are also affected including the eyes, which frequently have multiple retinal hamartomas. These astrocytic lesions do not become malignant and only rarely affect vision. In very rare cases, retinal astrocytic hamartomas can hemorrhage. Cardiac rhabdomyomas typically present in neonatal life in over 50% of children with TSC. Interestingly, these hamartomas can spontaneously regress and do not typically lead to cardiac disease in later life. In the neonatal period, these lesions can cause congestive heart failure and arrhythmias.

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Renal lesions, including angiomyolipomas (AMLs), renal cysts, and rarely, renal cell carcinoma, can cause morbidity and even mortality in TSC patients. AMLs consist of fat and smooth muscle tissue and are space occupying lesions. They compress the surrounding renal parenchyma and over time can enlarge. Rupture of an AML can cause retroperitoneal hemorrhage. Renal cysts do not typically cause hemorrhage but they disrupt the renal cytoarchitecture. Further, the renal system is frequently affected in a contiguous gene syndrome of TSC and results in polycystic kidney disease (PKD) due to the deletion of TSC2 and PKD1, which lies adjacent to TSC2. Renal failure or hypertension is possible as a complication of an AML or as a possible symptom of PKD. A small percentage of patients with TSC will develop renal cell carcinoma and thus clinical vigilance with serial imaging is indicated in TSC patients with AMLs. The rare pulmonary complication of pulmonary lymphangioleiomyomatosis (LAM) only affects female TSC patients. The progression of LAM can lead to end stage lung disease curable only by lung transplantation. Other pulmonary lesions can take the form of multifocal micronodular pneumocyte hyperplasia or pulmonary cysts. NEUROLOGICAL MANIFESTATIONS Morbidity from TSC is often a result of neurologic manifestations including epilepsy, mental retardation, and autism. There is a high concordance between these neurological disorders and the incidence of speciﬁc brain lesions in TSC patients known as tubers, subependymal nodules, and subependymal giant cell astrocytomas (SEGAs). More than 50% of TSC patients will be affected by autism, developmental delay, or learning disabilities. Over 70% of TSC patients have epilepsy. While seizures are very common, the spectrum of epilepsy in TSC patients can span more benign simple partial seizures to life threatening generalized tonic–clonic seizures. A signiﬁcant number of TSC patients suffer from infantile spasms (IS) occurring within the ﬁrst year of life. The most aggressive cases of epilepsy often start with IS and the appearance of IS in TSC patients may be linked to the subsequent development of severe cognitive delay or autism. IS caused by TSC responds to ACTH treatment but recent reports suggest that vigabatrin is highly effective in this situation. The reason for this is not yet elucidated. Many patients have focal epilepsies that may respond well to antiepileptic drugs, and drug free remission is not uncommon. Medically refractory epilepsy patients may be candidates for epilepsy surgery for removal of the epileptogenic zone, which typically is an identiﬁed tuber. Some have taken the approach of a staged epilepsy surgery in which the patient alternates between epilepsy monitoring unit and epilepsy surgery until the dominant epileptogenic zones have been removed. TSC patients have a higher incidence of Lennox–Gasteaux syndrome with multiple types of seizures,

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mental retardation, and a characteristically abnormal EEG. Seizures of Lennox–Gasteaux syndrome often are resistant to medical therapy. Surgical treatments include implantation of a vagus nerve stimulator and palliative corpus callosotomy to stop drop attacks. Another life threatening problem is that TSC patients occasionally develop hydrocephalus from obstruction of the foramen of Monro and lateral ventricle due to growth of a SEGA. Surgical resection is the best treatment option and one reason to resect what is usually a benign lesion. The signiﬁcant neuropsychological problems of developmental delay and autism are treated with early intervention with intensive behavior therapy. Behavioral problems are often treated with both modiﬁcation therapy and pharmaceutical intervention. NEUROPATHOLOGY There are three main types of brain lesions seen in tuberous sclerosis: cortical tubers, subependymal nodules, and SEGAs. The neuropathological hallmarks of TSC are sclerotic nodules in the cerebral cortex called tubers. Bourneville named the disease in 1880 ‘‘tuberous sclerosis of the cerebral convolutions’’ (1). On gross pathology, the brain exhibits small white nodules that project from the surface of the brain (2). These nodules can vary from involving one convolution to involving the convolutions of most of a hemisphere. The nodules are found at the gray–white junction of the cortex, and rarely involve the cerebellum or brainstem (3,4). Histologically, tubers are composed predominantly of glial cells in a perpendicular orientation to the pia mater. Interspersed among the glia are dysmorphic neurons and the hallmark TSC: giant cells. These different cells form dysplastic cortical tissue with aberrant columnar and laminar organization (4). The giant cells have no preference for deep or superﬁcial structures, and are not well differentiated (5,6). Morphologically, these giant cells are large with glassy eosinophillic cytoplasm and have short, thickened processes (7). Various groups have tried to characterize these cells with immunohistochemistry and mRNA proﬁling, but these studies have yielded conﬂicting results. Some giant cells are immunoreactive to glial cell markers and others to neuronal markers (8–12). Electron microscopic and mRNA data suggest that giant cell mRNA has a neuronal pattern (10,13–16). One possible unifying hypothesis is that giant cells are phenotypically immature. Alternatively, the cells could be of a mixed neuroglial phenotype (7). The earliest time that tubers have been demonstrated is at 19 weeks of gestation. On prenatal MRI, fetal tubers have been seen as early as 20 weeks. On MRI scanning in adults, tubers have a low signal lesion on T1 and high signal on T2-weighted images. While tubers are usually seen in the cerebral cortex, they can also be found in the cerebellum (17). In the white matter underlying a tuber, there are often high signal migration

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lines that are thought to represent dysplastic heterotopic neurons that failed to complete migration (17–19). An alternative hypothesis supported by signiﬁcant evidence is that migration of abnormal cells continues into adult life, leaving these telltale marks on MRI (7). Clinically, the signiﬁcance of the tubers is their epileptogenicity and relationship to developmental delay (20,21). Studies trying to elucidate the mechanism of epileptogenicity have focused on differences in excitatory and inhibitory systems between normal tissue and tuber. For example, there is variation in GAD65 and NR1 immunoreactivity (22), and recent studies have shown differences in mRNA expression in GABAA receptor subunits, glutamate receptor subunits, and neurotransmitter uptake sites. Further, protein upregulation of NMDA 2B receptor expression has been observed (16). Subependymal nodules are protrusions into the ventricular space that can be found in any ventricle, but are most prominent in the lateral ventricles. Grossly, they can line the ventricle and can be called ‘‘candle guttering.’’ These nodules are also comprised of dysplastic astrocytes with giant cells (5,6). Frequently, these nodules calcify over childhood and puberty and can be seen as hyperdense lesions on CT scan. On MRI scan, these nodules are hypointense on both T1 and T2 sequences (4). Subependymal nodules located near the Foramen of Monro tend to enlarge and are called giant cell astrocytomas (17,23,24). These can be seen in 10% to 15% of patients, and a portion of these will eventually need neurosurgery to prevent obstruction of cerebrospinal ﬂuid outﬂow, impingement on optic chiasm, or hypothalamic/pituitary involvement (4). Other neuropathologic ﬁndings in TSC can include partial agenesis of the corpus callosum, transmantle cortical dysplasia, schizencephaly, intracranial aneurysms, intracranial moyamoya vasculopathy, cerebellar hemisphere hyperplasia, and vermian agenesis (4,23–27). GENETICS Tuberous sclerosis is an autosomal dominant disease with highly variable expressivity; two different mutations that have been associated with the disease. One of the loci is the TSC1 gene at 9q34 that codes for the 130 kDa hamartin protein (28–30). It is a 23 exon gene product of which the ﬁrst two exons are not translated. Hamartin is hydrophilic, has a single putative transmembrane domain, and is widely expressed throughout the brain (31). The second locus is the TSC2 gene at 16p13, which encodes a 200 kDa protein (32). It has 42 exons with the ﬁrst exon being noncoding (33). There are alternative splice variants of TSC2 involving exons 25, 26, and 31 (34,35). The PKD1 gene is located immediately centromeric to the TSC2 gene, so patients with large deletions often have PKD in addition to tuberous sclerosis (36).

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Almost 500 different mutations have been reported. TSC2 mutations comprise ~70% of sporadic mutations, while TSC1 mutations account for ~10% to 15% (37–39). They include nonsense, splicing, deletion, insertion, and missense mutations, and large deletions and rearrangements (40,41). There are some clear patterns. For example, missense mutations are very rare in TSC1 but much more common in TSC2 (40,42). Also, there is a higher rate of large rearrangements and deletions in TSC2.

Biallelic Inactivation It increasingly appears as though cells that possess one normal TSC allele do not exhibit overt abnormalities. Instead the disease follows Knudson’s twohit hypothesis: there must be a second genetic mutation in the normal allele resulting in a loss of heterozygosity for a cell to be affected (43). The pattern that is seen clinically thus reﬂects a germline mutation followed by a somatic gene mutation in a dividing cell line. This combination of mutations results in the growth of clonal hamartomas in many areas of the body. This has been demonstrated in many of the hamartomas in TSC patients including LAM, chordomas, and SEGAs (44–46). It is likely to be true in cortical tubers as well. Because cells that are homozygous negative for TSC1 or TSC2 are hamartomatous, both of these genes are considered tumor suppressor genes. Genetic Counseling The diagnosis of TSC in a child leads directly to the difﬁcult problem of genetic counseling for the parents. This counseling is complicated by the existence of mosaicism, in which a somatic mutation early in development causes little or no disease in a parent but is passed on to the children (47). Unaffected parents of an affected child thus still have a 1% to 2% chance of having more children with TSC (48,49). Genetic heterogeneity may play a role as well, given the complex nature of the disease (41).

Genetic Testing There is a high incidence of new mutations in TSC, so lack of a diseased progenitor should not alter diagnostic decisions (1). Genetic tests are now available for conﬁrmation and for prenatal testing, but unfortunately, the test is still difﬁcult to use clinically. Genetic testing of the genes that are pathogenic in TS is complicated by the large size of the two genes (TSC1 and TSC2), the large number of disease-causing mutations, and the existence of somatic mosaicism. Clinical test methods can consist of sequencing of the entire coding region, targeted mutation analysis, or sequencing of select exons. Identiﬁcation of a known mutation has a high positive

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predictive value, but sequence analysis at best is 60% to 70% sensitive, so there is a high false negative rate (39). SIGNALING PATHWAY AND CELL BIOLOGY Hamartin and tuberin, the products of TSC1 and TSC2, respectively, are widely expressed throughout the body, including brain, cardiac muscle, skin, kidney, and liver (50–55). Mutation of either TSC1 or TSC2 produces a similar phenotype, and the generally accepted hypothesis for this phenomenon is that the two proteins work together in similar pathways (41). Hamartin and tuberin co-localize and co-immunoprecipitate with antibodies to either protein (50,54,56–60). They form a heterodimer in vivo and localize mostly to the cytosol (61,62). Smaller amounts can be found in the golgi, early endosomes, and the cytoskeleton or membrane fraction (56,62–64). The association of the two proteins is essential for the stability of each (54,65). mTOR Pathway Recently, it has been shown that the tuberin/hamartin complex is an integral part of a ubiquitous signaling pathway called the ‘‘mTOR Pathway’’ (Fig. 1). It is also called the ‘‘insulin signaling pathway’’ because it is responsible for transducing the presence of insulin into increased cellular protein expression.

Figure 1 The mTOR cascade in TS.

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This pathway is highly conserved evolutionarily, and studies in Drosophila have been essential for deﬁning the relationships between signaling molecules. A description of a simpliﬁed version of this pathway follows. Insulin and other growth factors such as IGF-1 start this cascade by binding to surface receptors. This activates the generation of phosphoinositol-3-phosphate (PI3) through PI3 kinase (PI3K) (66,67). This in turn activates protein kinase B, also called AKT (68). Activated AKT causes phosphorylation of the TSC2 (tuberin) part of the complex (69). Normally, the tuberin/hamartin complex exerts a tonic inhibitory effect on the mammalian target of rapamycin (mTOR), but when phosphorylated, the tuberin/hamartin complex dissociates, thus activating mTOR. mTOR activates cellular translation by phosphorylating two regulatory proteins, phospho-S6 Kinase (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) (70–73). In turn, these proteins activate the mammalian translation apparatus leading to increased protein synthesis and cell growth. Mutations in both alleles of either TSC1 or TSC2 result in a loss of inhibition of mTOR activity (74). Then abnormally active mTOR results in excessive protein synthesis and growth. From the tuberous sclerosis researcher’s point of view, the critical part of this pathway is the interaction between the TSC complex and mTOR. But the details of how the TSC complex inhibits mTOR are still being debated (74–78). mTOR is a complicated protein which acts as an integrator of growth factor and nutritional status. A leading theory suggests that tuberin (TSC2) has GTPase-activating protein activity which acts on a small G-protein called Ras homolog enriched in brain (Rheb) (79–83). Rheb in turn then acts on one of the cluster of proteins that associate with mTOR (84). The signiﬁcance of this pathway is that all of the elements downstream from the TSC complex are likely targets for reversing some of the molecular effects of the TSC mutations. Fortunately, many well-characterized agents antagonize elements of this pathway, including rapamycin, an mTOR inhibitor that has already been used in humans as an immunosuppressive drug. If upregulation of this pathway leads to clinical manifestations such as developmental delay or seizures, there is hope that antagonists of the mTOR pathway will provide new avenues to therapy. Cell Morphology While activation of protein synthesis likely contributes to alterations of cell morphology, other effects of TSC mutations may be involved as well. For example, hamartin (TSC1) binds to ezrin, radixin, and moesin (ERM) proteins, a family of proteins that links the plasma membrane to the actin cytoskeleton (85). Hamartin co-localizes with moezin and binds to intermediate neuroﬁlament light chain in cortical neurons (86). Experimental inhibition of hamartin function results in loss of cell adhesion and

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overexpression leads to focal adhesions (87). Thus, loss of the structural function of hamartin likely has a signiﬁcant effect on cell morphology resulting in the pathological ﬁndings of giant cells and atypical glia (56). Cell Cycle Regulation Tuberin and hamartin may have inhibitory effects on cell proliferation. Different mechanisms have been explored in the search to ﬁnd the signaling pathway responsible for the mitogenic properties of TSC mutations. Hamartin is highly expressed in the G0 phase of cell cycle, and indeed overexpression of hamartin attenuates cell proliferation (88). A second effect on cell cycle comes from the GAP activity of tuberin, which activates the mitogenic G-proteins rap1a and rap1b (89,90). One of the best characterized cell cycle functions of the TSC complex is to lengthen the G1 phase of the cell cycle. Therefore, loss of function of the TSC complex shortens this phase of cell cycle (56,76,91). The cycle through this checkpoint is regulated by pRB (retinoblastoma protein), which is activated by cyclin-dependent kinases, which in turn are inhibited by p27KIP1. The TSC complex stabilizes p27KIP1 and a loss of function mutation of a member of the TSC complex reduces the inhibition of the checkpoint (88,92–95). Finally, there is also evidence that TSC1 and TSC2 play a role in wnt-bcatenin signaling, a pathway known to affect cell proliferation (96). Following evidence that cyclin D1 expression is increased in TSC reduced tissues, Mak et al. (98) induced ectopic expression of hamartin and tuberin in renal tumors and found that expression was inversely related to b-catenin levels (10,97). Further investigation suggested that hamartin and tuberin were a part of the b-catenin degradation complex that includes GSK3. The participation of hamartin and tuberin in multiple major signaling pathways may indicate that these proteins have a role in coordinating signals between several pathways (99). These mitogenic properties of loss of function of the TSC likely contribute to the clonal proliferation of the hamartomatous malformations that characterize TSC. Further investigation into these processes may impact the neurological treatment of tubers and giant cell astrocytomas. Cell Migration Abnormally active cell migration may contribute to the process of tuber formation. An intriguing hypothesis is that tubers continue to grow because abnormal cells are continuously generated through adulthood in the subventricular zone and migrate to the cortex (7). Markers of distinct neurodevelopmental epochs were evaluated immunohistochemically in an effort to deﬁne the etiology of tubers. Dysmorphic neurons exhibited HuD and NeuN immunolabeling, consistent with a differentiated neural phenotype. Giant cells, in contrast, demonstrated reactivity to CRMP4, doublecortin

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and LIS-1 expression that would be expected from newly generated cells (12). In order for there to be newly generated cells, some of the cells must die over time, because tubers do not grow continuously through adulthood. These cells may be injured by ongoing inﬂammatory processes, and there is ongoing cell death in tubers, as evidenced by TUNEL and caspase 8 labeling (100). Thus, tubers may continuously change throughout life, though not in apparent size. If indeed there is continuous abnormal migration of cells throughout life, it might be possible to design small molecules to impede this process, leading to better long-term outcomes. CONCLUSION Much has been learned about TSC since it was ﬁrst deﬁned in 1880. TSC is now understood to be an autosomal dominant disease that manifests predominantly as multiple hamartomatous lesions, of which the tuber is just one type. As a consequence of loss of tuberin or hamartin, these hamartomatous tissues have an abnormally upregulated insulin signaling pathway, abnormal morphology due to structural protein disruption, and abnormal cell cycle regulation, in part due to alteration of wnt-b-catenin signaling. The loss of tuberin or hamartin is a result of a dividing progenitor cell suffering a somatic mutation that results in a second hit and complete knockout of one of the genes. Tuberin and hamartin form a heteromer in vivo, so elimination of either protein results in the same disease. While many tissues in TS patients have been shown to derive from cells that have a second hit, the tuber, the pathognomonic lesion, has resisted this easy characterization. It is possible that tubers are formed from a combination of wildtype tissue, and continuously migrating cells generated from per ventricular progenitor cells that have suffered a second hit. Despite the explosion of knowledge about the molecular biology of the disease, perfect genetic testing and molecular treatments are still not possible. Diagnosis is still made on the basis of clinical symptoms and lesions because the genetic tests for TSC1 and TSC2 have a high rate of false negatives. The current treatments for this syndrome frequently reduce symptoms of the disease to the point where many affected individuals can lead productive lives, but the vast majority of patients would beneﬁt from further developments in medical therapy. The near term goal of TSC research is to gain enough medical control over the disease to change the treatment regimen from the current combination of clinical diagnosis and symptomatic therapy to that of genetic diagnosis and disease-modifying therapy. Identiﬁcation of the genes responsible for TSC and ongoing investigations into the molecular and cellular effects of TSC1 and TSC2 dysfunction point toward the high likelihood of disease modifying pharmaceutical agents being available in the near future. The ﬁrst pharmaceutical targets that are being tested act on elements

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57. Johnson MW, et al. Co-localization of TSC1 and TSC2 gene products in tubers of patients with tuberous sclerosis. Brain Pathol 1999; 9(1):45–54. 58. Catania MG, et al. Hamartin expression and interaction with tuberin in tumor cell lines and primary cultures. J Neurosci Res 2001; 63(3):276–283. 59. Johnson MW, et al. Hamartin and tuberin expression in human tissues. Mod Pathol 2001; 14(3):202–210. 60. Murthy V, et al. Developmental expression of the tuberous sclerosis proteins tuberin and hamartin. Acta Neuropathol (Berl) 2001; 101(3):202–210. 61. Nellist M, et al. TSC2 missense mutations inhibit tuberin phosphorylation and prevent formation of the tuberin–hamartin complex. Hum Mol Genet 2001; 10(25):2889–2898. 62. Nellist M, et al. Characterization of the cytosolic tuberin–hamartin complex. Tuberin is a cytosolic chaperone for hamartin. J Biol Chem 1999; 274(50): 35647–35652. 63. Yamamoto Y, et al. Multicompartmental distribution of the tuberous sclerosis gene products, hamartin and tuberin. Arch Biochem Biophys 2002; 404(2): 210–217. 64. Plank TL, Yeung RS, Henske EP. Hamartin, the product of the tuberous sclerosis 1 (TSC1) gene, interacts with tuberin and appears to be localized to cytoplasmic vesicles. Cancer Res 1998; 58(21):4766–4770. 65. Benvenuto G, et al. The tuberous sclerosis-1 (TSC1) gene product hamartin suppresses cell growth and augments the expression of the TSC2 product tuberin by inhibiting its ubiquitination. Oncogene 2000; 19(54):6306–6316. 66. Endemann G, Yonezawa K, Roth RA. Phosphatidylinositol kinase or an associated protein is a substrate for the insulin receptor tyrosine kinase. J Biol Chem 1990 Jan 5; 265(1):396–400. 67. Ruderman NB, et al. Activation of phosphatidylinositol 3-kinase by insulin. Proc Natl Acad Sci USA 1990; 87(4):1411–1415. 68. Burgering BM, Coffer PJ. Protein kinase B (c-Akt) in phosphatidylinositol3-OH kinase signal transduction. Nature 1995; 376(6541):599–602. 69. Potter CJ, Pedraza LG, Xu T. Akt regulates growth by directly phosphorylating Tsc2. Nat Cell Biol 2002; 4(9):658–665. 70. Redpath NT, Foulstone EJ, Proud CG. Regulation of translation elongation factor-2 by insulin via a rapamycin-sensitive signalling pathway. EMBO J 1996; 15(9):2291–2297. 71. Graves LM, et al. cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells. Proc Natl Acad Sci USA 1995; 92(16):7222–7226. 72. Price DJ, et al. Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. Science 1992; 257(5072):973–977. 73. Chung J, et al. Rapamycin-FKBP speciﬁcally blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases. Cell 1992; 69(7): 1227–1236. 74. Gao X, et al. Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling. Nat Cell Biol 2002; 4(9):699–704.

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75. Gao X, Pan D. TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth. Genes Dev 2001; 15(11):1383–1392. 76. Potter CJ, Huang, H, Xu T. Drosophila Tsc1 functions with Tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell 2001; 105(3):357–368. 77. Inoki K, et al. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 2002; 4(9):648–657. 78. Inoki K, Corradetti MN, Guan KL. Dysregulation of the TSC-mTOR pathway in human disease. Nat Genet 2005; 37(1):19–24. 79. Manning BD, Cantley LC. Rheb ﬁlls a GAP between TSC and TOR. Trends Biochem Sci 2003; 28(11):573–576. 80. Stocker H, et al. Rheb is an essential regulator of S6K in controlling cell growth in Drosophila. Nat Cell Biol 2003; 5(6):559–565. 81. Saucedo LJ, et al. Rheb promotes cell growth as a component of the insulin/ TOR signalling network. Nat Cell Biol 2003; 5(6):566–571. 82. Zhang Y, et al. Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nat Cell Biol 2003; 5(6):578–581. 83. Garami A, et al. Insulin activation of Rheb, a mediator of mTOR/S6K/4EBP signaling, is inhibited by TSC1 and 2. Mol Cell 2003; 11(6):1457–1466. 84. Castro AF, et al. Rheb binds tuberous sclerosis complex 2 (TSC2) and promotes S6 kinase activation in a rapamycin- and farnesylation-dependent manner. J Biol Chem 2003; 278(35):32493–32496. 85. Lamb RF, et al. The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho. Nat Cell Biol 2000; 2(5):281–287. 86. Haddad LA, et al. The TSC1 tumor suppressor hamartin interacts with neuroﬁlament-L and possibly functions as a novel integrator of the neuronal cytoskeleton. J Biol Chem 2002; 277(46):44180–44186. 87. Astrinidis A, et al. Tuberin, the tuberous sclerosis complex 2 tumor suppressor gene product, regulates Rho activation, cell adhesion and migration. Oncogene 2002; 21(55):8470–8476. 88. Miloloza A, et al. The TSC1 gene product, hamartin, negatively regulates cell proliferation. Hum Mol Genet 2000; 9(12):1721–1727. 89. Maheshwar MM, et al. The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis. Hum Mol Genet 1997; 6(11):1991–1996. 90. Wienecke R, Konig A, DeClue JE. Identiﬁcation of tuberin, the tuberous sclerosis-2 product. Tuberin possesses speciﬁc Rap1GAP activity. J Biol Chem 1995; 270(27):16409–16414. 91. Tapon N, et al. The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation. Cell 2001; 105(3):345–355. 92. Olashaw N, Pledger WJ. Paradigms of growth control: relation to Cdk activation. Sci STKE 2002; 2002(134):RE7. 93. Miloloza A, et al. Evidence for separable functions of tuberous sclerosis gene products in mammalian cell cycle regulation. J Neuropathol Exp Neurol 2002; 61(2):154–163.

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23 Neurofibromatosis Amit Malhotra and David H. Gutmann Department of Neurology, Washington University School of Medicine and St. Louis Children’s Hospital, St. Louis, Missouri, U.S.A.

James Dowling The Division of Neurology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

INTRODUCTION Neuroﬁbromatosis can be divided into two phenotypically and genetically distinct disorders, both having tumor growth as a hallmark. Neuroﬁbromatosis 1 (NF1), once known as peripheral neuroﬁbromatosis or Von Recklinghausen’s disease, is characterized by skin lesions, multiple tumor types, and vascular lesions, while neuroﬁbromatosis 2 (NF2) is characterized by predominantly central nervous system (CNS) tumors, most notably bilateral vestibular schwannomas (1). There are also more rare variants of classic NF1, including segmental (or mosaic) NF1, in which pathological features are restricted to one area of the body, and spinal neuroﬁbromatosis, characterized by the late onset of paraspinal neuroﬁbromas (2). In this chapter, we shall focus on the clinical and pathogenic features of most common form of NF, NF1, with a limited discussion of the clinical features of NF2.

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NEUROFIBROMATOSIS 1 History The ﬁrst case report of probable NF1 was published in 1768 by Mark Akenside of the Royal College of London in a paper on cancers (3). Other case reports followed, but no clear association with nervous system tissue was noted until Robert W. Smith’s 1849 monograph on ‘‘neuromas’’ postulated a connective tissue or nerve origin for these tumors (4). Subsequently, Frederick Von Recklinghausen published a thorough review of the disease in 1882, detailing the autopsy ﬁndings in a 47-year-old male and 55-yearold female with NF1 (5). He also gave the ﬁrst clear description of the ‘‘neuromas’’ actually arising from nervous tissue. Since this time, there have been great advances in the genetics and pathophysiology of NF1, yet certain clinical and epigenetic phenomena remain elusive. Epidemiology NF1 is the most common of the neurocutaneous syndromes and one of the most common genetic syndromes overall (2,6,7). It results from a single gene defect and is transmitted in an autosomal dominant pattern. This said, approximately 50% of cases are the result of sporadic mutations, and the mutation rate has been estimated at one in 10,000 gametes per generation (6). Interestingly, greater than 90% of these new mutations are paternally derived (8). The prevalence of NF1 ranges in various studies from 1 in 2500 live births in the United Kingdom to 1 in 4000 elsewhere (6,7,9). NF1 has been described in all races and ethnic groups, and has no gender bias (10). While NF1 is 100% penetrant, there is wide variation in the clinical features, even between individuals with the identical genetic mutation. There has yet been no clear correlation between genotype (speciﬁc genetic mutation) and phenotype (speciﬁc clinical features), with the exception that large or complete deletions tend to be seen in children with a more severe clinical course. Clinical Features Individuals with NF1 present with multiple abnormalities of the skin, eyes, viscera, and nervous system. These clinical features are often noted in early childhood with full expression typically seen by age 20 (11). Certain clinical features follow a developmental timeline with age dependent expression. For example, while Lisch nodules (iris hamartomas) are seen in only about 10% of one-year-old children, greater than 95% of 21-year-old patients with NF1 have Lisch nodules (12). There are other features that are almost always congenital, such as plexiform neuroﬁbromas. Thus, while the diagnosis may be entertained in very young infants, it is often not deﬁnitively made until later in childhood. Multiple authors have recommended, as is our

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Table 1 National Institutes of Health (NIH) Diagnostic Criteria for NF1 Any two or more are required for the diagnosis of NF1 Six or more cafe´-au-lait macules Over 5 mm in greatest diameter in prepubertal individuals Over 15 mm in greatest diameter in postpubertal individuals Two or more neuroﬁbromas of any type or one plexiform neuroﬁbroma Freckling in the axillary or inguinal regions Optic glioma Two or more Lisch nodules (iris hamartomas) A distinctive osseous lesion such as sphenoid dysplasia or thinning of the long bone cortex with or without pseudarthrosis A ﬁrst degree relative (parent, sibling, or offspring) with NF1 by the above criteria

practice, that young children in which a deﬁnitive diagnosis cannot be established, but for whom there is a high index of suspicion, be followed as though they have NF1. In this regard, the vast majority of young children with six or more cafe´ au lait (CAL) macules greater than 5 mm will develop additional features of NF1 over time and will meet diagnostic criteria by age 21 (11–13). Table 1 lists the clinical criteria required to establish a deﬁnite diagnosis of NF1. Cafe´-au-lait Macules CAL macules, along with neuroﬁbromas, represent the classic features of NF1 (Fig. 1). These are almost always present at birth and are seen in nearly every patient. CAL macules derive their name from the hyperpigmented macules, which resemble ‘‘coffee with milk.’’ In Caucasian skin, their hue is more often a reddish brown color, while in darkly pigmented skin, it often is a more brown color. The borders of typical CAL macules are smooth and well-deﬁned, without notable asymmetry or irregularity. NF1-associated CAL macules are typically ﬂat and the pigmentation is uniform. CAL macules can be present in any location of the body, perhaps with the exception of the eyebrows, scalp, palm, and soles (14), and they typically increase in number and size during the ﬁrst few years of life. Pathologically, these lesions represent a collection of melanocytes containing giant melanosomes located in the basal layer of the epidermis (2). While it is theoretically possible to meet criteria for a diagnosis of NF1 without CAL macules, their absence ought to raise doubt regarding the diagnosis. Lisch Nodules Lisch nodules are iris hamartomas containing melanin and are pathognomic for NF1. They are seen in almost all affected individuals by age 21 and have no impact on visual function. Routine ophthalmologic examination will not adequately detect these lesions, nor will it differentiate them from iris nevi.

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Figure 1 Representative clinical features in NFI: (A) cafe´ au lait macule, showing smooth regular border and hyperpigmentation, (B) dermal neuroﬁbromas on the forearm of an adult with NF1, and (C) a right thigh plexiform neuroﬁbroma with overlying skin changes and abnormal pigmentation. Abbreviation: NFI, neuroﬁbromatosis.

Lisch nodules are best identiﬁed by slit lamp examination, through which the three-dimensional nature of these hamartomas can be appreciated. Neurofibromas Neuroﬁbromas are peripheral nerve sheath tumors consisting of a mixture of cellular elements, including Schwann cells, ﬁbroblasts, mast cells, and

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perineural cells (15). Local cutaneous neuroﬁbromas can be pedunculated/ sessile or subcutaneous lesions. Neuroﬁbromas are usually restricted to the dermis and subcutaneous tissue as seen in Figure 1, and harbor no increased risk for malignant transformation (11,14,15). Rarely, these tumors are seen in infancy, but are most often ﬁrst seen in early adolescence, and increase in number and size throughout adulthood. Hormonal changes appear to have a signiﬁcant impact on the appearance and growth of neuroﬁbromas, as evidenced by the increase in number and size during puberty and pregnancy (16,17). There is some data to suggest that the early development of neuroﬁbromas is predictive of a larger overall neuroﬁbroma burden and distribution but additional studies will be required (18). Neuroﬁbromas may be associated with signiﬁcant itching, suggesting that the mast cells present in these tumors may contribute to neuroﬁbroma growth. Finally, and often most important to affected patients, is the issue of cosmesis. Surgical excision of tumors by an experienced practitioner can result in a good outcome, but there are frequent instances where neuroﬁbromas may regrow after excision. Currently, there are no clear guidelines for when and how best to remove the neuroﬁbromas, though some authors advocate early removal (19). Diffuse cutaneous neuroﬁbromas are similar to localized ‘‘discrete’’ neuroﬁbromas with a few important exceptions. These tumors tend to be poorly demarcated and plaque-like in appearance, with a predilection for the head and neck regions. Furthermore, there may be rare malignant transformation of these tumors (15). Plexiform neuroﬁbromas are also peripheral nerve sheath tumors consisting of neoplastic Schwann cells, but differ from the cutaneous ‘‘discrete’’ neuroﬁbromas by the involvement of multiple nerve fascicles. Unlike the cutaneous neuroﬁbroma, plexiform neuroﬁbromas may be associated with soft tissue hypertrophy, bony deformities, and overlying hyperpigmentation or hypertrichosis, as shown in Figure 1 (20,21). Plexiform neuroﬁbromas, especially those involving the trigeminal nerve, tend to be present at birth, and are presumed to represent congenital tumors. On clinical exam, they are often described as ‘‘a bag of worms.’’ One of the morbid complications associated with plexiform neuroﬁbromas is transformation into a malignant peripheral nerve sheath tumor (MPNST). The lifetime risk of malignant transformation has been estimated at between 8 and 13%. Clinical clues to the development of a MPNST include the sudden onset of pain, an associated neurologic deﬁcit, and rapid tumor growth. Recent studies have suggested that positron emission tomography (PET) may be useful for distinguishing growing plexiform neuroﬁbromas from MPNSTs (22). The development of an MPNST is associated with high mortality, with ﬁve-year patient survival estimated at 21% (23). Management of these tumors by surgical means has proven unsatisfactory. Resection rarely is able to achieve tumor-free margins, and tumor recurrence

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or metastasis to bone or lung is common. Further management modalities involving anti-angiogenesis agents, mast-cell stabilizers, and farnesyl transferase inhibitors are under investigation. Optic Pathway Glioma Optic pathway gliomas (OPGs) are the most common intracranial tumor in NF1, with approximately 15% of children developing these neoplasms (24,25). OPGs are almost exclusively seen in childhood, with a median age of detection of 4.2 years. Most OPGs are seen within the ﬁrst decade of life (26,27). While most OPGs do not result in clinical symptoms, approximately one-third of children will develop visual or endocrinologic abnormalities, and many of these children will require treatment. NF1associated OPGs are generally slow growing tumors, and occasionally will regress spontaneously (24,27,28). Symptomatic tumors rarely develop after the age of 10 (27,29). The typical symptoms of optic pathway glioma progression are visual abnormalities and hypothalamic dysfunction. Visual signs and symptoms include visual ﬁeld abnormalities, decreased visual acuity, afferent pupillary defects, optic disc pallor or atrophy, and decreased color vision. Hypothalamic symptoms typically manifest as an isolated endocrinopathy, such as precocious puberty or a diencephalic syndrome. The rate of precocious puberty has been estimated at approximately 3% in NF1, with 100% of these individuals subsequently found to harbor a chiasmatic glioma (30). Histologically, NF1-associated OPGs are almost always World Health Organization grade I pilocytic astrocytomas, occurring anywhere along the optic pathway (18). Most OPGs are found in the anterior part of the pathway, including the prechiasmatic optic nerve, optic chiasm, and hypothalamus. Because of the risk of OPG in children with NF1, it is recommended that all children suspected of having NF1 obtain annual complete ophthalmologic evaluations until the age of 10 (31). We do not recommend baseline MRI scans, owing to their lack of predictive value for ﬁnding symptomatic tumors that eventually require treatment (32). Once symptomatic, NF1associated OPGs are most often treated using a combination of carboplatin and vincristine chemotherapy (15). Cognitive Deficits It has long been recognized that children with NF1 have a higher incidence of cognitive disorders than the general population. The exact type of cognitive deﬁcit, degree of impairment, and molecular pathogenesis, though, remain areas of fertile research. Before systematic reviews beginning in the 1980s, there was a perception that children with NF1 had a signiﬁcant degree of mental retardation. These early reports suffered from a lack of formalized consensus diagnostic criteria and incomplete neuropsychological testing.

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Mental retardation, as deﬁned by Weschler Full-Scale IQ of less than 70, is increased in incidence in NF1 patients. Incidence estimates vary between 4 and 8%, which is signiﬁcantly higher than in the general population (33–35). The mean IQ in these studies of children with NF1 demonstrated Wechsler Intelligence Scale for Children-Revised (WISC-R) scores between 89 and 98. This is within one standard deviation of the population norm, suggesting that while there is a lowering of IQ with NF1, as a group children with NF1 are not signiﬁcantly different from the normal population. Interestingly, when more sensitive measures were used, such as comparing NF1 patients to their unaffected siblings, clear IQ differences became apparent (36). In one study, children with NF1 had mean Wechsler Full-Scale scores in the normal range (96.5), but when compared to their siblings, they were signiﬁcantly lower (mean 108.5) (36). While some authors have suggested that the lower IQ mainly results from deﬁcits in the nonverbal domain (37,38), there is likely no consistent pattern of deﬁcit in verbal versus performance IQ (39). Further complicating matters are studies reporting a decrease in IQ with age and others reporting an increase in IQ with age (40,41). A study by Legius et al. (42) found the mean IQ for children between four and six years of age was 99 as compared to 87.7 for children 6 to 16. These results contrast with cross-sectional data reviewed by Riccardi and Eichner (43) revealing increasing IQ with age. Recently, Hyman et al. (44) found no signiﬁcant change in cognitive abilities over time as compared with an age matched control group. Lastly, children with NF1 and intracranial pathology (e.g., brain tumor), excluding unidentiﬁed bright objects (UBOs), perform worse on cognitive tasks than NF1 patients without intracranial pathology. Interestingly, patients with brain tumors, but not NF1, fare signiﬁcantly better than children with NF1 with or without intracranial pathology (45). Some authors advocate excluding NF1 patients with intracranial pathology (46), while others include them (47). Learning disabilities (LD) are the major cognitive disorder seen in children with NF1. Estimates of incidence range from 30% to 60% with a predominance of visuospatial deﬁcits (48–50). In this respect, the judgment of line orientation (JLO) task, a test of visuospatial ability, has been shown to be abnormal in children with NF1 (37,38). Only one report challenges the predominance of visuospatial learning disabilities (51), and other researchers found broader impairment in executive function, speciﬁcally organization, planning, and problem solving (51–55). While these abnormalities are evident on neuropsychological testing, the extent and range of the deﬁcits in the classroom has yet to be fully elucidated. Further, while many children with NF1 have LD, no longitudinal studies have rigorously examined academic performance and career choice in a cohort of NF1 patients with and without identiﬁed LD. Work over the past 20 years has also shown signiﬁcant impairment in various attention domains in children with NF1 (33,36,38). Speciﬁcally,

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there are deﬁcits in sustained attention (continuous performance tests), selective attention (ability to pay attention to one particular task in a sea of distractors), and divided attention (ability to attend to two different stimuli at the same time) (33,36). These deﬁcits have renewed interest in the diagnosis of attention deﬁcit hyperactivity disorder (ADHD) in NF1 patients. Thirteen studies of ADHD in children with NF1 have reported ﬁndings to date, with the predominance of data supporting an increased incidence. Estimates range from 30 to greater than 50% of NF1 children meeting diagnostic criteria for ADHD (39,56,57). Fortunately, it appears that children with NF1 and ADHD respond favorably to stimulant treatment and experience improvement in all cognitive and behavioral domains. The presence of T2 hyperintensities or UBOs has been proposed as a marker for the cognitive deﬁcits in NF1. UBOs represent areas of increased T2-weighted signal on brain MRI that are isointense on T1, show no mass effect or surrounding edema, and do not enhance with contrast. UBOs are seen in 40–80% of NF1 patients and tend to decrease in number with age (49,58,59). Early studies suggested that these lesions were areas of cortical dysplasia or heterotopia (60,61), but the only study with pathological conﬁrmation of MRI ﬁndings revealed atypical glial inﬁltrates with hyperchromatic nuclei, spongiform changes in the white matter, perivascular gliosis with areas of microcalciﬁcation, and areas of dysmyelination suggesting the T2 hyperintensities were the result of the intramyelinic edema in the vacuolar changes associated with the abnormal glial proliferation (62). Recent studies using functional imaging have not clariﬁed the picture. PET analysis has resulted in conﬂicting data, with some studies revealing the UBOs to be metabolically inactive, while other studies reveal normal metabolism (63,64). The problems in understanding the pathology of these lesions mirror the problems in understanding their relationship to cognitive deﬁcits. A number of studies have examined the relationship between UBOs and cognitive difﬁculties. Six studies have shown no relationship between the presence of UBOs and learning deﬁcits (40,65–69) while seven studies have shown decreased cognitive abilities in association with UBOs (44,52,54,70–73). Most of the negative studies are earlier and suffer from small study size, lack of rigorous psychometric testing, and lack of comparison of between the distributions of test scores. More recent work tends to show a relationship between UBOs and cognitive function. Feldman et al. in a study of 100 NF1 patients and 100 controls matched for age, sex, and socioeconomic status showed that the NF1 patients with UBOs had signiﬁcantly decreased IQ (84.1 compared to 102.2 for controls) and ﬁne motor performance as compared to controls and NF1 patients without UBOs (73). Another recent study followed a cohort of NF1 patients and their unaffected siblings over an eight-year period and found that there was no improvement in cognitive function with age despite the signiﬁcant decrease in number, size, and intensity of the T2 lesions (44). They also noted that the best predictor of adult cognitive function in their study was

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the presence of childhood UBOs. While the story on UBOs is not yet complete, the emerging data seems to support a relationship between UBOs in childhood and decreased cognitive performance throughout life. Other Features of NF1 While the above features are the predominant ones encountered in individuals with NF1, numerous other abnormalities can be seen. Bony abnormalities, including dysplastic scoliosis with vertebral scalloping, sphenoid wing dysplasia that occasionally can compromise the orbit, and bony dysplasias that can progress to pseudarthroses, are seen in a small percentage of individuals with NF1 (14,18). These lesions can become quite disabling, and often require special bracing, surgery, and occasionally amputation in the case of continued fracture. In addition, vascular abnormalities, including renal artery stenosis leading to hypertension, and carotid stenosis or occlusive disease leading to moya–moya and stroke, can be a signiﬁcant cause of morbidity and mortality in adults with NF1 (2,74–77). In addition, other malignancies, including juvenile chronic myelogenous leukemia, pheochromocytoma, Wilms tumor, and neuroblastoma, have been reported in individuals with NF1 (78–82). Lastly, the overall life expectancy of adults with NF1 is reduced by 10–15 years, often resulting from complications of hypertension and malignancy (74,81). Genetics and Molecular Pathogenesis Our understanding of NF1 has beneﬁted greatly from recent advances in genetics, culminating in the identiﬁcation of the NF1 gene in 1990 (83–85). The gene spans approximately 350 kb of genomic DNA and is composed of at least 60 exons with three different alternatively spliced exons. Analysis of the protein product, neuroﬁbromin, revealed that it contained a Ras-GTPase activating domain (GAP). Function of Neurofibromin Neuroﬁbromin is expressed at low levels in many different tissues but is predominantly found in Schwann cells, myeloid cells, oligodendrocytes, adrenal medullary cells, neurons, and astrocytes (86–90). Neuroﬁbromin has been shown both in vitro and in vivo to possess Ras-GAP activity, and in this fashion, inactivates Ras, reduces Ras-mediated proliferation, and inhibits tumor formation. Ras functions as an intracellular signaling molecule in a number of species including Drosophila, yeast, and mammals. Ras exists in two different conformations: active, in which it is bound to guanosine triphosphate, and inactive, in which it is bound to guanosine diphosphate. GAP molecules, such as neuroﬁbromin, act by promoting the conversion of the active form to the inactive form of Ras via the hydrolysis of guanosine triphosphate. In its active form, Ras promotes proliferation in many cell

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types, while in neurons, it promotes growth factor-independent differentiation and survival and plays a role in long-term potentiation, memory, and learning (91–94). While originally thought to function only as a negative regulator of RAS, recent studies have shown that neuroﬁbromin may be involved in other signaling pathways. Two recent studies have implicated neuroﬁbromin in cyclic adenosine monophosphate pathway regulation (cAMP) in both mammalian cells and Drosophila (95,96). Molecular Pathogenesis The functions of neuroﬁbromin have shed some light on the development of the clinical features seen in patients with NF1. As is true for any tumor predisposition syndrome, the NF1 gene functions as a tumor suppressor gene. While all individuals with NF1 are born with one mutated (nonfunctional) and one functional copy of the NF1 gene, tumor formation is associated with loss of the one remaining functional NF1 gene to result in no neuroﬁbromin expression, and increased cell proliferation (97–99). Loss of neuroﬁbromin expression as a consequence of NF1 inactivation results in increased RAS activity in NF1-associated tumors. In this respect, the neoplastic Schwann cells in neuroﬁbromas leukemic cells from children with NF1, and NF1-associated pilocytic astrocytomas all exhibit increased RAS activity (99–102). Mouse Models of NF1-Associated Clinical Features Several mouse models for plexiform neuroﬁbromas have been developed. The ﬁrst model involves the injection of Nf1/ embryonic stem cells into the developing mouse blastocyst (103). In a small number of these mice, tumors with the histologic and immunohistochemical appearance of human plexiform neuroﬁbromas developed. A second more tractable model of NF1-associated plexiform neuroﬁbroma was developed by Luis Parada and associates, in which Nf1þ/ mice were genetically engineered to lack neuroﬁbromin expression in Schwann cells. In these studies, plexiform neuroﬁbromas were observed. Interestingly, the plexiform neuroﬁbromas contained a large number of inﬁltrating mast cells, suggesting a role for mast cells in the development of these tumors (101,104,105). The requirement for Nf1þ/ cells also underlies the development of optic nerve gliomas where Nf1 inactivation alone in astrocytes is necessary, but not sufﬁcient for tumor formation (106). The cellular and molecular pathogenesis of the cognitive deﬁcits in NF1 was recently reviewed by Costa and Silva (91). Nf1þ/ mice exhibit speciﬁc learning deﬁcits, including visuospatial, but not simple associative learning similar to children with NF1 (92). These studies laid the foundations for understanding the cognitive deﬁcits operative in children with NF1. One of the most clinically relevant mouse learning studies has been

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the amelioration of the Nf1þ/ mouse cognitive deﬁcits by either genetic or pharmacologic manipulation of the Ras pathway (107,108). In contrast, in Drosophila, there is evidence that neuroﬁbromin modulates rutabaga-encoded adenylate cyclase (93,109) and that Nf1 null ﬂies have learning impairments in olfactory associative learning tasks (93). The Nf1/ ﬂy learning deﬁcits were reversed with inducible expression of the normal Nf1 transgene and with normalization of the adenylate cylase pathway. In Drosophila, the Ras-GTPase pathway seems to be involved in circadian rhythm regulation as circadian abnormalities can be rescued by Ras mutations (110). Collectively, these data point to Ras as an important component of learning and cognitive function. Recent work has expanded on this theme and shed light on other aspects of Ras involvement in cognitive function. Numerous studies have shown Ras signaling pathway involvement in synaptic plasticity, long-term potentiation (LTP) and contextual learning (111–114). These studies looked at mutational activation of RAS, but other reports have shown pharmacological alteration of the pathway, speciﬁcally via inhibition of PI3K and MEK, also results in learning deﬁcits and abnormal synaptic plasticity (114,115). In keeping with the involvement of Ras in these processes, electrophysiological studies on Nf1þ/ mice by Costa et al. (108) showed abnormalities in LTP, which could be reversed by decreasing Ras expression. Similarly, they examined evoked inhibitory postsynaptic potentials and found them to be increased in the Nf1þ/ mice, suggesting increased GABA inhibition. Further studies into the modulation of both GABA and Ras will help clarify not only NF1-associated cognitive pathology, but possibly also the general physiology of cognition. Future Questions The past decade has witnessed tremendous strides towards the elucidation of the molecular pathogenesis underlying the clinical features seen in individuals with NF1. With the identiﬁcation of the NF1 gene and its protein product, neuroﬁbromin, signiﬁcant attention has been focused on the biochemical pathways and cellular processes regulated by neuroﬁbromin, and the consequence of NF1 inactivation on cell growth, embryonic development, and adult tissue function. It is now possible, with the recent development of robust models of NF1 clinical disease, to design and test novel therapies for NF1 in preclinical studies. CLINICAL FEATURES OF NEUROFIBROMATOSIS 2 NF2 is an autosomal dominant condition in which affected individuals develop multiple distinct nervous system tumors. NF2 is caused by mutation of the NF2 gene on chromosome 22q12. The hallmark of NF2 is the presence

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of bilateral vestibular schwannomas, a ﬁnding observed in the vast majority of patients with NF2. The disease also predisposes to the development of other types of nervous system tumors, including meningiomas, schwannomas in other locations, ependymomas, and spinal astrocytomas (gliomas), as well as speciﬁc ophthalmologic abnormalities. NF2 is phenotypically and genetically distinct from NF1, and contrasts with NF1 in the infrequent occurrence of CAL macules and skinfold freckling, as well as the lack of Lisch nodules, bony abnormalities, and cognitive deﬁcits (116,117). NF2, unlike NF1, is a rare condition, with an estimated incidence of 1:40,000. The prevalence of the disease is 1:200,000, which underscores the accelerated mortality in patients with the diagnosis. In fact, the average estimated life span after diagnosis is 15 years, with the mean age of diagnosis between 18 and 24 and the average age of death 34-years-old (118). In addition, those individuals who begin to develop CNS tumors at an early age often suffer a more insidious clinical course. Patients with NF2 typically present with symptoms referable to their vestibular schwannomas, including deafness (the most common initial symptom), tinnitus, and balance problems. Other common presenting neurologic ﬁndings include weakness, pain, numbness, and seizures. It is worth noting that NF2 is commonly diagnosed in adults in their mid to late twenties, however, teenagers may develop symptoms in the early second decade of life and be given the diagnosis of NF2. This latter group of individuals frequently exhibit a larger tumor burden and die at an earlier age (119). In addition, about 1 in 10 patients will be diagnosed with NF2 during asymptomatic screening initiated because of an affected ﬁrst degree relative (120). Individuals with NF2 may also develop ocular or visual dysfunction. While patients with NF2 rarely present with blindness, eye ﬁndings are quite often present at the time of initial diagnosis, and become of increasing relevance as the disease progresses. The most common eye pathology is juvenile cataracts, and speciﬁcally posterior subcapsular lenticular opacities. In addition to cataracts, retinal tufts, epiretinal membranes, retinal dysplasia, iris nevoid hyperplasia, retinal hamartomas, and pseudophakia may be found (128). Patients can also develop optic pathway meningiomas, some of which may present in early childhood. Diagnostic criteria for NF2 were initially established in 1987, when a NIH consensus conference was convened to deﬁne the clinical features sufﬁcient for the diagnoses of NF1 and NF2 (121). Historically, the criteria grew out of a need to distinguish NF1 from NF2, but subsequently were used to identify at-risk individuals who would beneﬁt from close screening and follow up, and to separate NF2 patients from those with isolated nervous system tumors. The criteria have been modiﬁed since 1987 in an attempt to improve diagnostic accuracy (121), with the Manchester criteria for NF2 (Table 2) likely providing the highest degree of speciﬁcity and sensitivity (122).

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Table 2 Diagnostic Criteria for NF2 1991 NIH criteria

Manchester criteria

Bilateral vestibular schwannomas First degree relative with NF2 and unilateral vestibular schwannoma

Bilateral vestibular schwannoma First degree relative with NF2 and unilateral vestibular schwannoma or any two of meningioma, schwannoma, glioma, neuroﬁbroma, posterior subscapular lenticular opacities First degree relative with NF2 and any Unilateral vestibular schwannoma and any two of meningioma, schwannoma, one of meningioma, schwannoma, glioma, neuroﬁbroma, posterior glioma, neuroﬁbroma, juvenile subscapular lenticular opacities posterior subscapular lens opacity Multiple meningiomas and unilateral vestibular schwannoma or any two of schwannoma, glioma, neuroﬁbroma, cataract

Source: From Ref. 7.

NF2 is a dominantly inherited condition caused by a germline inactivating NF2 gene mutation. The NF2 gene encodes a protein, termed merlin (or schwannomin), which functions as a tumor suppressor (123). Approximately half of all NF2 patients represent sporadic cases of NF2 with de novo mutations in the NF2 gene, whereas the other half of cases represent inherited mutations. The nature of the mutation is important, as there may be a clinically signiﬁcant genotype/phenotype association. In general, persons with nonsense or frameshift mutations have signiﬁcantly more tumors than those with deletions, missense mutations, and splice site mutations (124). While genetic testing is available, there are no clear mutational hotspots, and the diagnosis is most often made on clinical grounds, with mutation testing reserved for screening of presymptomatic family members. The management of NF2, after the diagnosis is established, is largely centered on the expectant management of tumors. In this regard, presymptomatic radiologic screening is an essential element of patient care in NF2, and entails yearly neurologic exams, yearly ophthalmologic exams, serial BAERs and serial MRI scans. MR imaging of the entire neuroaxis is usually obtained every other year, but may be performed more frequently once tumors have been identiﬁed (116). Since nearly all NF2 patients have or develop vestibular schwannomas, their care is primarily focused on schwannoma management (125). The mainstay of treatment is surgical, with complete resection (and preservation of hearing and balance) possible for small vestibular tumors. Large tumors are managed with close observation, with debulking and other surgical interventions considered only when brainstem or other cranial nerve involvement is imminent. Hearing loss is a major morbidity associated with

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NF2, and may be present at the time of diagnosis. The use of hearing aids, cochlear implants, and brainstem implants is associated with limited success, but once hearing loss is severe, patients must utilize other communication modalities, such as lip reading and sign language. Importantly, another critical element of management with NF2 is eye care. As mentioned, there is a high incidence of cataracts, which requires early detection and prompt treatment to preserve vision (126). Patients with NF2 almost always have multiple CNS tumors in addition to bilateral vestibular schwannomas. Management of these tumors is based on the size, location, and their appearance on neuroimaging. Since the vast majority of these tumors are slow growing and not clinically detrimental, many are followed by serial MRI studies. Clinically signiﬁcant tumors can be treated with surgery. Unfortunately, the success of surgical treatment is often variable and is frequently associated with treatment-related comorbidities. In addition, there is no cure for NF2, and there are no speciﬁc chemotherapeutic agents currently available that are uniquely effective for the many tumor types arising in individuals with NF2. Moreover, the complex nature of NF2 requires a multidisciplinary approach at a specialized clinical center for NF (127). In this regard, early recognition and prompt referral of patients to such centers is associated with improved patient outcome. REFERENCES 1. Evans DGR, Huson SM, Donnai D, Neary W, Blair V, Newton V, Harris R. Clinical study of type 2 neuroﬁbromatosis. Q J Med 1992; 84:603–618. 2. Menkes JH, Maria BL. Neurocutaneous syndromes. In: Menkes JH, Sarnat HB, eds. Child Neurology. Philadelphia: Lippincott 2000:859–884. 3. Akenside M. Observations on cancers. Med Trans Roy Coll Phys Lond 1768; 1:64–92. 4. Smith R. Pathology, diagnosis and treatment of neuroma. Dublin: Dublin, Hodges & Smith, 1849. 5. Von Recklinghausen FD. Ueber die multiplen Fibrome der Haut und ihre Beziehung zu den multiplen Neuromen. Berlin: August Hirschwald, 1882. 6. Huson SM, Compston DA, Clark P, Harper PS. A genetic study of Von Reckinghausen neuroﬁbromatosis in Southeast Wales. I: Prevalence, ﬁtness, mutation rate, and effect of parental transmission on severity. J Med Genet 1989; 36:704–711. 7. Friedman JM. Epidemiology of neuroﬁbromatosis type 1. Am J Med Genet 1999; 89:1–6. 8. Stephens K, Kayes L, Riccardi VM, Rising M, Sybert VP, Pagon RA. Preferential mutation of the neuroﬁbromatosis type 1 gene in paternally derived chromosomes. Hum Genet 1992; 88:279–282. 9. Reynolds RM, Browning GGP, Nawroz I, Campbell IW. Von Reckinghausen’s neuroﬁbromatosis: neuroﬁbromatosis 1. Lancet 2003; 362:1552–1554. 10. Victor M, Ropper A, eds. Adams and Victor’s Principles of Neurology. 7th ed. New York: McGraw-Hill, 2001.

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24 Genetics of Parkinson’s Disease Sathya R. Sriram Institute for Cell Engineering and Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.

Valina L. Dawson Departments of Neurology, Neuroscience, and Physiology and Program in Cellular and Molecular Medicine, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.

Ted M. Dawson Departments of Neurology and Neuroscience and Program in Cellular and Molecular Medicine, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.

INTRODUCTION Parkinson’s disease (PD), ﬁrst described by James Parkinson in 1817 (1), is the second most common neurodegenerative disease in the United States. This debilitating movement disorder has an estimated prevalence of more than 0.1% of the population older than 40 years of age (2). The classical symptoms of patients with PD include bradykinesia, rigidity, involuntary tremors and postural instability, and progress towards a state of incapacitation and occasionally dementia over years (3). Neuropathologically, PD is characterized by a signiﬁcant preferential loss of dopaminergic neurons from the substantia nigra pars compacta. Concomitant with the nigral damage is extensive pathology in other areas of the brain such as the locus coerulus, cerebral cortex, thalamus, amygdala and basal forebrain (4). The resultant obliteration

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of the catecholaminergic neurotransmitters, such as dopamine, required for movement manifests as an impaired parkinsonian phenotype. Another pathological hallmark of PD is the presence of dystrophic neurites (Lewy neurites) and eosinophilic inclusions (Lewy bodies), comprised of a halo of radiating ﬁbrils and a less-deﬁned core (5,6). However, the identiﬁcation of some familial PD cases that lack Lewy bodies challenges the classically held belief that these cytoplasmic inclusions are a pathognomonic feature of the disease (7). PD is primarily a sporadic disease with no known etiology. Hypotheses on the causes for the observed pathology have focused on environmental factors such as oxidative stress (8), xenobiotic metabolism (9), and mitochondrial dysfunction (10). Until about a decade ago, PD was thought to have little or no genetic component as the extended pre-clinical phase made segregation of family history difﬁcult. Further, concordance between siblings was deemed insigniﬁcant when using solely clinical criteria to deﬁne PD (11). More accurate imaging techniques such as PET studies to reveal the number of dopaminergic neurons in the substantia nigra has subsequently resulted in a signiﬁcant concordance between twins (12). Segregation analysis and epidemiological studies of PD patients provide further evidence for a genetic component for PD (13,14). Over the past few years, the identiﬁcation of several genes for monogenically inherited forms has revolutionized the ﬁeld of PD research. Although the familial forms of PD may account for less than 10% of all cases of PD, the study of these rare genes has provided considerable insight into the pathogenesis of PD. This chapter reviews the different genes implicated in the rare Mendelian forms of PD. GENES LINKED TO FAMILIAL PD At least ten distinct loci have been linked to PD (Table 1)—ﬁve of the genes have been identiﬁed and four of these have been clearly linked to PD. The ﬁrst locus, PARK1, linked to an autosomal dominant (AD) form of familial PD, was mapped to the long arm of human chromosome 4 in 1996 and was identiﬁed subsequently as the gene encoding the presynaptic protein, a-synuclein (15,16). The second PD locus, PARK2, for an autosomal recessive (AR) form of juvenile parkinsonism (ARJP), was localized to human chromosome 6 (17). By positional cloning, the gene responsible for ARJP in Japanese patients was identiﬁed as parkin (18). The third locus, PARK 7, results from mutations in DJ-1 and is responsible for a minority of ARJP cases described (19–22). A missense mutation in the ubiquitin carboxyterminal hydrolase L1 (UCH-L1) gene at the PARK5 locus was described in a German family with AD PD (23); however, since the mutation has not been found in any other family as yet and linkage analysis was not used for identiﬁcation, it is unclear whether this is a pathogenic mutation (24). The PARK6 locus (1p35–36) was identiﬁed in several European families with

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Table 1 Loci Linked to Familial PD Locus

Chromosomal location

Genes

PARK1 PARK2 PARK3 PARK5 PARK6 PARK7 PARK8 PARK9 PARK10

4q21.3 6q25.2-q27 2p13 4p14 1p35–36 1p36 12p11.2-q13.1 1p36 1p32

a-synuclein Parkin Unknown UCHL-1 PINK1 DJ-1 Unknown Unknown Unknown

PARK11

2q36–37

Unknown

Mode of inheritance AD AR AD AD AR AR AD AR Late-onset susceptibility AD

Reference (15,16) (17,18) (27,28) (23) (25,26) (19–22) (29,115) (30) (31,116) (32)

Abbreviations: AD, autosomal dominant; AR, autosomal recessive.

AR early-onset parkinsonism and recently mapped to the PINK1 gene (25,26). Five other loci (8–11) have been mapped by linkage analysis to familial forms of PD, but the genes that are mutated in these cases are yet to be identiﬁed (27–32). PARK1: a-Synuclein a-synuclein is a 140-amino acid protein that belongs to a family of three highly homologous synucleins (designated a-, b- and g-synuclein) made from three different genes (33). a-synuclein contains imperfect repeats of KTKEGV in the amino-terminal half that have the potential to form amphipathic a-helices, a hydrophobic region, and an acidic carboxy-terminal region (33). This small protein is abundantly expressed in many parts of the brain and localizes primarily to presynaptic nerve terminals (34,35). The physiologic function of a-synuclein is unknown, although it has been suggested that it may have a role in synaptic plasticity. Mutations in the a-synuclein gene appear to be a very rare cause of PD (Fig. 1A) (36). The ﬁrst mutation identiﬁed in a-synuclein was an A53T mutation resulting from a G!A transition at nucleotide position 209 in a large kindred of Italian descent (16). Subsequently, this mutation was identiﬁed in a number of Greek kindreds as well (37,38). Haplotype analyses showing allele sharing in a small region surrounding the a-synuclein gene in families carrying the A53T mutation suggests a founder effect (37). A second mutation, A30P, resulting from a G!C transition at nucleotide position 88 was identiﬁed in a small German kindred (39). Further, signiﬁcant differences in haplotypes between PD cases and controls indicate that genetic variability

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Figure 1 Schematic representation of known pathogenic mutations in genes linked to familial PD. Point mutations (!) that result in amino acid changes in the proteins, ), duplications (———) and triplications (&&&&&) of as well as deletions ( exons (not to scale) that have been identiﬁed for (A) a-synuclein, (B) parkin, (C) PINK1 and (D) DJ-1 in PD patients are indicated.

in the a-synuclein gene may be a risk factor for the development of PD (40). Gene triplication of the a-synuclein gene has been associated with early-onset familial PD in two independent families with cornu ammonis 2/3 (CA2-3) hippocampal neuronal loss as a deﬁning pathological feature (41,42). A third

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point mutation, E46K, in a-synuclein has been recently discovered in a Spanish family with AD parkinsonism with dementia (43). The clinical features of individuals with a-synuclein mutations mirror those of idiopathic PD, including the presence of Lewy bodies and some cognitive impairment, although the age of onset is somewhat lower and there is rapid disease progression (44). The resemblance, both clinically and pathologically, of this rare form of familial PD to the more frequent sporadic form, highlights the importance of studying a-synuclein’s role in causing PD. Immunohistochemical data showing that phosphorylated and unphosphorylated a-synuclein appear to be one of the principal components of Lewy bodies in both the sporadic and familial forms of the disease further validates the signiﬁcance of this molecule in PD (45–47). a-synuclein inclusions have also been described in other neurodegenerative disorders with parkinsonian phenotypes (48). Several model systems have been studied to understand the pathogenic role for a-synuclein in PD patients. The ﬁrst a-synuclein transgenic mice, over-expressing the wild-type human a-synuclein gene, had progressive accumulation of a-synuclein and ubiquitin-immunoreactive inclusions in neurons along with selective dopaminergic terminal loss in the basal ganglia and motor impairments (49). Several other transgenic mouse models showed that the mutant a-synuclein causes signiﬁcantly greater neurotoxicity in vivo, associated with appearance of detergent insoluble aggregates of a-synuclein in the brain (50). To address the question of selective neuronal vulnerability, transgenic mice over-expressing mutant and wild-type a-synuclein under the control of the rat tyrosine hydroxylase promoter were developed. Early studies in these mice showed accumulation of a-synuclein in the substantia nigra neurons but no neurodegeneration or motor deﬁcits (50,51). Subsequently, however, overexpression of human a-synuclein in nigrostriatal terminals resulted in increased density of the dopamine transporter, enhanced toxicity to the neurotoxin MPTP, and age-related declines in motor coordination (50,51). To further study the role of selective neuronal vulnerability in the substantia nigra, mutant and wild-type a-synuclein were over-expressed in rat using lentiviral and adenoassociated viral vectors. Selective loss of dopaminergic neurons in these animals was observed associated with denervation of the dopaminergic input to the striatum and correlated with the appearance of abundant a-synuclein-positive inclusions and extensive neurite pathology (50–52). Extension of this model to small primates has produced some promising preliminary observations including a-synuclein-positive inclusions concomitant with a 40–75% loss of tyrosine hydroxylase-positive neurons in the substantia nigra and motor impairments (52). While none of the mammalian transgenic models fully recapitulate PD, they have been useful for studying synucleinopathy-induced neurodegeneration. A comprehensive list of known a-synuclein transgenic animal models has been compiled allowing for comparison between the various models (53). Transgenic Drosophila models of a-synuclein-induced PD by over-expressing mutant and wild-type a-synuclein throughout the

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brain develop a progressive and selective degeneration of dopaminergic neurons with ﬁbrillar inclusions as well as a locomotor disorder compatible with a human PD phenotype (54). These transgenic animal models support the hypothesis that a-synuclein-induced PD is caused by a gain-of-function (or over-expression of the wild-type protein) versus a loss-of-function mutation. This hypothesis is further strengthened by the recent discovery of parkinsonism in a family with an a-synuclein gene (originally, PARK4) triplication, suggesting that an increased dosage of a-synuclein maybe the cause of PD in this family (55). While in most transgenic models the development of ﬁbrillar a-synucleinpositive inclusions is associated with neurodegeneration, one line of mice has motoric impairments and loss of dopamine terminals even in the presence of non-ﬁbrillar a-synuclein inclusions (49). This observation raises the possibility that the transient a-synuclein protoﬁbrils, an intermediate in the ﬁbrillization process, may be pathogenic (56). Further, an a-synuclein-dopamine adduct, formed under oxidizing conditions, stabilizes these protoﬁbrils, providing a plausible explanation for the dopamine-selective degeneration in PD (57). However, the absence of any neurodegeneration in transgenic mice that over-express the protoﬁbrillogenic A30P mutant a-synuclein provides some in vivo evidence that protoﬁbrillar a-synuclein may not be the primary pathogenic entity (58). Indeed, neurodegeneration is observed in transgenic mice and Drosophila only when A30P a-synuclein forms inclusions and ﬁbrils (54,59,60). Another ﬁnding in support of the notion that the ﬁbrillar form of a-synuclein is more toxic is that b-amyloid promotes a-synuclein ﬁbrillar inclusions, resulting in a more severe phenotype in double transgenic mice overexpressing mutant human b-amyloid precursor protein and a-synuclein (61). Familial-associated mutants of a-synuclein have a greater propensity to aggregate than wild-type a-synuclein, resulting in the development of PD (11,61). Thus, ﬁbrillization and aggregation of a-synuclein may play a role in neuronal dysfunction and death in PD. Finally, mitochondrial inhibitors, such as MPTP and rotenone, as well as proteasome inhibitors induce the formation of a-synuclein-positive inclusions (62,63), the latter being especially interesting in the light of other PD genes (PARK2 and PARK5) that encode proteins in the ubiquitin–proteasome degradation pathway. PARK2: Parkin Linkage analysis of 13 Japanese families with ARJP localized the gene (PARK2) for the most common form of familial PD to chromosome 6q25.2-27 (17). The gene encodes a 465-amino acid protein called parkin, which shows moderate homology to ubiquitin at the N-terminus and contains two RING-ﬁnger motifs, named for Really Interesting New Gene, and an

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in between RING ﬁnger (IBR) domain at the C-terminus (18,64,65). This domain motif is common to several E3 ligases, enzymes that catalyze the conjugation of activated ubiquitin to target proteins for degradation via the proteasome. Polyubiquitination requires the ATP-dependent activation of an E1 ubiquitin-activating enzyme and an E2 ubiquitin-conjugating enzyme that covalently attaches an activated 8 kDa ubiquitin molecule to a protein substrate. An E3 ubiquitin ligase, which is responsible for providing speciﬁcity to ubiquitin conjugation, brings the E2 enzyme with the activated ubiquitin in close proximity to the substrate and facilitates the transfer of ubiquitin. E3s also catalyze the formation of chains of ubiquitin molecules on the substrate, targeting it for degradation by the 26S proteasome. While ubiquitin chains formed by linkage through the lysine residue at position 48 in the ubiquitin molecule signal degradation, linkage through other lysine residues (at position 29, 33 and 63) serves as a signal for different cellular processes. Parkin functions as an E2-dependent E3 ubiquitin protein ligase in the ubiquitin–proteasome degradation pathway (64–66). It appears to use both UbcH7 and UbcH8 as its E2s, as well as the ER-associated Ubc6 and Ubc7 (67). A wide variety of mutations have been described in parkin ranging from point mutations and frameshifts to complex DNA rearrangements, including deletions and multiplications of complete exons (Fig. 1B). Although parkin mutations predominantly cause AR parkinsonism, the described mutations cover a broad range of phenotypes and inheritance patterns. While most of the 40 different point mutations that have been identiﬁed are homozygous, a number of compound heterozygous and heterozygous mutations have also been reported, leading to much debate in the ﬁeld about the clinical consequence of carrying only a single parkin mutation. A number of polymorphisms in the parkin gene have also been identiﬁed that may confer increased susceptibility to (Ser167Asn and, Val 380Leu) or protection from (Arg366Trp) developing PD (68). The contribution of parkin mutations or polymorphisms to late-onset PD may be in the form of susceptibility alleles and cannot be completely dismissed, especially in the cases that have a family history of the disease (69,70). Clinical symptoms and PET imaging studies are relatively similar between idiopathic PD and parkin-associated PD (71). Pathologically, PARK2 brains have severe neuronal loss with gliosis in the substantia nigra and locus coeruleus (7). Parkin-associated PD is distinctly different from sporadic PD pathology in that Lewy bodies are lacking implying that parkin may be necessary for the formation of these inclusions. However, Lewy body pathology has been documented in one patient with a compound heterozygous mutation in the parkin gene (deletion of 40bp in exon 3 and Arg275Trp) (72).

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The molecular pathway by which parkin mutations result in selective dopaminergic cell loss remains elusive. In an effort to study the effect of parkin dysfunction, a number of animal models have been created, primarily by disrupting the parkin gene. The ﬁrst published model was in Drosophila, where parkin-null mutants showed reduced lifespan, locomotor deﬁcits with mitochondrial pathology and male sterility (73). Two mouse models have been published since that show normal brain morphology and no aberrant dopaminergic cell morphology, but both groups found neurochemical abnormalities that may affect dopamine release/transmission (74,75). A third parkin knockout mouse model shows signiﬁcant cell loss in the locus coeruleus along with reduced levels of norepinephrine (76), highlighting the importance of this brain area in PD pathology. Recent proteomic analysis of the brains of parkin knockout mice reveals changes in levels of proteins involved in mitochondrial function or oxidative stress, which suggest a role for parkin in normal mitochondrial function (77). As the loss of E3 ubiquitin ligase activity in parkin may cause PD, it is logical to evaluate the protein substrates of parkin since dysfunction of the proteasomal processing of parkin substrate(s) may lead to an abnormal accumulation of the substrate and subsequent dopaminergic cell loss. The ﬁrst substrate identiﬁed was the synaptic vesicle-associated protein CDCrel-1, which has a suggestive role in synaptic vesicle release (64). Synphilin-1, which interacts with a-synuclein and is present in Lewy bodies, is also ubiquitinated by parkin (78,79). To date, six other parkin substrates have been identiﬁed— p38 subunit of the aminoacyl-tRNA synthetase, Parkin-associated endothelin receptor-like receptor (Pael-R), glycosylated a-synuclein, synaptotagmin XI, cyclin E and b-tubulin—a high number for any E3 ligase, which usually dictates substrate speciﬁcity in the ubiquitin-degradation pathway (80–85). A true parkin substrate would accumulate in the absence of parkin, unable to be targeted for degradation. Further it may be possible that the parkin substrate relevant to dopaminergic function is yet to be identiﬁed. Parkin has been shown to be protective under stress conditions, and such as kainate toxicity, Pael-R-induced ER stress, and a-synuclein-induced toxicity (81,84,86,87). A potential role for parkin in synaptic transmission and plasticity has also been described by virtue of its association with post-synaptic density complexes (88). A very interesting post-translational modiﬁcation of parkin has been recently reported that may explain the single parkin heterozygous mutations associated with ARJP-S-nitrosylation of parkin under nitrosative stress conditions inhibit both parkin’s ubiquitination activity and its protective function (89). The deleterious effect of a single heterozygous mutation that results in haploinsufﬁciency could be enhanced in the presence of environmentally induced nitrosative stress, thus accounting for the association of these mutations with PD. The mechanisms leading to neurodegeneration, cell death, and disease in parkin-associated PD are not yet well understood. Since parkin-associated

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PD is largely characterized by an absence of Lewy bodies, there may be a direct relationship between the formation of these cytoplasmic inclusions and the parkin protein that is yet to be revealed. On the other hand, parkin’s function as an E3 ligase may have an impact on protein levels and stability that results in an increased susceptibility of dopaminergic neurons. Further research is warranted to understand the role of parkin in familial and, perhaps, sporadic PD. PARK5: UCHL-1 In a German family with an AD form of PD, a missense mutation (I93M) was identiﬁed in the ubiquitin C-terminal hydrolase (UCHL-1) gene at the PARK5 locus (23). The group also reported a partial loss of catalytic activity in the enzyme in vitro, implying that an aberrant proteasome degradation pathway may be affected. This was particularly exciting with the discovery of parkin, an E3 ubiquitin ligase, strongly linking the proteasome pathway to PD. However, the I93M mutation does not inﬂuence the risk of idiopathic PD (90). A protective polymorphism, S18Y, has been described that is also controversial (91,92). Extensive searches for more mutations in the UCHL-1 gene and sporadic PD cases with UCHL-1 mutations have not yielded any positive results, indicating that if this is truly a PD gene, then it is a very rare cause (93). PARK6: PINK1 The PARK6 locus (1p35–36) was identiﬁed by linkage analysis and haplotype construction in a large family from Sicily with AR early-onset parkinsonism (25). Subsequently, the identiﬁcation of additional European families with linkage to PARK6 reduced the candidate interval from 12.5 to 9 cM (94,95). Fine mapping of the locus in three families deﬁned a 2.8 Mb region of homozygosity, containing approximately 40 genes. Bioinformatics analysis and exon ampliﬁcation of candidate genes led to the association of PINK1 (PTEN-induced kinase 1) with the PARK6 locus (26). The PINK1 gene contains eight exons that encode a 581 amino acid protein, which has a mitochondrial targeting motif and a highly conserved protein kinase domain sharing homology with the serine/threonine kinases of the Ca2þ/Calmodulin family. Two homozygous mutations in the PINK1 kinase domain have been identiﬁed in three consanguineous PARK6 families—a truncating nonsense mutation W437OPA and a missense mutation at a highly conserved amino acid G309D (Fig. 1C) (26). These mutations could potentially affect the kinase activity or substrate recognition leading to a pathogenic phenotype. It is hypothesized that there may be a link between mutations in PINK1 and mitochondrial dysfunction that needs to be further explored (26).

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PARK7: DJ-1 The PARK7 locus, localized to chromosome 1p36, was identiﬁed by homozygous mapping in a genetically isolated family with several consanguinity loops affected by early-onset PD (22). In 2003, Bonifati et al. (19,20) identiﬁed a second family of Italian origin with autosomal recessive earlyonset PD and eventually linked mutations in DJ-1 to the PARK7 locus. First cloned as an oncogene product, this multi-functional protein was subsequently identiﬁed as a regulatory subunit of an RNA-binding protein, an infertility-related protein as well as a modulator of the androgen receptor via sumoylation at lysine 130 (96–99). Recent studies on the crystal structure of DJ-1 have shown that the protein may function as a dimer (100–102). DJ-1 has also been shown to be a hydroperoxide-responsive protein, with a modiﬁed pI under stress conditions induced by H2O2 or paraquat, indicating that it may function as an oxidative stress indicator (103,104). This is especially interesting since oxidative stress and damage have been implicated in the death of neurons and in the pathogenesis of PD (105). A number of mutations in DJ-1 have been described including base changes, deletions and splicing mutations (Fig. 1D) (106). The ﬁrst identiﬁed mutation was a large homozygous genomic deletion leading to an absence of the gene product, while the second identiﬁed mutation was a homozygous L166P point mutation (19,22). Another homozygous mutation, M26I, results in a missense product. A heterozygous single base deletion and a base change in exon 2, c.56delC and c.57G!A, results in a truncated protein, and a base change in IVS6-1G!C leads to an aberrant RNA because of defective splicing (107). Recently another point mutation, E64D, was found in a small kindred of Turkish ancestry (108). Three heterozygous mutations in DJ-1— D149A, R98Q, and A104T—have also been documented; however, it is unclear whether these mutations confer susceptibility to the disease by themselves or cause disease synergistically with other unknown factors (109). The frequency of pathogenic DJ-1 mutations is very low in PD (110). Mutations in DJ-1 have been linked to AR early-onset PD. The clinical symptoms are similar to that seen in parkin-linked PD except for foot dystonia and sleep beneﬁt (111). Ongoing research on the properties of mutated DJ-1 protein that may lead to PD has revealed that the L166P mutation reduces the stability of the protein by rendering it more susceptible to degradation and abolishes its ability to dimerize (112). Co-localization of DJ-1 with tau inclusions in tauopathies has deﬁned a link between DJ-1-linked PD and other neurodegenerative diseases (113). Further, DJ-1’s ability to function as an antioxidant and/or a chaperone may be disrupted in the disease state. Studying the effects of a knockdown or knockout of the DJ-1 protein in in vivo models would be an interesting approach to elucidate the pathogenic mechanism in PD.

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Other Genes and Loci Linked to PD A susceptibility locus for late-onset autosomal dominant PD in a group of European families was mapped to chromosome 2p13 (28). The PARK3 locus has subsequently been reported to be in linkage disequilibrium with a mutation inﬂuencing age at onset of PD (27). More recently, another study of North American PD patients suggests strong evidence for a gene at the PARK3 locus that contributes to both PD susceptibility and age at onset (114). The PARK8 locus was identiﬁed in a genome wide linkage analysis of a Japanese family with AD parkinsonism (29). Although the clinical features were compatible with sporadic PD, cases with and without Lewy body pathology were observed. Additionally, a recent study linked two Caucasian families with AD PD to this same region on chromosome 12 (115). Kufor– Rakeb syndrome is an early onset, AR nigro-striatal-pallidal-pyramidal neurodegenerative disorder with clinical features including PD. Linkage in patients with this disorder to a 9 cM region of chromosome 1p36 that is distinct from the 1p35-36 locus was reported and designated as PARK9 (30). A population-wide study of Icelandic late-onset PD patients revealed linkage to a region on chromosome 1p32, assigned PARK10 (31). Signiﬁcant evidence of linkage to the same area was found in a different study with families from the US and Australia, contributing to both disease susceptibility as well as age at onset (116). Variation in a gene on chromosome 2q36-37 (PARK11) was linked to PD susceptibility, with suggestive epistatic or additive interaction between parkin and this locus (32). Although the mechanisms underlying the neurodegeneration in PD are not well understood, considerable evidence suggests that genetic factors may inﬂuence susceptibility to the disease. In addition to linkage analysis studies, numerous association studies have been reported that use either single nucleotide polymorphisms or repeat polymorphisms on genetic markers. Association studies are more useful than linkage analysis for diseases attributable to multiple genes with small effects. Since PD is clearly a multi-factorial disease, the simplicity of association studies has resulted in their frequent use to investigate various candidate genes and proteins, including those involved in dopamine metabolism (monoamine oxidases, dopamine transporter, dopamine receptors, catechol-O-methyltransferase, and tyrosine hydroxylase); detoxiﬁcation of xenobiotics and other metabolites (cytochrome-P450 enzymes, N-acetyltransferase 2, glutathione transferase, human heme oxygenase 1, and manganese superoxide dismutase); lipoproteins (APOE-e4), mitochondrial-associated genes (mitochondrial tRNAGlu, mitochondrial DNA, and subunits of Complex I) and other putative genes (tau, synphilin-1, nuclear receptor related-1, angiotensin-converting enzyme, alpha 1-antichymotrypsin, and brain derived nerve growth factor) (117). However, no consistent ﬁndings have emerged and no speciﬁc gene polymorphism has been unequivocally associated with PD as yet. Two

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heterozygous mutations have been identiﬁed in the nuclear receptor related-1 (NR4A2) gene in 10 out of 107 individuals with familial PD (118). A transcription factor with a role in the differentiation and maintenance of dopaminergic neurons, NR4A2 is an attractive candidate gene for PD, but the search for mutations has been unsuccessful so far, indicating that the contribution of NR4A2 mutations to familial PD, if any, is low (119,120). An association between tau and frontotemporal dementia and parkinsonism (FTDP) has been proposed with the identiﬁcation of mutations in exon 10 of the tau gene. Four families with FTDP have an asparagine to lysine mutation at position 279 of tau (121). In addition, two independent groups have observed a heterozygous deletion of a highly conserved asparagines residue at position 296 linked to probable sporadic PD (122). Mutations in the exon 10 of tau results in both a splicing and functional deficit, leading to reduced microtubule assembly and increased tau aggregation in these patients. Co-incubation of tau and a-synuclein synergistically promotes the ﬁbrillization of both proteins (123). Amyloid-like a-synuclein and tau ﬁlamentous inclusions occur in PD as well as transgenic a-synuclein models suggesting an interaction between these two proteins, which promotes their ﬁbrillization and drives the formation of pathologic inclusions. Select polymorphisms in the tau gene may also be associated with the pathogenesis of PD; however, the mechanism of susceptibility remains unknown (124,125). One of the more widely studied PD susceptibility candidate genes is cytochrome P450 2D6 (CYP2D6), an enzyme involved in the biotransformation of various chemicals including MPTP. It is hypothesized that people with a signiﬁcantly reduced enzyme activity due to polymorphisms in the CYP2D6 gene are genetically susceptible to PD because of an impaired ability to detoxify xenobiotics and toxins (117,126). Yet another suspected risk for PD is a deﬁciency in the activity of complex I in the mitochondrial respiratory chain, as reduced activity has been reported in a proportion of PD patients (127). Mitochondrial DNA encodes some subunits of complex I and a high rate of mutations has been observed in mitochondrial DNA of PD patients in addition to one family having a matrilineal inheritance of the disease with complex I dysfunction (128). No speciﬁc mutations have been identiﬁed in complex I as yet, although a number of environmental toxins, including MPTP and rotenone, have been implicated in causing complex I deﬁciency and thus may result in PD (126). Signiﬁcant associations with PD were found in polymorphisms of N-acetyltransferase 2 (NAT2), monoamine oxidase B (MAOB), glutathione transferase (GSTT1), and mitochondrial tRNAGlu. While this signiﬁcant association does not imply a causal relationship between the presence of the polymorphism and the pathogenesis of PD, it certainly warrants further study (117).

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Recently, serial analysis of gene expression (SAGE) was used along with linkage analysis in an interdisciplinary approach to identify susceptibility genes for complex diseases like PD (129). Some 400 substantia nigra genes have been identiﬁed within ﬁve large genomic linkage regions that represent excellent candidates for PD susceptibility alleles and further analyses. CLINICAL TESTING FOR GENETIC FORMS OF PD As the phenotype of genetic forms of PD generally matches that of sporadic PD, diagnosis of PD remains clinical. Genetic forms of PD are rare, with all of the loci except for PARK2 (parkin) being implicated in less than 10

Figure 2 Converging model for dopaminergic cell death in PD. Four proteins – a-synuclein, parkin, PINK1 and DJ-1 – have strong genetic evidence linking them to PD. These proteins, individually involved in functionally different pathways, converge to a common end point, when mutated or disrupted. The mechanism by which each results in an environment selectively toxic to dopaminergic neurons is not well understood. This schematic outlines the pathways that each protein has been implicated in, both under normal conditions (!) and in the disease state ( >).

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families worldwide. While research testing is available in selected situations, clinical testing is available only for PARK2 (parkin). However, the situations in which testing for parkin mutations is useful are relatively limited. The treatment of genetic and sporadic PD is identical at this time, so that identiﬁcation of inherited forms does not convey a distinct therapy. Occasionally, the unusual presentations of PD in children (with a greater degree of dystonia) may make parkin testing diagnostically valuable in speciﬁc situations. Clinically available genetic testing can be used for genetic counseling and carrier identiﬁcation. However, as PD from parkin mutation is so uncommon and the carrier frequency is likely to be well less than 1 in 100 in North American populations, there will be limited situations where such testing is useful. At present, presymptomatic identiﬁcation of affected individuals is possible in some situations where other family members have been identiﬁed but must be performed with appropriate genetic counseling (and never in children). Future identiﬁcation of protective therapies could change this perspective.

CONCLUSION Molecular genetic analysis has led to signiﬁcant progress in understanding the cause of familial PD cases with Mendelian inheritance. However, PD is a largely sporadic disease, and the genetic contribution to non-Mendelian PD is controversial. A growing number of susceptibility genes have been documented that may increase the risk for developing PD and assist in generating hypotheses on the etiology of this complex disease. The enormous number and inconsistency of association studies make it difﬁcult to obtain any conclusive ﬁndings. The search for PD-linked genes has evolved from studying single families to large population-based studies, leading to the identiﬁcation of several genes that may contribute to the pathogenic mechanisms in this devastating neurodegenerative disorder. The similarity of pathological presentation and the identiﬁcation of genes that normally function in overlapping pathways suggest a common end pathway of degeneration in the different forms of PD. The challenge in the ﬁeld lies in investigating the role of the numerous genes and loci identiﬁed so far (and those yet to be identiﬁed) to determine the missing links of this complex genetic puzzle (Fig. 2). REFERENCES 1. Parkinson J. An essay on the shaking palsy. 1817. J Neuropsychiatry Clin Neurosci 2002; 14(2):223–236 (Discussion 2). 2. Siderowf A, Stern M. Update on Parkinson disease. Ann Int Med 2003; 138(8):651–658.

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110. Healy DG, Abou-Sleiman PM, et al. DJ-1 mutations in Parkinson’s disease. J Neurol Neurosurg Psychiatry 2004; 75(1):144–145. 111. Riess O, Kruger R, Schulz JB. Spectrum of phenotypes and genotypes in Parkinson’s disease. J Neurol 2002; 249(suppl 3):III/15–20. 112. Moore DJ, Zhang L, Dawson TM, Dawson VL. A missense mutation (L166P) in DJ-1, linked to familial Parkinson’s disease, confers reduced protein stability and impairs homo-oligomerization. J Neurochem 2003; 87(6):1558–1567. 113. Rizzu P, Hinkle DA, et al. DJ-1 colocalizes with tau inclusions: a link between parkinsonism and dementia. Ann Neurol 2004; 55(1):113–118. 114. Pankratz N, Uniacke SK, et al. Genes inﬂuencing Parkinson disease onset: replication of PARK3 and identiﬁcation of novel loci. Neurology 2004; 62(9):1616–1618. 115. Zimprich A, Muller-Myhsok B, et al. The PARK8 locus in autosomal dominant parkinsonism: conﬁrmation of linkage and further delineation of the diseasecontaining interval. Am J Hum Genet 2004; 74(1):11–19. 116. Li YJ, Scott WK, et al. Age at onset in two common neurodegenerative diseases is genetically controlled. Am J Hum Genet 2002; 70(4):985–993. 117. Tan EK, Khajavi M, Thornby JI, Nagamitsu S, Jankovic J, Ashizawa T. Variability and validity of polymorphism association studies in Parkinson’s disease. Neurology 2000; 55(4):533–538. 118. Le WD, Xu P, et al. Mutations in NR4A2 associated with familial Parkinson disease. Nat Genet 2003; 33(1):85–89. 119. Zimprich A, Asmus F, et al. Point mutations in exon 1 of the NR4A2 gene are not a major cause of familial Parkinson’s disease. Neurogenetics 2003; 4(4):219–220. 120. Wellenbrock C, Hedrich K, et al. NR4A2 mutations are rare among European patients with familial Parkinson’s disease. Ann Neurol 2003; 54(3):415. 121. Soliveri P, Rossi G, et al. A case of dementia parkinsonism resemblingprogressive supranuclear palsy due to mutation in the tau protein gene. Arch Neurol 2003; 60(10):1454–1456. 122. Oliva R, Pastor P. Tau gene delN296 mutation, Parkinson’s disease, and atypical supranuclear palsy. Ann Neurol 2004; 55(3):448–449. 123. Giasson BI, Forman MS, et al. Initiation and synergistic ﬁbrillization of tau and alpha-synuclein. Science 2003; 300(5619):636–640. 124. Zappia M, Annesi G, et al. Association of tau gene polymorphism with Parkinson’s disease. Neurol Sci 2003; 24(3):223–224. 125. Kwok JB, Teber ET, Loy C, et al. Tau haplotypes regulate transcription and are associated with Parkinson’s disease. Ann Neurol 2004; 55(3):329–334. 126. Foltynie T, Sawcer S, Brayne C, Barker RA. The genetic basis of Parkinson’s disease. J Neurol Neurosurg Psychiatry 2002; 73(4):363–370. 127. Warner TT, Schapira AH. Genetic and environmental factors in the cause of Parkinson’s disease. Ann Neurol 2003; 53(suppl 3):S16–S23 (Discussion S-5). 128. Swerdlow RH, Parks JK, et al. Matrilineal inheritance of complex I dysfunction in a multigenerational Parkinson’s disease family. Ann Neurol 1998; 44(6): 873–881. 129. Hauser MA, Li YJ, et al. Genomic convergence: identifying candidate genes for Parkinson’s disease by combining serial analysis of gene expression and genetic linkage. Hum Mol Genet 2003; 12(6):671–677.

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25 Alzheimer’s Disease R. Scott Turner Department of Neurology and Neurology Service and Neuroscience Program and VA Geriatric Research Education and Clinical Center, Institute of Gerontology, University of Michigan, Ann Arbor, Michigan, U.S.A.

INTRODUCTION Although age related and progressive cognitive decline has been known since antiquity, Dr. Alois Alzheimer reported the ﬁve-year clinical course of a 51-year-old woman with progressive dementia and autopsy ﬁndings of neuronal loss, neuroﬁbrillary tangles, and miliary amyloid plaques upon light microscopic examination of Bielshowsky silver-stained brain sections. Thus, Dr. Alzheimer was the ﬁrst to suggest a link, perhaps causal, between a dementing disease and these abnormal proteinaceous aggregates in brain (1). EPIDEMIOLOGY What became known as Alzheimer’s disease (AD) now afﬂicts 2–3% of individuals at age 65, with an approximate doubling of incidence for every ﬁve years of age afterward. The prevalence of AD in one study approaches 50% of those over age 85 (2). Although AD is the most common cause of dementia in the elderly in the United States, it is not inevitable with aging, and escapees warrant further epidemiologic and genetic study. In 1990, there were an estimated four million individuals in the United States with AD. Because of an expanding population and increasing life expectancy, the number of affected individuals is projected to rise to 14 million in 2050.

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CLINICAL CRITERIA The Diagnostic and Statistical Manual, 4th edition (DSM-IV) (3) or National Institute of Neurologic, Communicative Disorders and StrokeAD and Related Disorders Association (NINCDS-ADRDA) (4) criteria are used for the clinical diagnosis of AD (Table 1). These criteria are similar and require a gradually progressive dementia severe enough to impair social or occupational functioning with other etiologies, including depression and delirium, excluded (5). By deﬁnition, dementia requires a decline in memory and at least one other cognitive domain—visuospatial skills, language and calculation, praxis, gnosis, or frontal and executive function. A diagnosis of deﬁnite AD requires light microscopic examination of brain sections at necropsy, or rarely by brain biopsy. Thus, only possible and probable AD are routinely diagnosed clinically. Possible AD is diagnosed when uncertainty arises from an additional secondary etiology or the dementia has an atypical onset, course, or presentation. Diagnostic accuracies, compared to autopsy, for possible and probable AD by NINCDS-ADRDA criteria are approximately 50–60% and 80–90% respectively, at specialized centers. Confounding diagnoses of subjects who present to cognitive disorders clinics at academic medical centers are often Lewy body dementia, frontotemporal dementias, or vascular dementia—in that order.

PATHOLOGIC CRITERIA The Khachaturian pathologic criteria for AD require that the density of amyloid plaques and neuroﬁbrillary tangles in brain sections exceed a given threshold that increases with age (6). In contrast, the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) criteria focus exclusively on the density of amyloid plaques (the sine qua non pathologic marker of AD), in brain sections compared to given (published) high-power microscopic ﬁelds. Because neuroﬁbrillary tangles are not speciﬁc to AD, they were not considered essential to the diagnosis (7). The current Reagan criteria for AD, however, require both amyloid plaques and neuroﬁbrillary tangles in multiple brain regions, and declare all such pathology abnormal (8). These criteria incorporate the semi-quantitative CERAD plaque density scale (7) as well as Braak and Braak staging of the density and distribution of pathologic abnormalities in AD brain (9). Despite numerous other profound neuropathologic changes in brain, including neuronal and synaptic loss, gliosis, inﬂammation, cholinergic and other neurotransmitter deﬁcits, microvascular amyloid angiopathy, oxidative damage, and mitochondrial dysfunction, the mainstay of pathologic diagnosis remains silver staining of brain sections and light microscopic examination of the density and distribution of neuritic plaques and neuroﬁbrillary tangles in brain—in other words, methods used by Dr. Alzheimer in 1906. The neuropathology of

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Table 1 Criteria for Diagnosis of Alzheimer’s Disease Criteria for the clinical diagnosis of PROBABLE Alzheimer’s disease Dementia established by clinical examination and documented by the mini-mental test; blessed dementia scale, or some similar examination, and conﬁrmed by neuropsychological tests Deﬁcits in two or more areas of cognition Progressive worsening of memory and other cognitive functions No disturbance of consciousness Onset between ages 40 and 90, most often after age 65 Absence of systemic disorders or other brain diseases that in and of themselves could account for the progressive deﬁcits in memory and cognition The diagnosis of PROBABLE Alzheimer’s disease is supported by Progressive deterioration of speciﬁc cognitive functions such as language (aphasia), motor skills (apraxia), and perceptions (agnosia) Impaired activities of daily living and altered patterns of behavior Family history of similar disorders, particularly if conﬁrmed neuropathologically; and laboratory results of Normal lumbar puncture as evaluated by standard techniques Normal pattern or nonspeciﬁc changes in EEG, such as increased slow-wave activity Evidence of cerebral atrophy on CT with progression documented by serial observation Features consistent with the diagnosis of PROBABLE Alzheimer’s disease (after exclusion of causes of dementia other than Alzheimer’s disease) Plateaus in the course of progression of the illness Associated symptoms of depression, insomnia, incontinence, delusions, illusions, hallucinations Catastrophic verbal, emotional, or physical outbursts, sexual disorders, and weight loss Other neurologic abnormalities in some patients, especially with more advanced disease and including Motor signs (muscle tone, myoclonus, or gait disorder) Seizures in advanced disease CT normal for age Features making the diagnosis of PROBABLE Alzheimer’s disease uncertain/unlikely Sudden onset Focal neurologic ﬁndings Seizures or gait disorder early in the disease process Clinical diagnosis of POSSIBLE Alzheimer’s disease May be made based on dementia in the absences of other disorders sufﬁcient to cause dementia, and in the presence of variations in the onset, presentation or clinical course May be made when a second disorder sufﬁcient to cause dementia is present, but is not felt to be the cause of dementia Criteria for DEFINITE Alzheimer’s disease are Presence of clinical criteria for PROBABLE Alzheimer’s disease Pathological evidence form biopsy or autopsy Source: NINCDS-ADRDA Criteria; from Ref. 4.

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AD is anatomically stereotypic and ﬁrst affects entorhinal cortex and hippocampus followed by other limbic structures and neocortex (9). This is consistent with the initial amnestic clinical presentation of patients with AD followed by an average of 8–12 years of progressive decline in multiple cognitive domains leading to mutism, inanition, a vegetative state, and death. Hospice care is available but underutilized for terminal patients with dementia. As with other neurodegenerative diseases, the etiology of selective vulnerability of brain regions to AD pathologies remains obscure. RISK FACTORS The major risk factor for AD is aging. Even subjects with Down’s syndrome and individuals with genetic polymorphisms or mutations that increase risk of AD require a degree of aging before signs and symptoms commence. It is unclear, however, what speciﬁc factors associated with aging increase risk of AD. Having a ﬁrst-degree relative with AD increases risk approximately 2- to 4-fold, and this grows higher with increasing numbers of affected ﬁrst-degree relatives. These data clearly implicate genetic factors in AD pathogenesis. However, several environmental factors also affect AD risk. For example, a low level of education or a history of head trauma severe enough to induce loss of consciousness both increase risk. Conversely, advanced educational and occupational attainment may be protective. Female gender slightly increases risk of AD; this may be due to a lack of postmenopausal estrogen (10,11). Risk factors for stroke, such as hypertension, diabetes mellitus, smoking, hypercholesterolemia, and hyperhomocysteinemia may also increase risk of AD. Whether these putative risk factors act directly on AD pathogenic mechanisms, indirectly by vascular compromise (infarcts), or both is unclear (12). Down’s syndrome (trisomy 21) including translocation Down’s (21q) is clearly a risk factor for AD (13). The high prevalence of progressive dementia in individuals with Down’s syndrome led to autopsy ﬁndings of typical AD pathologies, including neuroﬁbrillary tangles and amyloid plaques, in aging brain. However, the onset of dementia typically occurs in the third to ﬁfth decade of life, and neuropathology begins even sooner. The major disease mechanism is likely a gene dosage effect since amyloid precursor protein (APP) is encoded on chromosome 21q. Cells from Down’s syndrome subjects express about 1.5 times the normal level of APP, and thus secrete higher levels of Ab derived from APP by the proteases b- and g-secretase (Fig. 1). Ab peptides, including Ab40 and Ab42, spontaneously alter their conformation to become insoluble neurotoxic aggregates, and are the major component of amyloid plaques in AD brain (14). Although BACE-2 is also encoded on chromosome 21, the b-secretase active in brain (BACE-1) is encoded on chromosome 11.

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Figure 1 Amyloid precursor protein (APP) processing. APP is a type I transmembrane protein that may be cleaved either by a- and g-secretases to release p3 peptides and a large N-terminal ectodomain (APPsa) or by b- and g-secretases to release Ab40 or Ab42 and a large N-terminal ectodomain (APPsb). The latter is referred to as the amyloidogenic pathway. While the generic terms a-, b-, and g-secretase remain in use, the proteases are all now molecularly identiﬁed. BACE1 is b-site APP cleaving enzyme 1. The a-secretases are members of a disintegrin and metalloprotease family (ADAM10 and ADAM17/TACE, or tumor necrosis factor-a converting enzyme). While p3 peptides are relatively benign, Ab peptides are the major component of amyloid plaques in AD brain. The g- or e-cleaved C-terminal fragment of APP (gCTF, or APP intracellular domain, AICD) may translocate to the nucleus to regulate gene transcription (analogous to the g-cleaved Notch1 fragment NICD). APP and Notch1 are two members of a family of transmembrane proteins sequentially cleaved by a- or b- and then g-secretases to mediate cell signaling.

GENETICS OF FAMILIAL AD Mutations in Amyloid Precursor Protein Mutations Causing Alzheimer’s Disease Like many other human diseases, the identiﬁcation and analysis of probands and pedigrees with rare genetic forms of a far more common sporadic

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disease proved to be informative with regard to disease causation and development of potential therapeutic strategies. Thus, much has been learned about the putative cause of AD by the study of rare patients with early onset familial AD (OMIM #104300) (14). There are now 158 known familial AD mutations in three genes in 326 families. Other than pedigree analyses showing early onset (presenile) highly penetrant autosomal dominant patterns of inheritance, these genetic forms of AD are similar, both clinically and pathologically, to the overwhelming majority (>95%) of patients with sporadic (senile) AD. Thus, proposed pathogenic mechanisms of familial AD may be extrapolated to sporadic AD, and vice versa. The most aggressive earliest onset forms of familial AD, however, may also cause myoclonus, seizures, and spastic paraparesis—signs and symptoms atypical in patients with sporadic AD. The ﬁrst mutations discovered in familial AD were missense mutations in APP. Not coincidentally, these mutations cluster near the b- and g-cleavage sites that release Ab from APP (Table 2). The location of these APP mutations suggested a disease mechanism favoring amyloidogenic (producing Ab) over nonamyloidogenic APP catabolism (a toxic gain of function). For example, a double missense mutation (K670N/M671L in the APP770 isoform) near the b-cleavage site promotes Ab40 and Ab42 generation by enhancing BACE-1 enzyme kinetics. In contrast, any one of several single missense mutations near the g-cleavage site (APP T714I or A, V715M or A, I716V or T, V717I, G, F, or L, and L723P) speciﬁcally promotes Ab42 secretion. Ab42 is more spontaneously amyloidogenic than Ab40, again suggesting a disease mechanism. The identiﬁcation of these APP mutations led to the notion that the progressive accumulation and deposition of Ab/amyloid in brain causes AD—the amyloid hypothesis (Fig. 2)—and allowed the generation of human APP transgenic mice that develop age-dependent behavioral decline in learning and memory and progressive CNS Ab/amyloid accumulation and deposition (15,16). However, these mice do not develop neuroﬁbrillary tangles, loss of synaptic cholinergic markers, or neuronal loss, making them at best partial AD-like models of human disease (17). Mutations Causing Cerebral Amyloid Angiopathies and/or Dementia Pathogenic APP mutations within the Ab sequence usually result in a different phenotype, and present clinically with a combination of lobar hemorrhagic strokes or microvascular ischemic strokes and/or progressive dementia (OMIM #104760). The pattern of inheritance is also autosomal dominant with a high degree of penetrance. Pathologically, these disorders are characterized by a much greater Ab/amyloid burden within blood vessel walls in addition to parenchymal deposits as found with AD. Pathogenic missense mutations are known within the Ab sequence at positions 692,

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Table 2 Amyloid Precursor Protein (APP) Mutations Causing Alzheimer’s disease K670N/M671L D678N T714I T714A V715M V715A I716V I716T V717I V717G V717F V717L L723P Causing cerebral amyloid angiopathy and/or dementia A692G E693Q E693K E693G D694N APP cleavage sites

1 1 1 1 1 1 1 1 1 1 1 1 1

NL I A M A V T I G F L P

1 1 1 1 1 b

G Q K G N aa

770 770 770 770 770 770 770 770 770 770 770 770 770

770 770 770 770 770 gg e

Note: The sequential activities of b- and g-secretases on human APP (the APP770 isoform numbering is shown here) release Ab peptides (in gray). A double missense mutation at the b-cleavage site promotes Ab40 and Ab42 secretion. Several missense mutations near the g-cleavage sites speciﬁcally promote Ab42 secretion. Mutations causing a mix of cerebral amyloid angiopathy and/or dementia cluster near the a-cleavage site. The e-cleavage is also mediated by the presenilin/g-secretase complex to release the g-cleaved C-terminal fragment (gCTF) also known as the APP intracellular domain (AICD).

693, and 694 near the a-cleavage site (Table 2). Because these mutations are intrinsic to Ab they may alter its propensity to form insoluble amyloid ﬁbrils and shorter protoﬁbrils. The APP A692G (Flemish) mutation promotes Ab production but retards its ﬁbrillogenesis; this mutation leads to a combination of microvascular amyloidopathy and AD-like pathology, and presents with dementia and cerebral hemorrhages. The APP E693Q (Dutch) and APP D694N (Iowa) mutations promote amyloid ﬁbril formation from Ab and also present clinically with dementia and lobar cerebral hemorrhages; the APP E693K (Italian) mutation presents similarly, possibly by related mechanisms. The APP E693G (Arctic) mutation retards Ab40 and Ab42 production but enhances protoﬁbril formation and presents clinically as

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Figure 2 The amyloid cascade hypothesis of Alzheimer’s disease (AD). Ab, derived from APP, forms neuritic plaques (NP) in brain leading to neuroﬁbrillary tangles (NFT), and neuronal morbidity and mortality. A ghost tangle remains when a neuron containing a NFT dies. Genetic risks for AD (Down’s syndrome, mutations in APP, presenilin-1 or -2 (PS-1, PS-2), and the apolipoprotein E4 (ApoE4) polymorphism) are positioned proximally, implying causal roles. Although Ab/amyloid promotes NFT and Lewy body pathologies, mutations in tau or a-synuclein may cause familial frontotemporal dementias or Parkinson’s disease/Lewy body dementia, respectively. The mechanisms whereby age, female gender, and head trauma may increase AD risk are unclear. In parallel with these pathologic events in brain, cognitively normal elderly subjects become progressively amnestic and demented for 10 to 20þ years until death. MRI of the brain reveals diffuse cortical atrophy and white matter changes. Although shown as a linear cascade, several vicious cycles may become established that promote AD. Current drug treatments for cognitive and functional decline in patients with AD include cholinesterase inhibitors, the NMDA receptor antagonist memantine, and antioxidants (vitamin E, selegiline) that act on putative distal or downstream targets and thus provide only modest, palliative, and temporary beneﬁts. Potential disease-modifying treatments may prevent or treat AD by targeting more proximate events—for example, inhibiting b- or g-secretase to retard Ab generation, metal chelation to prevent Ab/amyloid deposition, and active or passive immunization to promote Ab/amyloid clearance.

AD, suggesting that protoﬁbril formation from Ab may be the unifying event in AD pathogenesis (18). Like all amyloidopathies, the b-pleated sheet conformation of Ab in amyloid plaques and blood vessel walls results in their ﬂuorescence with thioﬂavin-S staining and apple-green birefringence in Congo red-stained brain sections visualized by polarized light microscopy. Hereditary cerebral congophilic angiopathies with dementia are not limited to mutations in APP, but include other amyloidogenic proteins such as cystatin C (Icelandic) (OMIM#105150) and transthyretin (OMIM #105210). Similar to AD, other cerebral amyloidopathies may also cause progressive dementia, such as British familial dementia (OMIM #176500)

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or Danish familial dementia (OMIM #117300) caused by a point mutation or 10 base pair duplication, respectively, of the BRI gene on chromosome 13. Mutations in Presenilin-1 and Presenilin-2 Most early onset familial AD pedigrees do not have a mutation in APP, implicating other affected genes. By study of these families, mutations were identiﬁed in presenilin-1 (PS-1, for presenile dementia, or onset less than 60–65 years of age) on chromosome 14 and the homologous gene presenilin-2 (PS-2) on chromosome 1 [see Ref. (19) for a complete list of familial AD mutations]. Of the rare early onset familial forms of AD (<5%), the most commonly found mutations (84%) are in PS-1. The identiﬁcation of these mutations provided a test for the amyloid hypothesis. Do they alter APP metabolism and Ab generation? Again, studies of cells in culture, samples from affected patients (skin ﬁbroblasts, serum, etc.), and transgenic mice reveal that PS mutations speciﬁcally promote Ab42 generation from APP (a toxic gain of function) (20). Transgenic mice expressing both human mutant APP and human mutant PS-1 exhibit accelerated amyloid deposition in brain compared to transgenic mice expressing only mutant human APP. Thus, common to all familial AD mutations is an increased generation of Ab42 from APP. PS mutations may have other detrimental effects promoting AD, for example, by increasing susceptibility of neurons to apoptosis. GENETICS OF SPORADIC (LATE-ONSET) ALZHEIMER’S DISEASE Apolipoprotein E Polymorphisms Apolipoprotein E (ApoE) polymorphisms on chromosome 19 are associated with risk of sporadic late-onset AD (21). ApoE is synthesized in the liver and plays a role in lipid and cholesterol transport in lipoprotein particles in blood. In the brain ApoE is secreted by glia, with receptors on neurons. Thus, ApoE may be involved in CNS lipid and cholesterol metabolism. The three major ApoE polymorphisms—2, 3, and 4—result in six possible genotypes (Table 3). The gene frequency in the U.S. population is 3 > 4 > 2. Having one or two ApoE4 alleles increases the risk of late-onset AD and

Table 3 Apolipoprotein E (ApoE) Polymorphisms ApoE2 ApoE3 ApoE4

1 1 1

C C R

C R R

299 299 299

Note: The three major human ApoE alleles differ by encoding either C or R at amino acid residues 112 and 158 (as shown). Several mutations in ApoE (not shown) cause familial hypercholesterolemia (OMIM #107741).

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lowers the average age of onset with a gene dosage effect. Thus, the hierarchy of individual risk is ApoE4/4 > ApoE4/x > ApoEx/x. The ApoE2 allele is slightly protective, and ApoE3 is intermediate in risk. Genetic risks may be additive; for example, the ApoE4 allele lowers the age of AD onset in subjects with Down’s syndrome or with APP or PS-1 mutations. The mechanisms whereby ApoE polymorphisms affect AD risk are unknown. ApoE does not signiﬁcantly inﬂuence cellular APP metabolism. Rather, ApoE4 may promote the formation of insoluble ﬁbrillar amyloid from soluble Ab. Double transgenic mice expressing human mutant APP causing AD and human ApoE4 develop a greater amyloid burden in brain compared to mice coexpressing human ApoE3. ApoE-deﬁcient mice expressing human mutant APP develop few amyloid plaques, again suggesting a pathologic chaperone role for ApoE in Ab/amyloidogenesis. Additional mechanisms whereby ApoE4 increases risk of AD have not been excluded. The ApoE4 allele is also detrimental to subjects with a variety of brain insults including ischemic stroke, head trauma, multiple sclerosis, and non-AD neurodegenerative dementias. In fact, the ApoE2 allele is a human longevity gene. Other Genetic Polymorphisms Other than ApoE, 100þ genetic associations to sporadic AD remain controversial and often conﬂicting. Candidate genes on chromosome 12 include a2-macroglobulin (a2-M) and low-density lipoprotein-related protein (LRP). Perhaps not coincidentally, a2-M, LRP, and ApoE may all play a role in Ab clearance and/or deposition in brain. Candidate genes on chromosome 10 include insulin degrading enzyme (IDE) and urokinase-type plasminogen activator (PLAU) that directly or indirectly may promote Ab degradation. Ubiquilin, a PS-interacting protein, maps close to a 9q22 linkage peak. Other associations are to genes involved in cholesterol metabolism (Cyp46, or cholesterol 24-hydroxylase, and the cholesterol transporter ABCA1) (22), adaptor proteins that bind the cytoplasmic tail of APP to modulate its cellular trafﬁcking and processing (23), BACE-1 variants, and promoter region variants of PS-1 or ApoE (24,25). False positive and false negative associations in this literature would be minimized by ensuring adequate sampling, replication in an independent sample prior to publication, and thoroughly assessing haplotype structure of a candidate locus (25). THE AMYLOID HYPOTHESIS In support of the amyloid cascade hypothesis of AD is evidence that Down’s syndrome, APP, PS-1, and PS-2 mutations, and the ApoE4 polymorphism either promote Ab generation, especially Ab42, or its deposition in brain (Fig. 2). Immunohistochemical stains reveal that the earliest Ab deposits

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in aging brain are primarily Ab42. These pre-amyloid deposits (diffuse plaques) are thought to evolve into mature neuritic plaques, and thus have been likened to the benign fatty streaks that evolve into deleterious atherosclerotic plaques in blood vessels. Diffuse Ab/amyloid plaques, unlike neuritic plaques, are also thought to be benign because they are not surrounded by dystrophic neurites (swollen and deformed axonal and dendritic neuronal processes), reactive gliosis (microglial and astrocytic), and inﬂammation, and are not associated with clinical dementia (14). Evidence against the amyloid hypothesis of AD is the poor correlation of neuritic plaque burden, compared to neuroﬁbrillary tangle density or synaptic loss, to dementia severity, and weak evidence for linkage to putative downstream pathologies, such as apoptosis, oxidative injury, inﬂammation, and mitochondrial dysfunction. For example, Ab aggregates and ﬁbrils are thought to be neurotoxic, but in vivo evidence is suggestive, and mechanisms, perhaps involving elevated intracellular Ca2þ, are unclear. There is also poor linkage of amyloid plaques to the other major hallmark neuropathology of AD—neuroﬁbrillary tangles that are composed primarily of phosphorylated tau. Recent studies, however, including studies of mutant human tau and APP double transgenic mice and humant mutant tau, PS-1, and APP triple transgenic mice, support the notion that Ab pathologies precede and promote tau pathologies in brain (26–28). In support of this notion, a recently identiﬁed PS-1 mutation may be associated with familial Pick-type tauopathy but not b-amyloid plaques (29). Amyloid may be necessary but not sufﬁcient to cause AD. Neuroﬁbrillary tangles, neuronal and synaptic loss, gliosis, inﬂammation, and other pathologies downstream of CNS Ab/amyloid accumulation and deposition are likely to be of equal importance in inducing progressive dementia. However, tau (MAPT) mutations and polymorphisms on chromosome 17 cause different phenotypes, including Pick’s disease, collectively referred to as frontotemporal dementias (OMIM #600274 and others) (see chap. 26). Similarly, Ab/amyloid promotes a-synuclein pathologies but mutations in a-synuclein on chromosome 4 (or triplication of the a-synuclein gene) cause Parkinson’s disease/Lewy body dementia (OMIM #168600, #168601, and #605543) (see chap. 24). GENETIC TESTING PS-1 genetic testing is commercially available (30) and may be diagnostic for AD in the rare patient with early onset dementia (usually age 30–55) and a pedigree showing an autosomal dominant pattern of inheritance. Because 133 different mutations in PS-1 are known to cause AD (almost all single missense mutations) the entire PS-1 gene is sequenced. In contrast, only nine missense mutations in PS-2 are known to cause AD. Due to their extreme rarity, genetic testing for APP or PS-2 mutations is not commercially

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available. To date APP, PS-1, and PS-2 mutations are the only proven biomarkers of AD (31), but genetic testing is indicated only for the diagnosis of rare dementia patients with early onset AD and a suggestive pattern of inheritance. As is true in general for genetic markers of adult-onset diseases, PS-1 testing should not be obtained in minors, and with consenting symptomatic adults (or their legal guardian) testing must be accompanied by genetic counseling. Prenatal screening and testing of asymptomatic individuals in affected pedigrees raises ethical concerns as long as there is no cure for AD (see chaps. 2–4). ApoE genotyping is also commercially available (30) but its application should be limited to research studies since this information adds little to the predictive value of clinical diagnosis, adds cost, and requires genetic counseling for the subject and family members (32). Individuals with ApoE4 may not necessarily develop AD with aging and 60% of patients with AD do not carry an ApoE4 allele. A package of ApoE genotyping with tau and Ab42 levels in cerebrospinal ﬂuid is also marketed as a diagnostic test of AD but has unproven clinical utility (30). The International Huntington’s Disease (HD) Society and the World Federation of Neurology have issued guidelines for presymptomatic diagnosis for HD, and these guidelines have been adopted for testing in familial AD.

CURRENT TREATMENT STRATEGIES The Cholinergic Hypothesis A profound cholinergic deﬁcit was discovered in human AD cerebral cortex in 1976. This deﬁcit is partly due to loss of cholinergic neurons in the basal forebrain (nucleus basalis of Meynert) that project to the hippocampus and neocortex and play a role in learning and memory (33). This observation led to the notion that supplementation of central cholinergic systems may be effective for the treatment of AD—the cholinergic hypothesis—analogous to the dopaminergic supplementation strategies proven effective for patients with Parkinson’s disease. The ﬁrst drug with proven efﬁcacy for the treatment of cognitive and functional decline in patients with AD was the cholinesterase inhibitor tacrine (Cognex, FDA approved 1993) (34–36). However, this medication is limited by its four times a day dosing, multistep titration, and side effects, especially nausea, vomiting, diarrhea, and hepatotoxicity necessitating serum alanine aminotransferase monitoring. Thus, second generation cholinesterase inhibitors without hepatotoxicity have eclipsed tacrine—namely, donepezil (Aricept, approved 1996), rivastigmine (Exelon, approved 2000), and galantamine (Reminyl, approved 2001, now Razadyne) (37). There are few direct comparisons of these drugs, but efﬁcacy in improving or maintaining cognitive, functional, behavioral and global outcome measures over months to years appears comparable. Other

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central cholinergic supplementation strategies (cholinergic precursors and muscarinic cholinergic receptor agonists) have failed to prove efﬁcacy. Other Strategies Memantine (Namenda, FDA approved 2003) is an N-methyl-D-aspartate (NMDA) receptor antagonist with proven beneﬁt for patients with moderate to severe dementia due to AD (38,39). Memantine may protect vulnerable neurons from glutamate-induced excitotoxic morbidity and mortality. The antioxidants a-tocopherol (vitamin E) and selegiline (Deprenyl) delay functional decline and death in patients with AD (40). No cognitive beneﬁts were found in this study, but these were secondary endpoints. There was no additive effect of the two compounds, but either was superior to placebo. Despite ﬂaws in this study, vitamin E (2000 International Units daily) may be recommended to patients with AD. Clinical trials of Ginkgo biloba extract in patients with dementia are inconclusive. In addition to drugs to treat cognitive and functional decline in patients with AD, antidepressants, anxiolytics, neuroleptics, and anticonvulsants are often prescribed for management of a plethora of behavioral and psychiatric signs and symptoms including apathy, depression, anxiety, hallucinations, delusions, agitation, pacing, sleep/wake cycle disturbances, and catastrophic reactions (34,35). The availability of newer atypical neuroleptics and serotonin-modulating antidepressants also represents advances in the treatment of AD. FUTURE TREATMENT STRATEGIES b- and c-Secretase Inhibitors To date, drug treatments for AD are symptomatic with no beneﬁcial effect on the progressive underlying disease processes. In support of this notion, cessation of donepezil (Aricept) results in acute loss of clinical beneﬁts (37). The molecular identiﬁcation of b- and g-secretases that release Ab from APP has intensiﬁed development of small molecular weight inhibitors of these proteases as potential drugs to prevent or treat AD. Inhibitors of g-secretase are now being studied in clinical trials. PS-1 or PS-2 is an important component of the g-secretase complex, which also consists of nicastrin, pen-2, and aph-1 (Fig. 1) (14). However, neither b- nor g-secretase is speciﬁc to APP. For example, g-secretase also cleaves Notch1, a protein essential to embryonic development and adult processes such as hematopoiesis and gut epithelial cell differentiation (41). Underscoring its importance in development, PS-1 knockout (/) mice are lethal in utero and resemble the lethal Notch1 / phenotype. A perhaps more promising approach to AD treatment is inhibition of b-secretase, since BACE-1 deﬁcient mice are viable and

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appear normal. Inhibitors of BACE-1 are in preclinical stages of investigation. Due to the lack of substrate speciﬁcity of b- and g-secretases, however, drugs that inhibit these proteases may have dose-limiting toxic or intolerable side effects (42). Active and Passive Immunization Because of extensive gliosis and inﬂammatory responses to Ab/amyloid in AD brain, it was hypothesized that immunization of human APP transgenic mice with Ab may exacerbate or accelerate disease. In contrast, immunized mice develop little or no CNS amyloid deposition, indicating a novel therapeutic strategy (43). Immunization may promote Ab and amyloid clearance by anti-Ab immunoglobulin (IgG) complexes and phagocytic cells (microglia) in brain. Peripheral immune-mediated Ab clearance (a sink) may be an additional therapeutic mechanism. Immunization not only prevents but removes established amyloid plaques in human APP transgenic mouse brain. In support of the amyloid hypothesis, immunization prevents both plaque deposition in transgenic mouse brain and behavioral decline in learning and memory tasks. Recruitment into a clinical trial of Ab42 immunization in patients with dementia due to AD was halted due to the development of aseptic meningoencephalitis in 6% of subjects. However, the development of serum anti-Ab antibodies slows cognitive decline (44) and human autopsy data suggest that the immune response generated against the peptide elicits clearance of Ab plaques in brain (45). Novel active and passive immunization strategies are being pursued in nonhuman primates and transgenic mouse models of AD. Furthermore, a phase I clinical trial of passive immunization with a humanized anti-Ab monoclonal antibody is underway. Other Strategies Although epidemiologic and pilot data are promising, treatment trials with estrogens have shown no beneﬁt in postmenopausal women with AD. Likewise, despite a considerable inﬂammatory response in AD brain and promising pilot studies of older nonsteroidal anti-inﬂammatory inhibitors (NSAIDs), drugs inhibiting cyclooxygenase-2 speciﬁcally (COX-2 inhibitors) are also ineffective in patients with AD. Prednisone treatment is also without cognitive beneﬁt. Since retrospective epidemiologic studies appear promising, statins that lower serum cholesterol are also being explored for potential beneﬁt in prevention or treatment of AD. Estrogenic compounds, NSAIDs, and statins may be effective for the prevention or treatment of AD by inhibiting Ab42 generation from APP, in addition to their having other potential therapeutic effects. However, there is no evidence to support prescribing these drugs to patients with AD currently. Some clinical trials focus on more proximate events with the notion that treatment of AD patients may be too little, too late. Thus, clinical trials

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are enrolling subjects with mild cognitive impairment (MCI), a predementia syndrome with a 5–10% annual risk of conversion to dementia and AD (46). For example, a trial of vitamin E or donepezil (Aricept) in subjects with amnestic MCI found that donepezil therapy was associated with a lower rate of progression to AD during the ﬁrst 12 months; however, the rate of progression to AD after three years was not lower among patients treated with donepezil than among those given placebo (47). The exact neuropsychometric boundaries between normal aging, MCI, and dementia, are controversial. Orally administered iodochlorhydroxyquin (Clioquinol), a metal chelator, prevents age-dependent CNS amyloid deposition in human APP transgenic mice (48). A clinical trial of iodochlorhydroxyquin (with vitamin B12 supplementation) also reveals beneﬁts in patients with AD (49). Ab binds selectively to Cu2þ and Zn2þ and thus chelators of these ions solubilize Ab amyloid deposits. Finally, gene therapy for AD faces enormous barriers, such as access to the central nervous system, limited duration and extent of gene expression, and detrimental host responses to the vector or gene product (see chapter 5). Nevertheless, a phase I clinical trial of nerve growth factor (NGF)-expressing ﬁbroblasts injected into the brain of AD patients is underway. NGF promotes survival of neurons, including cholinergic neurons in the basal forebrain. Other gene therapy strategies with the Ab-proteases endothelin-converting enzyme, neprilysin, and insulin-degrading enzyme (IDE) show promising results in transgenic mouse models of AD (50,51).

PERSPECTIVES AD research has become a model of how biochemical pathology and molecular genetics have brought us to the threshold of safe and effective potentially disease-modifying clinical therapies. After decades of anosagnosia writ large and therapeutic nihilism, the identiﬁcation of rare probands and pedigrees with familial AD led to the discovery of genetic mutations, new hypotheses regarding pathogenesis, transgenic animal models of disease, and novel therapeutic strategies. The challenge remains, however, to return to the clinic with treatments for familial and sporadic AD based on recent advances in our genetic and molecular understanding of this devastating neurodegenerative disorder. If the cholinergic hypothesis may be used as a reference, we may anticipate the debut of Ab/amyloid-based treatments for patients with AD by 2008.

ACKNOWLEDGMENTS This work was supported by the VA Geriatrics Research Education and Clinical Center and NIA Grant P50 AG08671. The author has no disclosures to report.

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31. Turner RS. Biomarkers of Alzheimer’s disease and mild cognitive impairment: are we there yet? Exp Neurol 2003; 183:7–10. 32. The Ronald and Nancy Reagan Research Institute of the Alzheimer’s Association and the National Institute on Aging Working Group. Consensus report of the Working Group on: ‘‘Molecular and biochemical markers of Alzheimer’s disease.’’ Neurobiol Aging 1998; 19:109–116. 33. Davies P, Maloney AJF. Selective loss of central cholinergic neurons in Alzheimer’s disease. Lancet 1976; 2:1403. 34. Doody RS, Stevens JC, Beck C, Dubinsky RM, Kaye JA, Gwyther L, Mohs RC, Thal LJ, Whitehouse PJ, DeKosky ST, Cummings JL. Practice parameter: Management of dementia (an evidence-based review). Neurology 2001; 56: 1154–1166. 35. Scarpini E, Scheltens P, Feldman H. Treatment of Alzheimer’s disease: Current status and new perspectives. Lancet Neurol 2003; 2:539–547. 36. Knapp MJ, Knopman DS, Solomon PR, Pendlebury WW, Davis CS, Gracon SI. A 30-week randomized controlled trial of high-dose tacrine in patients with Alzheimer’s disease. JAMA 1994; 271:985–991. 37. Rogers SL, Friedhoff LT. The efﬁcacy and safety of donepezil in patients with Alzheimer’s disease: results of a US Multicentre, Randomized, Double-Blind, Placebo-Controlled Trial. The Donepezil Study Group. Dementia 1996; 7: 293–303. 38. Reisberg B, Doody R, Stofﬂer A, Schmitt F, Ferris S, Mobius HJ. Memantine in moderate-to-severe Alzheimer’s disease. N Engl J Med 2003; 348:1333–1341. 39. Tariot PN, Farlow MR, Grossberg GT, Graham SM, McDonald S, Gergel I, Memantine Study Group. Memantine treatment in patients with moderate to severe Alzheimer disease already receiving donepezil: a randomized controlled trial. JAMA 2004; 291:317–324. 40. Sano M, Ernesto C, Thomas RG, Klauber MR, Schafer K, Grundman M, Woodbury P, Growdon J, Cotman CW, Pfeiffer E, Schneider LS, Thal LJ. A controlled trial of selegiline, a-tocopherol, or both as treatments for Alzheimer’s disease. The Alzheimer’s Disease Cooperative Study. N Engl J Med 1997; 336:1216–1222. 41. Selkoe D, Kopan R. Notch and presenilin: Regulated intramembrane proteolysis links development and degeneration. Annu Rev Neurosci 2003; 26:565–597. 42. Dewachter I, Van Leuven F. Secretases as targets for the treatment of Alzheimer’s disease: The prospects. Lancet Neurol 2002; 1:409–416. 43. Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H, Guido T, Hu K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M, Liao Z, Lieberburg I, Motter R, Mutter L, Soriano F, Shopp G, Vasquez N, Vandevert C, Walker S, Wogulis M, Yednock T, Games D, Seubert P. Immunization with amyloid-b attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 1999; 400:173–177. 44. Hock C, Konietzko U, Streffer JR, Tracy J, Signorell A, Muller-Tillmanns B, Lemke U, Henke K, Moritz E, Garcia E, Wollmer MA, Umbricht D, de Quervain DJ, Hofmann M, Maddalena A, Papassotiropoulos A, Nitsch RM. Antibodies against b-amyloid slow cognitive decline in Alzheimer’s disease. Neuron 2003; 38:547–554.

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26 Tauopathies John C. van Swieten Department of Neurology, Erasmus Medical Center, Rotterdam, The Netherlands

INTRODUCTION Filamentous tau inclusions in neurons and glial cells of the brain are the pathological hallmark of a number of neurodegenerative disorders (designated ‘‘tauopathies’’) clinically characterized by dementia or parkinsonism. Alzheimer’s disease (AD) is the most common tauopathy, and is characterized by neuroﬁbrillary tangles and the deposition of b-amyloid in senile plaques. Neuroﬁbrillary tangles in AD consist predominantly of paired helical ﬁlaments and contain all six isoforms of hyperphosphorylated tau (1–3). Other tauopathies such as progressive supranuclear palsy, corticobasal degeneration, Pick’s disease, and argyrophilic grain disease show tau pathology in the absence of senile plaques, and are pathologically distinguished from each other by different types and distribution of tau-positive inclusions in the brain. The etiological role of the tau protein in neurodegeneration has been questioned for a long time, but has been convincingly demonstrated by the identiﬁcation of mutations in the tau gene in a number of familial disorders with dementia and/or parkinsonism (4–6). All disorders with mutations in the tau gene show accumulation of abundant ﬁlamentous tau protein in the brain.

TAU GENE—TAU PROTEIN Six different tau isoforms in the adult human brain are produced from the single tau gene by alternative splicing of exons 2, 3, and 10 (Fig. 1). The tau

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Figure 1 Schematic representation of the tau gene and the six different isoforms. (A) The human tau gene contains 16 exons, of which exons 2, 3 and 10 (dark grey boxes) are alternatively spliced. Exons 4A, 6 and 8 (light grey boxes) are not transcribed in the human brain. (B) The six different tau isoforms generated by alternative mRNA splicing of exons, 3 and 10 (dark grey boxes) vary in size from 353 to 441 amino acids. The black boxes represent microtubule binding domains. Source: Courtesy of Dr. E. van Herpen.

isoforms contain three or four amino-acid repeat regions constituting the microtubule binding domains of tau (7,8). Three isoforms contain three amino acid repeats, encoded by exons 9, 11, and 12, whereas the inclusion of the amino acid repeat encoded by exon 10 gives rise to the other three isoforms with four repeats (9,10). In adult human brain the ratio between three repeat and four repeat tau isoforms is close to one. Most mutations in the tau gene are located in the carboxy-terminal part of tau where the repeat regions are encoded (Fig. 2). Missense mutations or deletions in this part of the tau gene may differentially affect microtubule assembly and binding (11,12), but also enhance ﬁlament assembly of the tau protein itself in vitro (13,14). The aggregation of the tau protein into paired helical ﬁlaments by the formation of b-sheet structure is much faster for the P301L and DK280 mutations than for other mutations (15,16). Several other factors, such as glycosaminoglycans, heparin sulfate, and hyperphosphorlyation of the tau protein, may also inﬂuence the rate of self-assembly into ﬁlaments. Some mutations may also change the alternative splicing of exon 10, resulting in overrepresentation of 4-repeat or 3-repeat tau isoforms. This occurs with mutations of the splice-donor site in the intron following exon 10, as well as in some mutations in exon 10 itself.

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Figure 2 Schematic overview of mutations in the tau gene, identiﬁed in FTDP-17. All missense mutations identiﬁed up until now are located in exon 1, 9 to 13. The alternatively spliced exon 10 is depicted in grey. Mutations are numbered according to codon number of the longest tau isoform (441 amino acids). Source: Courtesy of Dr. E. van Herpen.

FRONTOTEMPORAL DEMENTIA Frontotemporal dementia (FTD) is much less common than AD. The prevalence is estimated between 5 and 15 per 100,000 persons in the age group 45–65 years, although exact data are lacking in the most countries/populations (17–19). The relative frequency of FTD in autopsy series of dementia under the age of 70 years is between 5% and 10% (20). This condition is clinically characterized by severe behavioral changes, language difﬁculties, and other cognitive dysfunctions (21). International consensus criteria are designed to establish the clinical diagnosis and to differentiate it from AD. These criteria comprise behavioural or cognitive deﬁcits manifested by either (1) early and progressive change in personality, characterized by difﬁculty in modulating behaviour (2) early and progressive change in language, characterized by problems with expression of language or severe naming difﬁculty and problems with word meaning. These deﬁcits interfere with the previous level of social and occupational functioning, and are not due to other systemic or nervous system conditions (i.e., psychiatric) or environmental factors (21,22). FTD is nonfamilial or sporadic in 70–80% of patients, whereas the remaining 20–30% is hereditary (17,23). The pathological hallmark of FTD is circumscribed atrophy of the frontotemporal cortex, as described by Arnold Pick (24), with neuronal loss, gliosis, and spongiosis of the superﬁcial layers of the cortex in all cases. FTD is pathologically heterogeneous and can be divided into three subtypes: with tau pathology, with ubiquitin pathology, and without distinctive histology (22,25). The entity Pick’s disease is now exclusively designated for FTD with intraneuronal argyrophilic inclusions, so-called Pick bodies, which consist

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of abnormal tau protein. Pick bodies are present in 10–30 percent of sporadic FTD (17,22,26). In contrast to this, 30–60% of familial FTD is pathologically characterized by tau pathology (27). Mutations in the tau gene have been identiﬁed in nearly all familial FTD patients with tau pathology (17,23,27). A considerable proportion (20–40%) of the total group of hereditary FTD shows neither tau mutations nor tau pathology (28–30). A few of these families are pathologically characterized by ubiquitin-positive, taunegative inclusions in the brain and are linked to chromosome 17q21–22; however, the genetic defect has still to be identiﬁed. Two other types of hereditary FTD are linked to chromosome 9, one to the genetic locus 9q21–22, and the other to 9p13.2–p12 (31). Mutations in the Valosin-containing protein gene have very recently been identiﬁed for the 9q21–22 type of hereditary FTD with inclusion body myositis and Paget’s disease (32). Finally, there is a Danish family with hereditary FTD with linkage to chromosome 3, for which the genetic defect is unknown (33,34).

FTDP-17 Genetic Epidemiology The etiological role of the tau gene was suspected for the ﬁrst time when a family with so-called dementia-disinhibition-parkinsonism-amyotrophy complex (DDPAC) showed signiﬁcant linkage to the tau gene-containing region on chromosome 17q21–22 (35,36). Subsequently, this linkage was conﬁrmed for several familial disorders known under different nosological entities in older literature, including hereditary Pick’s disease, familial subcortical gliosis or autosomal dominant dementia with widespread neuroﬁbrillary tangles (37–42). The descriptive term Frontotemporal Dementia and Parkinsonism linked to chromosome 17q21–22 (FTDP-17) denotes all these nosological entities with linkage to chromosome in 17q21–22, since the consensus meeting in Ann Arbor in 1996 (37). Pathological tau changes are present in neurons and glia in most of these disorders. In 1998, three research groups identiﬁed mutations in the tau gene in eight families (4–6); more than 30 different tau mutations have been recognized in families in Europe, North America, Japan, and Australia over the last years (see below). Interestingly, Pick-type pathology without b-amyloid plaques was recently described in a patient with a novel Presenilin 1 mutation (43). FTDP-17 usually shows an autosomal dominant mode of inheritance, but recessive forms have been recently described (44,45). The mode of inheritance is less obvious in a few families due to the lack of information on family history or the possibility of nonpaternity (46). The phenomenon of incomplete penetrance has been convincingly demonstrated with the L315R mutation (47). Some families with P301L and N279K mutations are very large with more than 20 affected members over several generations

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(48,49). Other much smaller families consist of a few affected members only, and sometimes the mutation (for example, the L266V mutation) is identiﬁed in the proband only (50). Sometimes affected relatives from earlier generations had clinical diagnoses other than FTD during life, including multiple sclerosis or Parkinson’s disease (48,50,51). A few mutations have been identiﬁed worldwide, in Europe, United States, Australia, and Japan (5,52–58), whereas several other are described in a single family (47,50,59–62). Intronic mutations are almost exclusively found outside Continental Europe (5,63,64). The P301L appears to be the most common mutation, with more than 20 families in United States, France, the Netherlands, Japan, Italy, and Poland (5,23,54,56,65–67). It is still unclear whether this high frequency could be explained by a common ancestor for all P301L patients. Haplotype analysis has shown that the R406W and N279K mutations have occurred as independent events in several families (58,68,69). In contrast to this, all families with the þ16 intronic mutation share a common haplotype, probably from a common ancestor in Wales, although recombinational events have resulted in reduced size of shared alleles in some of them (70). The frequency of tau mutations in FTD varies between different studies (17,23,56,71–74). The high prevalence of tau mutations in individuals with the clinical diagnosis of FTD in the Netherlands (14%) and in Northwest Britain (10%) contrasts with the much lower identiﬁcation in the United States (6%) (72,74,75). The frequency is obviously higher in familial cases of FTD, but even then varies considerably between the United States (8%) and Europe (20–30%). It is unclear whether this large geographical variation reﬂects true differences in prevalence or different ascertainment methods. A selection bias towards familial cases series is unlikely, since the percentage of familial cases in this series is approximately equal in these studies (17,23). The frequency of tau mutations is higher in patients with familial FTD with tau pathology in the brain (23,27). All series are consistent in the observation that the absence of tau pathology in familial FTD cases excludes the presence of tau mutations (23,27). Age at Onset The clinical presentation correlates to some extent with the type or location of the mutation within the tau gene. However, the inter- and intrafamilial variation in age at onset may be considerable. On the other hand, the average age at onset in families with the same mutation from different continents (Japan, United States, and Europe) may be remarkably similar, despite different genetic background and environment (52–55). The age at onset for the most common P301L and several other mutations is usually between 45 and 65 years. Dementia may occasionally occur between 65 and 70 years of age in patients with these mutations, but never after the age of 70 years (48,55). Intronic mutations show the same general

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distribution of age at onset, although a few cases have an earlier onset (around the age of 40) (41,63). Clinical symptoms may develop even earlier, between 25 and 30 years, in patients with the P301S and L315R mutations (44,47,76,77), or between 30 and 44 years for the L266V and N279K mutations (49,50,78). Late-onset dementia after the age of 70 years may also occur, as observed in cases with the R5H and I260V mutations (79,80). The occurrence of a healthy status in an 82-year-old carrier of the L315R mutation suggests that other genetic or environmental factors also play a role in determining the age of onset (47). The duration of illness for most mutations usually is between 8 and 10 years. An exception is the R406W mutation, which is characterized by a slow rate of disease progression lasting up to 25 years (48,69). Patients with an earlier onset of the disease often show a more aggressive disease leading to death within ﬁve years (47,76,77,81). Clinical Presentation Two major clinical subtypes can be distinguished among FTDP-17 patients: parkinsonism predominant and dementia predominant (57). Both types can occur in patients with the same mutation and even in the same family. The dementia phenotype is usually associated with the P301L mutation, as well as in the G272V (48,55,82). Personality changes are characteristic, with disinhibition, jocularity, and asocial behavior as salient features. However, apathy and loss of initiative are also prominent in the initial presentation of the disease in these mutations. Obsessive–compulsive behavior in some patients, or the occurrence of paranoid delusions and hallucinations in others may initially suggest a psychiatric disorder (42,48,81). Memory problems may also dominate the clinical presentation to such an extent that the clinical diagnosis of AD is considered during life (59,69). Patients may have increasing difﬁculty with planning, loss of concentration, and distractibility, and can develop impaired judgment and loss of insight disrupting their social and professional life. Emotional bluntness is often very embarrassing for family members, especially at majorlifeevents. Other common clinical features are hyperorality and roaming behavior. Patients develop early language difﬁculties consisting of word ﬁnding problems, and stereotyped words or phrases are frequently used (48–50,53,77,83). Semantic paraphasias, and impaired language comprehension are sometimes observed (63), but a true semantic dementia has not been reported yet. Paucity of speech results in mutism within ﬁve years in all patients, except for the long preservation of language abilities in some patients with R406W mutation (48). Partial or generalized epileptic seizures are a speciﬁc clinical feature of the P301S mutation, whereas mental retardation has been reported in a patient with the þ11 intronic mutation (76,84).

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While Parkinsonism is the dominant clinical presentation in some, but not all, patients with the N279K and intronic mutations (49,57,63,84,85), it may also occur in mutations with the predominant dementia subtype (86). Characteristic features include gait impairment, rigidity, bradykinesia, postural instability, and resting tremor, with no or only transient beneﬁts from levodopa treatment. Corticospinal tract signs are occasionally present in the parkinsonism-dominant type of FTD (52). Vertical gaze palsy, saccadic eye movements, and axial rigidity are early signs in a few speciﬁc mutations (S305N and delN296) and are consistent with the clinical diagnosis of progressive supranuclear palsy (PSP) (45,87). Patients with parkinsonism may develop these symptoms later in the disease (41,49). Unilateral rigidity, dystonia and contractures in combination with impaired eye movements occur in some mutations and suggest the clinical diagnosis of corticobasal degeneration (CBD) (53,77,87). Observations in genotype–phenotype studies indicate that there is clinical overlap between FTDP-17, PSP, and CBD (77). At present, a debate about whether to consider these conditions as clinical phenotypes of a single disease or as distinct clinical entities is irrelevant; the main issue for researchers is to determine whether FTDP-17, sporadic and familial PSP, and CBD all have the same underlying pathophysiology. Neuropsychology Patients with FTDP-17 differ in cognitive functioning in daily life from those with AD. They have relatively intact episodic memory and do not lose their way. Orientation and visuoconstructive functions are usually fully intact. Intelligence scores are often low. Verbal ﬂuency, abstractive thinking, and executive functions including planning and mental set-shifting (Wisconsin Card Sorting Test) is nearly always impaired, reﬂecting frontal dysfunction. Attention and concentration are usually decreased, often contributing to low test results (48,55). Poor performance on formal memory tests may be present, but often immediate and delayed recall in verbal and nonverbal memory tests are relatively preserved (76). Language dysfunction may consist of inefﬁcient word retrieval, anomia, and sometimes mildly impaired comprehension, although never consistent with the cognitive proﬁle of semantic dementia. The pattern of cognitive dysfunction is similar for all mutations. A very interesting observation is that cognitive functioning may be impaired decades before the presentation of dementia, as asymptomatic mutation carriers have already shown reduced verbal ﬂuency, attention, and motor speed and set shifting in their twenties and early thirties (18,88,89). Investigations The clinical diagnosis of FTDP-17 can be supported by neuroimaging ﬁndings. Frontotemporal atrophy is the most common neuroradiological feature

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(48,55,64). Some patients show a predominantly temporal pattern of atrophy, often asymmetric (77,79,87), and occasionally have hippocampal atrophy as well (in patients with the G389R mutation) (46). Diffuse cerebral atrophy is a common ﬁnding in some patients, especially those with intronic mutations (41,45,53,69,84,90). Single-photon emission computed tomography (SPECT) shows hypoperfusion of the anterior part of the brain early in the disease, even in patients with normal brain morphology (82). Glucose metabolism is reduced in frontal and temporal lobes of the brain on positron emission tomography (PET) scan (46). In patients with parkinsonism, FluoroDopa metabolism in the globus pallidus is signiﬁcantly impaired on PET (49). FP-CIT scan shows a severe symmetrical decrease of presynaptic dopamine transporter binding to the striatum in the P301S mutation compared to controls (76). Electroencephalography is usually normal (55), except for interictal epileptic discharges described in patients with the P301S mutation (76). Denervation potentials and fasciculations on electromyography reﬂecting anterior horn cell disease have been found in one series, but understanding their signiﬁcance awaits systematic investigation in other mutations (82). Normal levels of total and hyperphosphorylated tau are found in cerebrospinal ﬂuid in FTD with tau pathology (91). This ﬁnding has yet to be explained in the perspective of increased levels of CSF in other tauopathies such as AD and PSP (92,93). Genetic Testing in FTDP-17 Patients with FTD and their family are usually very concerned about the possibility of hereditary FTD. Detailed family history and clinical information about affected relatives is often very helpful for the genetic counselor or clinician. Genetic screening of tau gene can be very useful to establish the diagnosis in patients and to inform family members about their own risk to develop the disease. If a patient carries a mutation, asymptomatic at-risk individuals may request genetic testing to relieve anxiety and uncertainty or to plan their futures (94,95). It is important to emphasize and discuss the emotional impact of unfavorable results with healthy relatives requesting DNA testing. Potential risks are emotional depression, insurance, and employment problems. Although a considerable proportion would consider genetic testing, one studies show that only a small number of patients decide to pursue such testing (96). Another important implication of genetic testing might be to learn more about the clinical phenotype and the penetrance of mutations. Recent observations of incomplete penetrance in the L315R mutation and the occurrence of autosomal recessive transmission with the S352L mutation have important implications for genetic counselling (44,47). The recent identiﬁcation of a PS1 mutation in a patient with Pick type pathology implies that it may be worthwhile to screen the PS1 gene in patients with familial FTD without tau mutations (43).

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PATHOLOGY OF FTDP-17 Macroscopic Features FTDP-17 is characterized pathologically by neuronal loss and gliosis in the cerebral cortex, subcortical nuclei, white matter, and brainstem. For the most common mutations (P301L, N279K, intronic) the pathological variation is known, whereas for many other mutations the pathological observation has been conﬁned to a single patient. The brain weight at autopsy is often reduced, and is frequently less than 1000 g. Lateral ventricles are often grossly enlarged (63). Macroscopically, the brain shows frontal and temporal atrophy in most mutations, sometimes in a knife-edge pattern. The temporal atrophy is most prominent in the anterior part, with relative preservation of the posterior part of the superior temporal gyrus (55,97,98). The atrophy occasionally extends into the parietal lobe (50,76). Frontotemporal atrophy may be mild in some mutations (41,97,99,100). Atrophy of the hippocampus and amygdala often accompanies temporal atrophy (50,55,63), although these structures may also be normal (42,65). The involvement of the subcortical nuclei is variable, ranging from normal in some (69,100,101) to severely atrophic in other mutations (50,65,84,97,98,102). The substantia nigra and locus coeruleus show depigmentation in some mutations (69,82,85,97,102), but may be normal in other mutations outside exon 10 (42,59,60,98). The brainstem and pons are sometimes atrophic, whereas the dentate nucleus of the cerebellum usually has a normal appearance (18,46,82). Microscopy Severe neuronal loss and gliosis is seen in the frontal or temporal cortex of patients with most mutations, but may be absent or mild in a few other mutations (97,99,100). Ballooned cells may be present in deep cortical layers (85,97,98). The hippocampus usually shows loss of pyramidal cells in cornu ammonis (CA) and the subiculum, although there is no or only focal neuron loss in several cases (42,87,97,102). The amygdala is affected in most cases. Subcortical nuclei, except for the thalamus, usually show neuronal loss (60,61,64), but may be normal (81,85,87,90,99,101–106). Gliosis and/or demyelination of the subcortical white matter may be present (46,76,98), occasionally with degeneration of the corticospinal tracts (85). The substantia nigra and locus coeruleus often have severe neuronal loss (63), but may be normal in exon 12 and 13 mutations (42,48,83,98). Neuronal loss may also be present in other brainstem nuclei, the dentate nuclei and spinal cord (41,84,97,100). The presence of diffuse senile, and sometimes neuritic, plaques in the cortical regions is a remarkable ﬁnding in several cases (48,65,79,102,107), but it might be that their presence is a secondary and coincident pathological feature in patients at older age (108).

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Different silver staining techniques (Bodian, Bielschowski, methanamine silver or Gallyas) are used to visualize neuronal and glial inclusions. These techniques positively stain for NFT, neuropil threads, dystrophic neurites, coiled bodies and astrocytic processes (6,46,48,50,77,79,80,83,97,100,104). Pick or Pick-like bodies are mostly positive with silver staining, except for Gallyas staining in patients with the K369I mutation (85,98). Immunohistochemistry Immunohistochemistry with phosphorylation-dependent anti-tau antibodies shows more widespread tau pathology in both neurons and glial cells than silver staining. A large number of phosphorylation-dependent tau-antibodies are available, but AT8 and PHF1 antibodies produce the strongest staining. Distinct tau mutations are associated with different types of neuronal tau inclusions, such as diffuse or punctate staining, Pick bodies, neuroﬁbrillary tangles, and pretangles. Pick bodies usually do not stain with the 12E8 antibody speciﬁc for the 262 phosphorylation site, except for those from patients harboring G389R mutation (46). Tau-positive inclusions are most often found in the frontotemporal cortex and subcortical nuclei, but can also occur in the midbrain, pons, brainstem, cerebellum, and spinal cord. Tau-positive inclusions often parallel the severity of neuronal loss, but may be sometimes more prominent in regions with less severe neuronal loss (47,97). Tau pathology may be conﬁned to nerve cells (60) or it may be more severe in oligodendroglia and astrocytes dependent on the type of the mutation (77,80,97,104). NFT are often numerous in the frontotemporal cortex, but may be more widespread in subcortical nuclei in some mutations (85,97,105). Flame-shaped or globose neuroﬁbrillary tangles are associated with intronic mutations, and some mutations of exon 12 and 13 (40,42)(97,102). Extracellular tangles are occasionally seen (101). The granule cells of the dentate gyrus of the hippocampus frequently contain Pick-like bodies, and the pyramidal cells of CA and subiculum often show NFT (102,109); however, occasionally the hippocampus is free of tau pathology (42). The substantia nigra and locus coeruleus often show NFT, glial inclusions, or neuropil threads (97,102), but lack tau pathology in some patients with exon 12 and 13 mutations (42,59,69,83,98). Cerebellar nuclei, brain stem, and spinal cord may show a few NFT, glial inclusions, or neuropil threads. Neuropil threads are often present in variable severity and distribution, whereas thick axonal swellings are occasionally seen (46). Pretangles are characteristically found in patients with the most common P301L mutation, but may also occur in patients with other mutations (48,109). Several mutations are associated with Pick bodies varying in severity and distribution in cortex, hippocampus, amygdala, and subcortical nuclei (47,50,60,61,103,109). These Pick bodies usually stain

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positive with antibodies speciﬁc for 3-repeat tau and negative for 4-repeat speciﬁc antibodies. Glial tau pathology in patients with some mutations is more severe than neuronal pathology. Glial tangles or coiled bodies are more abundant in the white matter than in the cortex in cases with predominantly 4-repeat tau isoforms (77,84,85,102,105). Tufted astrocytes speciﬁc for PSP are found in the cortex and subcortical nuclei several exonic and intronic mutations (50,64,78,100,102). Their presence in brains from patients with mutations affecting all six isoforms (L266V, L315R, R5H, S305N) contrasts with the observation of abundant glial tau pathology usually associated with mutations that only affect 4R tau or that increase the relative amount of 4R tau. Tufted astrocytes are usually more abundant in cortical areas with severe neuronal loss than in areas less severely involved (47,78) suggesting that astrocytic inclusions develop later than neuronal deposits, or are longer lived. Phagocytosis of neuronal deposits by astrocytes provides a possible explanation, but the exact pathophysiological relationship between tau mutations and astrocytic tau pathology awaits further clariﬁcation. Biochemistry Immunoblotting of sarkosyl-insoluble tau from the brain shows distinct proﬁles according to the location of the mutation in the tau gene (Table 1). Tau deposits in exon 10 and intronic mutations at the splice donor site following exon 10 consist predominantly or exclusively of 4-repeat tau isoforms. Two different pathophysiological mechanisms underlie the predominance of 4-repeat isoforms. Firstly, the intronic and some exon 10 mutations (at position 279, 296, and 305) produce a 2–10 fold increase of exon 10þ transcripts over exon 10 transcripts (6,12,110). In individuals with these mutations, overexpression of 4-repeat isoforms leads to the deposition of 4-repeat tau isoforms. Secondly, missense mutations in exons 9–13 result in reduced binding to microtubules (11,47,59). In tau containing the P301L mutation in exon 10, this functional change affects the 4-repeat tau isoforms only and results in the selective aggregation of this isoform. The sarkosyl-insoluble tau deposits in patients with the P301L mutation also consist predominantly of the mutated tau isoforms, with a selective depletion of the mutated protein in the soluble fraction, while the overall ratio (1:1) of 4-repeat versus 3-repeat isoforms is normal (111). Neuronal and glial tau pathology is usually associated with the deposition of 4-repeat tau isoforms, while neuronal tau pathology is associated with all six isoforms. Neuroﬁbrillary tangles in the exon 12 and 13 mutations, V337M and R406W, contain all six hyperphosphorylated tau isoforms as expected (40,48), since the location of mutation outside exon 10 results in the expression of equal amounts of mutated 4-repeat versus 3-repeat isoforms and subsequent aggregation of these isoforms. Pick bodies consist exclusively or predominantly of 3-repeat isoforms in two exon

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Table 1 Biochemical Composition of Sarkosyl-Soluble and -Insoluble tau into Different tau Isoforms for Distinct Mutations in the tau Gene Soluble tau

Insoluble tau Before After dephosphorylation dephosphorylation

exon 1 exon 9

exon 10

intronic

exon 11 exon 12

exon 13

R5L R5H K257T

3R ¼ 4R 3R ¼ 4R 3R ¼ 4R

L260I L266V

3R ¼ 4R 3R ¼ 4R (4R > 3R) 3R ¼ 4R 60, 64 n.a. 64, 68 n.a. n.a. n.a. 64,68 > 72

G272V N279K DK280 L284L N296N, DN296, N296H P301L P301S S305N S305S þ3, þ11, þ12, þ13, þ14, þ16 L315R S320F Q336R V337M E342V S352L K369I G389R R406W

3R ¼ 4R n.a. n.a. n.a. 4R > > 3R

3R ¼ 4R 3R ¼ 4R n.a. 3R ¼ 4R 4R > 3R n.a. 60, 64, 68 3R ¼ 4R 3R ¼ 4R

64, 68 60, 64 > 68, 72 kDa 64,68 60, 64

4R > 3R 4R > 3R 3R > 4R 4R 3R > 4R 3R > 4R 4R

4R

64, 68 > 72

4R > 3R

64,68 > 72

4R

60,64 > 68 60,64

4R 3R ¼ 4R

60, 64, 68, 72 n.a.

3R ¼ 4R 4R > 3R

60, 64, 68 60, 64 > 68, 72 60, 64, 68, 72

n.a. 3R ¼ 4R 3R ¼ 4R

9 mutations (50,60,78), whereas other mutations with Pick bodies show both 3- and 4-repeat tau isoforms on western blots (46,47,59,61,62,98,103). Although associated glial tau pathology in these cases may be responsible for the presence of 4-repeat tau isoforms, staining with speciﬁc tau antibodies has also shown a mixture of 3- and 4-repeat tau in Pick bodies associated with the Q336R mutation (101). The association between the location of the mutation and tau isoform proﬁle is less clear for other mutations (47,59,83). In contrast to the

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observations of predominant 3-repeat tau isoforms in patients with some exon 9 mutations, exclusively 4-repeat isoforms are found in patients carrying another exon 9 mutation (80). The neuronal and glial tau pathology in the R5L mutation of exon 1 consist mainly of 4R isoforms, despite the fact all six isoforms contain this amino-acid change (100). Electron Microscopy Tau lesions consist of abnormal ﬁlaments made from hyperphosphorylated tau protein (41). These ﬁlaments can be studied in sarkosyl-insoluble brain homogenates, or in epon-ﬁxed brain tissue (81). In these studies on FTDP-17, tau ﬁlaments have speciﬁc morphologies that vary among the different tau gene mutations. Paired helical ﬁlaments with a diameter of 8–20 nm and periodicity of 80 nm are found in AD, but also in FTDP with exon 12 and 13 mutations, which have all six tau isoforms (40,81,83,109). Slender twisted ﬁlaments or ribbons with irregular periodicity of 90– 130 nm are characteristic for intronic and some exonic mutations with a predominance of 4-repeat tau isoforms, together with a minority of straight ﬁlaments (41,52,97,99). Narrow irregularly twisted ﬁlaments of 15 nm and periodicity of 130 nm or more are found in other mutations, including P301L (60,65,98,109). Several mutations produce predominantly straight ﬁlaments (50,61,77,101). These ﬁlaments can be decorated by phosphorylation dependent and -independent tau antibodies. Functional Studies The primary effect of tau mutations is either at the protein level or at the premRNA transcription level. Missense mutations in exon 9–12 of the tau gene reduce the ability of the tau protein to promote microtubule assembly, with stronger effect on the 3-repeat isoforms than on the 4-repeat tau isoforms (11,112). The reduction in the rate of microtubule assembly varies between different mutations, and is largest for the P301L mutation. This functional change is probably caused by the introduction of new phosphorylation sites in some mutations (11,60) or removal of such sites (as in the S320F mutation) (59). Mutations in exon 1 and exon 13 also change the biochemical properties of the tau protein, but produce less change in microtubule binding (12). The intronic mutations are located at several positions (þ3, þ11, þ12, þ13, þ16) within a stem loop structure following exon 10. This stem loop acts as a splice-donor site and regulates the alternative splicing of exon 10. The intronic mutations destabilize the secondary structure of the stem loop and result in an increased splicing of exon 10 (5,6,113) and a change in the ratio of 3-repeat versus 4-repeat tau isoforms. Position 305 codes for the last amino acid in exon 10, and mutations at this position (S305S, S305N) destabilize the stem loop and lead to alterations in the ratio of

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3-repeat to 4-repeat isoforms. In addition, some coding region mutations in exon 10 (at position 279, 284, and 296) alter alternative splicing by destroying a splicing silencer element or by creating a splicing enhancer sequence (85,105,107). These mutations create tau that has normal or slightly increased microtubule binding (110). The change in splicing of exon 10 differs between these mutations and depends on their position, varying between a 2- and a 26-fold increase in the ratio of 4-repeat versus 3-repeat isoforms (6,14,84,87,110). The idea is that the overproduction of 4-repeat tau isoforms produces an excess of these isoforms over available binding sites on microtubules, resulting in hyperphosphorylation of tau and its assembly into ﬁlaments (2,114). The state of hyperphosphorylation of tau and its interactions with other proteins have been investigated in several in vitro studies, which will not be discussed here in detail. Transgenic Model Studies Mutant human tau protein has been expressed in transgenic mouse models. These mouse models differ from each other in tau mutation, expression level of the tau protein (depending on the promoter used), tau isoform expression (shortest 4R versus longest 4R isoform), and genetic background. Expression levels of tau protein also vary between neurons in different brain regions and spinal cord, and result in an age-dependent neurological phenotype. The JNPL3 transgenic mouse expressing the shortest 4-repeat tau isoform (4R0N) with the P301L mutation shows motor weakness of all limbs and behavioural changes by the age of six months (115). Neuropathologically, it is characterized by neuronal loss in the spinal cord, ﬁbrillary gliosis in the anterior horns, and axonal degeneration in the anterior roots. Neuroﬁbrillary tangles are found in the spinal cord, brain stem, and cerebellar nuclei of this transgenic mouse model, whereas pretangles are widely distributed in the hippocampus, basal ganglia, and entorhinal cortex. NFT are immunoreactive to phosphorylation-dependent and independent tau antibodies, and consist of straight and twisted ﬁlaments. Skeletal muscle shows neurogenic atrophy. Western blots of sarkosyl-insoluble tau have a 64 kDa band, containing the human hyperphosphorylated 4R0N tau isoform (115). The clinical and pathological phenotype varies between different transgenic mouse models with speciﬁc tau mutations. The transgenic mouse expressing the P301S mutation develops severe paraparesis and tremor at the age of 12–14 months (116), whereas expression of the mutant (R406W) human tau in a transgenic mouse model results in associative memory impairment (117). The G272V transgenic mouse model is characterized by high expression levels of tau protein and accumulation of hyperphosphorylated tau protein in oligodendrocytes throughout the brain, without neuronal tau pathology (118). P301L transgenic mice have tau

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inclusions not only in neurons, but also in oligodendrocytes and astrocytes. Mouse models also enable us to investigate different aspects of the pathophysiology. Recent observations in V337M transgenic mice support the idea that accumulation of mutant tau, and subsequent accumulation of RNA and phosphorylated tau, precedes the loss of microtubules (119,120). Other studies on transgenic mice have investigated the relationship between tau pathology and extracellular deposition of amyloid b-protein, which co-exist in AD. Double (P301L/mutant APP) transgenic mice expressing mutant tau and APP have shown that abnormal amounts of mutant b-amyloid precursor protein (APP) enhance the formation of neuroﬁbrillary tangles in the limbic system. This observation has been conﬁrmed in experiments with injection of b-amyloid ﬁbrils into the brains of P301L mutant transgenic mice (121,122). However, at the other end, aggregation of tau protein in FTDP-17 brains with tau mutations may also be responsible for increased levels of soluble Ab-40 and Ab-42 protein (123). Other Tauopathies Progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick’s disease, and argyrophilic grain disease are sporadic late-onset neurodegenerative disorders that differ from each other by the distribution and biochemical composition of tau inclusions. These disorders are mostly sporadic, although the predominance of speciﬁc tau isoforms andthe signiﬁcant association of the H1 haplotype indicate that a genetic factor in or around the tau gene plays a role in their pathophysiology. PSP and CBD are both late-onset extrapyramidal disorders with tau pathology in subcortical nuclei, brainstem, and neocortex. Pick’s disease usually has a more cortical distribution of lesions, and the lesions in argyrophilic grain disease are predominantly conﬁned to hippocampus and amygdala. Progressive Supranuclear Palsy PSP is a slowly progressive condition with a prevalence of approximately 5 per 100 habitants (124–126). The age at onset is between 60 and 70 years in 50% of patients, and below the age of 60 years or above the age of 70 years in 25% each (127). The clinical picture is characterized by supranuclear gaze palsy, postural instability with frequent falls, axial rigidity, and cognitive impairment (128). Patients with PSP may show behavioral changes that vary in severity and correlate with loss with frontal grey matter on MRI (129). The International Consensus criteria from 1996 specify the diagnosis possible, probable, deﬁnite PSP. However, PSP shows considerable clinical and pathological heterogeneity (130), and approximately 20% of pathological PSP cases do not satisfy the criteria for probable PSP (131,132). Atypical patients may

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present with levodopa responsive parkinsonism or resemble idiopathic Parkinson’s disease, and are pathologically associated with more prominent mesial temporal AD pathology with NFT (130). Other atypical clinical presentations include primary progressive aphasia and unilateral limb dystonia (133–135). The pathological diagnosis is based on the presence of globoid NFT and neuropil threads in the basal ganglia and brainstem, and the presence of tau-positive tufted astrocytes as supportive feature (131,136). Tufted astrocytes are usually most abundant in the caudate nucleus, putamen, precentral gyrus, and premotor cortex (131,137). Ballooned cells may be present in limbic structures in some PSP cases in which argyrophilic grain disease cooccurs (138). Some brains show Lewy bodies in a pattern similar to that in Parkinson’s disease, although it remains unclear whether this is only a coincidental phenomenon (139). NFT in PSP consist of straight ﬁlaments, in contrast to the paired helical ﬁlaments in AD. The pattern of tau isoforms on Western blots of sarkosylinsoluble tau has two major bands of 64 and 68 kDa in most (140,141), but not all, PSP brains, whereas a more diffuse staining on Western blots is seen in some PSP cases (142). NFT in PSP contains predominantly 4-repeat isoforms, and ultrastructurally consists of straight ﬁlaments in the sarkosyl-insoluble fraction. The 4-repeat to 3-repeat ratio is increased in a proportion of PSP cases, with a decreased amount of the smallest 3R0N isoform (141,143,144). It is uncertain whether the change in 4 - repeat versus 3 - repeat ratio is due to an decrease in the amount of the smallest 3R0N isoform or to an increase in the amount of 4 repeat isoforms (141,144). Some brain regions (globus pallidus, brainstem, cerebellum) have higher expression levels of mRNA of 4-repeat tau than of 3-repeat tau isoforms (145,146). The role of the tau protein in the pathogenesis of PSP is further supported by the observation of a strong association with a dinucleotide polymorphism (A0) in the intron between exon 9 and 10 of the tau gene (147). The A0 allele is in linkage disequilibrium for PSP with several other single nucleotide polymorphisms within the tau gene including its promoter (148,149). Only two different haplotypes of the tau gene are present in the general population, H1 and H2; PSP patients are signiﬁcantly more often homozygous for the H1 haplotype than controls (80–90% in PSP vs. 50– 60% in controls). This H1 haplotype has been further extended to a 2 Mb region including the tau gene and several other candidate genes (150), and speciﬁc subhaplotypes of H1E and H2E have been identiﬁed that modify the risk for PSP (151). Very recently, a reﬁned physical map has shown that a 900-kb region of the H2 haplotype appears to be an inversion of the H1 haplotype (152). The genetic factor responsible for PSP may be a polymorphic or pathogenic variant in or outside the tau gene or in one of the other candidate genes, and may have an effect on tau splicing, expression, or both. The so-called Saitohin gene nested within the intron between exon 9 and 10 of the tau gene may be an interesting gene, since its Q allele is associated with occurrence of PSP

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although the protein still has an unknown function (153). One candidate pathogenic variant within this gene may be the Q allele, which is associated with the occurrence of PSP (154). Future studies may elucidate the relevance of Q allele and other polymorphisms within the haplotype block for the pathogenesis of PSP. Corticobasal Degeneration Corticobasal degeneration is a rare neurodegenerative disorder clinically characterized by a progressive late-onset parkinsonism associated with apraxia, alien limb, dystonia, and/or myoclonus. The clinicopathological correlation is poor. Pathologically conﬁrmed cases have clinically diagnosed with other diagnoses, including primary progressive aphasia (155,156), while clinically diagnosed CBD may pathologically be deﬁned as PSP, AD, Pick’s disease or Creutzfeldt–Jakob disease, (157–159). Pathologically, the main macroscopic ﬁndings are cortical atrophy, often asymmetric, of the posterior part of the frontal lobe, and pallor of the substantia nigra. Tau pathology in CBD consists of neuronal, oligodendroglial, and astrocytic lesions (160,161). Affected cortical areas, hippocampus, and striatum in CBD have numerous pretangles, and only occasionally a few NFT. Astrocytic plaques and coiled bodies in caudate nucleus, putamen, and prefrontal cortex are characteristic pathological features for CBD (137,156,162–164). The presence of ballooned or swollen neurons is an important histological feature, and their wide distribution in cingulate gyrus, amygdala, insular cortex, and claustrum distinguish CBD from other neurodegenerative disorders (156). Sarkosyl-insoluble tau in CBD consists predominantly of 4-repeat tau isoforms, as in PSP (143,165). Recent studies suggest that CBD and PSP (as disorders with 4-repeat isoforms) may be differentiated from each other by distinct tau fragments, reﬂecting a difference in proteolytic processing of abnormal tau in the two conditions (166). Similar to PSP, pathological proven cases of CBD have shown a signiﬁcantly higher frequency of the H1 hapotype and H1/H1 genotype compared to controls (0.92 and 0.84 vs. 0.77 and 0.60) (167). Sporadic Pick’s disease Slowly progressive aphasia in a patient with temporal lobe atrophy of the brain at autopsy is the ﬁrst description of the disease by Arnold Pick in 1892. Round, argyrophilic neuronal inclusions, (so-called Pick bodies), were described by Onari and Spatz in 1926 for the ﬁrst time, and their presence is required for the diagnosis of Pick’s disease (168). The age at onset is usually between 50 and 70 years. Pick’s disease is indistinguishable from other FTD forms and may present with primary progressive aphasia or a frontal lobe syndrome. Hereditary Pick’s disease is associated with several mutations in the tau gene (46,98,109,169), whereas the etiology in the sporadic form of Pick’s disease is still unknown. The two forms of Pick’s disease are clinically indistinguishable. Frontotemporal cortex, hippocampus, substantia nigra,

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and less often basal ganglia show severe neurodegenerative changes. The disease is pathologically characterized by ballooned cells and round, argyrophilic neuronal inclusions (Pick bodies) mainly seen in the dentate gyrus of the hippocampus, and in layers II and VI of the frontal and temporal neocortex (160,170,171). Pick bodies stain positive with tau- and ubiquitin-antibodies, and consist of straight and twisted ﬁlaments (171). Pick bodies in sporadic Pick’s disease usually consist exclusively of 3-repeat tau isoforms (172–174), although recent studies have shown a mixture of 3- and 4-repeat tau isoforms or even predominantly 4-repeat tau in some cases (175). In contrast to the signiﬁcant association between 4-repeat tauopathies and H1 haplotype, Pick’s disease (as a 3-repeat disorder) is not associated with H2 haplotpye (176). Argyrophilic Grain Disease Argyrophilic grain disease (AGD) is a sporadic late-onset dementia with a prevalence that increases with age (177). It is characterized by abundant argyrophilic grains in neuronal processes throughout sector CA1 of the hippocampus, entorhinal cortex, and amygdala (178,179). Coiled bodies in oligodengroglia are usually present in deeper cortical layers and white matter (180). Although argyrophilic grains co-occur in several other neurodegenerative diseases, like AD, PSP, and multiple system atrophy, they exist also in pure form (181). Argyrophilic grains stain positive with Gallyas silver staining and AT8 tau-antibody, and consist of 9–19 nm straight ﬁlaments (180). The amygdala usually has aB-crystallin-expressing ballooned cells and tau-positive astrocytes (138). Biochemically, sarkosyl-insoluble tau in AGD mainly consists of 4-repeat tau isoforms similar to PSP and CBD, whereas the soluble fraction shows normal expression of all six isoforms (179,180,182). The frequency of the H1/H1 genotype in AGD subjects is siginifcantly higher than in controls (182,183), although this association has not been conﬁrmed in all studies (184). The Q7 polymorphism in the Saitohin gene, coding for arginine in a protein of 128 amino acids, is overrepresented in AGD subjects (185).

CONCLUSIONS The recognition of the etiological role of the tau gene in disorders with dementia or parkinsonism has had important implications for clinical practice, i.e., genetic counselling. Studies on tau mutations in FTDP-17 have shown that dysfunction or dysregulation of the tau protein can cause neurodegenerative changes in the presence of accumulation of hyperphosphorylated tau protein in neurons and glial cells. In addition, clinical and pathological observations in other tauopathies as PSP, CBD, Pick’s disease and argyrophilic grain disease support the hypothesis that the tau protein also plays an important role in the pathogenesis of the disorders (186). Some

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functional effects of tau mutations have been clariﬁed, for example, their effect on microtubule binding, and the change in ratio of 4- versus 3-repeat tau isoforms. However, it is unknown how this change in isoform composition leads to neuronal and glial tau pathology. The correlation between the location of the mutation within tau gene at one end and tau pathology or tau isoform proﬁle at the other end is weaker than considered earlier. It is unknown how missense mutations outside the exons 9–12 lead to a reduction of microtubule binding activity. Also, the pathophysiological signiﬁcance of glial pathology in both FTDP-17 and related disorders awaits further explanation. The factors contributing to the variation in clinical phenotype, including the phenomenon of incomplete penetrance have to be determined. Perhaps, the identiﬁcation of the genetic defect responsible for the hereditary non-tau FTDP-17 will provide more understanding of tau-related neurodegeneration, although it might also reﬂect a completely independent pathophysiological mechanism. Finally, the elucidation of the genetic factor that is necessary but not sufﬁcient to cause PSP and CBD will be a major step forward in the understanding of tauopathies. REFERENCES 1. Crowther RA. Straight and paired helical ﬁlaments in Alzheimer disease have a common structural unit. Proc Natl Acad Sci USA 1991; 88:2288–2292. 2. Goedert M, Spillantini MG, Cairns NJ, Crowther RA. Tau proteins of Alzheimer paired helical ﬁlaments: abnormal phosphorylation of all six brain isoforms. Neuron 1992; 8:159–168. 3. Flament S, Delacourte A, Hemon B, Defossez A. Characterization of two pathological tau protein, variants in Alzheimer brain cortices. J Neurol Sci 1989; 92:133–141. 4. Poorkaj P, Bird TD, et al. Tau is a candidate gene for chromosome 17 frontotemporal dementia. Ann Neurol 1998; 43:815–825. 5. Hutton M, Lendon CL, et al. Association of missense and 50 -splice-site mutations in tau with the inherited dementia FTDP-17. Nature 1998; 393:702– 705. 6. Spillantini MG, Murrell JR, et al. Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc Natl Acad Sci USA. 1998; 95:7737–7741. 7. Lee G, Neve RL, Kosik KS. The microtubule binding domain of tau protein. Neuron 1989; 2:1615–1624. 8. Kar S, Fan J, et al. Repeat motifs of tau bind to the insides of microtubules in the absence of taxol. Embo J 2003; 22:70–77. 9. Goedert M, Spillantini MG, et al. Multiple isoforms of human microtubuleassociated protein tau: sequences and localization in neuroﬁbrillary tangles of Alzheimer’s disease. Neuron 1989; 3:519–526. 10. Goedert M, Jakes R. Expression of separate isoforms of human tau protein: correlation with the tau pattern in brain and effects on tubulin polymerization. EMBO J 1990; 9:4225–4230.

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130. Morris HR, Gibb G, et al. Pathological, clinical and genetic heterogeneity in progressive supranuclear palsy. Brain 2002; 125:969–975. 131. Josephs KA, Dickson DW. Diagnostic accuracy of progressive supranuclear palsy in the Society for Progressive Supranuclear Palsy brain bank. Mov Disord 2003; 18:1018–1026. 132. Osaki Y, Ben-Shlomo Y, et al. Accuracy of clinical diagnosis of progressive supranuclear palsy. Mov Disord 2004; 19:181–189. 133. Oide T, Ohara S, et al. Progressive supranuclear palsy with asymmetric tau pathology presenting with unilateral limb dystonia. Acta Neuropathol (Berl) 2002; 104:209–214. 134. Mochizuki A, Ueda Y, et al. Progressive supranuclear palsy presenting with primary progressive aphasia–clinicopathological report of an autopsy case. Acta Neuropathol (Berl) 2003; 105:610–614. 135. Litvan I, Agid Y, et al. Clinical research criteria for the diagnosis of progressive supranuclear palsy (Steele-Richardson-Olszewski syndrome): report of the NINDS-SPSP international workshop. Neurology 1996; 47:1–9. 136. Hauw JJ, Verny M, et al. Constant neuroﬁbrillary changes in the neocortex in progressive supranuclear palsy. Basic differences with Alzheimer’s disease and aging. Neurosci Lett 1990; 119:182–186. 137. Hattori M, Hashizume Y, et al. Distribution of astrocytic plaques in the corticobasal degeneration brain and comparison with tuft-shaped astrocytes in the progressive supranuclear palsy brain. Acta Neuropathol (Berl) 2003; 106:143–149. 138. Togo T, Dickson DW. Ballooned neurons in progressive supranuclear palsy are usually due to concurrent argyrophilic grain disease. Acta Neuropathol (Berl) 2002; 104:53–56. 139. Mori H, Oda M, et al. Lewy bodies in progressive supranuclear palsy. Acta Neuropathol (Berl) 2002; 104:273–278. 140. Flament S, Delacourte A, et al. Abnormal Tau proteins in progressive supranuclear palsy. Similarities and differences with the neuroﬁbrillary degeneration of the Alzheimer type. Acta Neuropathol (Berl) 1991; 81:591–596. 141. Liu WK, Le TV, et al. Relationship of the extended tau haplotype to tau biochemistry and neuropathology in progressive supranuclear palsy. Ann Neurol 2001; 50(4):494–502. 142. Arai T, Ikeda K, et al. Different immunoreactivities of the microtubule-binding region of tau and its molecular basis in brains from patients with Alzheimer’s disease, Pick’s disease, progressive supranuclear palsy and corticobasal degeneration. Acta Neuropathol (Berl) 2003; 105:489–498. 143. Arai T, Ikeda K, et al. Distinct isoforms of tau aggregated in neurons and glial cells in brains of patients with Pick’s disease, corticobasal degeneration and progressive supranuclear palsy. Acta Neuropathol (Berl) 2001; 101(2): 167–173. 144. Gibb GM, de Silva R, et al. Differential involvement and heterogeneous phosphorylation of tau isoforms in progressive supranuclear palsy. Brain Res Mol Brain Res 2004; 121:95–101.

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145. Takanashi M, Mori H, et al. Expression patterns of tau mRNA isoforms correlate with susceptible lesions in progressive supranuclear palsy and corticobasal degeneration. Brain Res Mol Brain Res 2002; 104:210–219. 146. Chambers CB, Lee JM, et al. Overexpression of four-repeat tau mRNA isoforms in progressive supranuclear palsy but not in Alzheimer’s disease [In Process Citation]. Ann Neurol 1999; 46:325–332. 147. Conrad C, Andreadis A, et al. Genetic evidence for the involvement of tau in progressive supranuclear palsy. Ann Neurol 1997; 41:277–281. 148. Baker M, Litvan I, et al. Association of an extended haplotype in the tau gene with progressive supranuclear palsy. Hum Mol Genet 1999; 8:711–715. 149. Pastor P, Ezquerra M, et al. Further extension of the H1 haplotype associated with progressive supranuclear palsy. Mov Disord 2002; 17:550–556. 150. Pittman AM, Myers AJ, et al. The structure of the tau haplotype in controls and in progressive supranuclear palsy. Hum Mol Genet 2004; 13:1267–1274. 151. Pastor P, Ezquerra M, et al. Novel haplotypes in 17q21 are associated with progressive supranuclear palsy. Ann Neurol 2004; 56:249–258. 152. Stefansson H, Helgason A, et al. A common inversion under selection in Europeans. Nat Genet 2005. 153. Conrad C, Vianna C, Freeman M, Davies P. A polymorphic gene nested within an intron of the tau gene: implications for Alzheimer’s disease. Proc Natl Acad Sci USA 2002; 99:7751–7756. 154. de Silva R, Hope A, et al. Strong association of the Saitohin gene Q7 variant with progressive supranuclear palsy. Neurology 2003; 61:407–409. 155. Ferrer I, Hernandez I, et al. Primary progressive aphasia as the initial manifestation of corticobasal degeneration and unusual tauopathies. Acta Neuropathol (Berl) 2003; 106:419–435. 156. Dickson DW, Bergeron C, et al. Ofﬁce of Rare Diseases neuropathologic criteria for corticobasal degeneration. J Neuropathol Exp Neurol 2002; 61: 935–946. 157. Boeve BF, Maraganore DM, et al. Pathologic heterogeneity in clinically diagnosed corticobasal degeneration. Neurology 1999; 53:795–800. 158. Lang AE. Corticobasal degeneration: selected developments. Mov Disord 2003; 18(Suppl 6):S51–S560. 159. Kertesz A, Munoz D. Relationship between frontotemporal dementia and corticobasal degeneration/progressive supranuclear palsy. Dement Geriatr Cogn Disord 2004; 17:282–286. 160. Iwatsubo T, Hasegawa M, Ihara Y. Neuronal and glial tau-positive inclusions in diverse neurologic diseases share common phosphorylation characteristics. Acta Neuropathol (Berl) 1994; 88:129–136. 161. Mori H, Nishimura M, Namba Y, Oda M. Corticobasal degeneration: a disease with widespread appearance of abnormal tau and neuroﬁbrillary tangles, and its relation to progressive supranuclear palsy. Acta Neuropathol (Berl) 1994; 88:113–121. 162. Komori T. Tau-positive glial inclusions in progressive supranuclear palsy, corticobasal degeneration and Pick’s disease. Brain Pathol 1999; 9:663–679. 163. Feany MB, Dickson DW. Widespread cytoskeletal pathology characterizes corticobasal degeneration. Am J Pathol 1995; 146:1388–1396.

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164. Katsuse O, Iseki E, et al. 4-repeat tauopathy sharing pathological and biochemical features of corticobasal degeneration and progressive supranuclear palsy. Acta Neuropathol (Berl) 2003; 106:251–260. 165. Buee Scherrer V, Hof PR, et al. Hyperphosphorylated tau proteins differentiate corticobasal degeneration and Pick’s disease. Acta Neuropathol (Berl) 1996; 91:351–359. 166. Arai T, Ikeda K, et al. Identiﬁcation of amino-terminally cleaved tau fragments that distinguish progressive supranuclear palsy from corticobasal degeneration. Ann Neurol 2004; 55:72–79. 167. Houlden H, Baker M, et al. Corticobasal degeneration and progressive supranuclear palsy share a common tau haplotype. Neurology 2001; 56(12):1702–1706. 168. Rossor MN. Pick’s disease: a clinical overview. Neurology 2001; 56:S3–S5. 169. Schenk VW. Re–examination of a family with Pick’s disease. Ann Hum Genet 1959; 23:325–333. 170. Probst A, Tolnay M, et al. Pick’s disease: hyperphosphorylated tau protein segregates to the somatoaxonal compartment. Acta Neuropathol (Berl) 1996; 92:588–596. 171. Dickson DW. Neuropathology of Pick’s disease. Neurology 2001; 56:S16–2S0. 172. Sergeant N, David JP, et al. Different distribution of phosphorylated tau protein isoforms in Alzheimer’s and Pick’s diseases. FEBS Lett 1997; 412: 578–582. 173. Delacourte A, Robitaille Y, et al. Speciﬁc pathological Tau protein variants characterize Pick’s disease. J Neuropathol Exp Neurol 1996; 55:159–168. 174. Delacourte A, Sergeant N, et al. Vulnerable neuronal subsets in Alzheimer’s and Pick’s disease are distinguished by their tau isoform distribution and phosphorylation. Ann Neurol 1998; 43:193–204. 175. Zhukareva V, Mann D, et al. Sporadic Pick’s disease: a tauopathy characterized by a spectrum of pathological tau isoforms in gray and white matter. Ann Neurol 2002; 51:730–739. 176. Morris HR, Baker M, et al. Analysis of tau haplotypes in Pick’s disease Corticobasal degeneration and progressive supranuclear palsy share a common tau haplotype. Association of an extended haplotype in the tau gene with progressive supranuclear palsy. Neurology 2002; 59:443–445. 177. Braak H, Braak E. Argyrophilic grain disease: frequency of occurrence in different age categories and neuropathological diagnostic criteria. J Neural Transm 1998; 105:801–819. 178. Braak H, Braak E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol (Berl) 1991; 82:239–259. 179. Tolnay M, Sergeant N, et al. Argyrophilic grain disease and Alzheimer’s disease are distinguished by their different distribution of tau protein isoforms. Acta Neuropathol (Berl) 2002; 104:425–434. 180. Zhukareva V, Shah K, et al. Biochemical analysis of tau proteins in argyrophilic grain disease, Alzheimer’s disease, and Pick’s disease : a comparative study. Am J Pathol 2002; 161:1135–1141. 181. Martinez-Lage P, Munoz DG. Prevalence and disease associations of argyrophilic grains of Braak. J Neuropathol Exp Neurol 1997; 56:157–164.

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182. Togo T, Sahara N, et al. Argyrophilic grain disease is a sporadic 4-repeat tauopathy. J Neuropathol Exp Neurol 2002; 61:547–556. 183. Ishizawa T, Ko LW, et al. Selective neuroﬁbrillary degeneration of the hippocampal CA2 sector is associated with four-repeat tauopathies. J Neuropathol Exp Neurol 2002; 61:1040–1047. 184. Miserez AR, Clavaguera F, et al. Argyrophilic grain disease: molecular genetic difference to other four-repeat tauopathies. Acta Neuropathol (Berl) 2003; 106:363–366. 185. Conrad C, Vianna C, et al. Molecular evolution and genetics of the Saitohin gene and tau haplotype in Alzheimer’s disease and argyrophilic grain disease. J Neurochem 2004; 89:179–188. 186. Goedert M. Tau protein and neurodegeneration. Semin Cell Dev Biol 2004; 15:45–49.

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27 Amyotrophic Lateral Sclerosis Teepu Siddique Davee Department of Neurology and Clinical Neurosciences and Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, U.S.A.

Lisa Dellefave Davee Department of Neurology and Clinical Neuroscience, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, U.S.A.

ALS BACKGROUND Amyotrophic lateral sclerosis (ALS), also called Charcot’s disease or Lou Gehrig’s disease, belongs to a larger group of disorders affecting the motor neurons. A relay of two systems of motor neurons, the upper motor neuron (UMN) and lower motor neuron (LMN), controls voluntary muscles, and symptoms can vary based on the degree of involvement of each component of this relay system. Disorders of the motor neuron can be classiﬁed based on the involvement of one or both components of the relay system (Tables 1a and 1b). Other overlapping classiﬁcations are based on the topography of involvement (primary lateral sclerosis, spastic paraparesis, spinal muscular atrophy, and progressive bulbar palsy) (Table 2). ALS involves both the UMN and LMN. Clinical Symptoms of ALS ALS is a progressive paralytic disorder caused by degeneration of UMNs of the motor cortex in the brain and LMNs in the brain stem and spinal cord. The annual incidence of ALS worldwide is 1 to 3 per 100,000 (1,2).

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Motor neuron disease

694

Table 1a Categories of Disorders of the Motor Neuron: Lower Motor Neuron Involvement Only Inheritance

Gene

Protein

Clinical features of disease

References

SMA1

AR

SMN

Survival motor neuron protein

112

SMA2

AR

SMN

Survival motor neuron protein

SMA3

AR, AD

SMN

Survival motor neuron protein

SMA4

AR

SMN

Survival motor neuron protein

AD

VAPB

VAPB

AR

IGHMP2

Immunoglobulin mu binding protein 2

AR

Loci: 11q13.3

Unknown

AD

GARS

Glycyl tRNA synthase

Onset in utero to 6 months. Presents with hypotonia and weakness; problems with sucking, swallowing, and breathing. Never able to sit. Onset between 3 and 15 months. Proximal leg weakness, fasciculations, ﬁne hand tremor. Never able to stand. Facial muscles spared. Onset 15 months to teen years Proximal leg weakness, delayed motor milestones. Onset is median age of 37 years. Proximal weakness; variable within families. Late onset SMA Finkel type atypical ALS in seven families. Infantile onset. Distal muscle atrophy and weakness with severe respiratory involvement. Childhood onset. Distal muscle atrophy and weakness. Adult onset. Slowly progressive distal amyotrophy, upper limb predominant.

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112

112

87 113

114 115

Siddique and Dellefave

Distal spinal muscular atrophy (SMA5)

112

BSCL2

Seipin

AD

Loci: 12q2-q24

Unknown

AD

Loci: 2q14

Unknown

AD

Loci: 12q24.3

Unknown

X-linked recessive

Loci: Xq13.1-q21

Unknown

X-linked recessive

AR

Androgen Receptor

AD

DCTN1

Dynactin

Adult onset. Slowly progressive distal amyotrophy. Congenital, non-progressive with contractures. Adult onset. Slowly progressive distal amyotrophy with vocal cord paralysis. Adult onset. Slowly progressive distal amyotrophy. Childhood onset pes cavus or varus. Gait instability with distal amyotrophy. Onset teens to adulthood. Proximal muscle weakness, muscle atrophy, and fasciculations. Onset is early adulthood. Progressive weakness of facial muscles, hands, and distal lower extremities without sensory symptoms. Respiratory muscles involved. Single family identiﬁed.

116,117 118 119

120 121

Amyotrophic Lateral Sclerosis

Spinal bulbar muscular atrophy Spinal bulbar muscular atrophy

AD

122

123

Abbreviations: SMA, survival muscular atrophy; SMN, survival motor neuron; AR, autosomal recessive; AD, autosomal dominant.

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695

Motor neuron disease

696

Table 1b Categories of Disorders of the Motor Neuron: Upper Motor Neuron Involvement Only Inheritance

Type Gene/Loci

Protein

Clinical features of disease

References

Juvenile primary lateral sclerosis JPLS1 (ALS2)a

AR

ALS2

Alsin

Progressive ascending UMN disorder starting in infancy with lower extremities and eventually involving the arms and bulbar regions.

19,124

Hereditary spastic paraplegia (HSP) pure

AD

SPG3A: SPG3A gene

SPG4: SPG4 gene

SPG8: 8q23-q24

SPG10: KIF5A gene

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125

126

127

128

129

Siddique and Dellefave

SPG6: NIPA1 gene

In general, symptoms of AD HSP pure are: Onset varies intra- and interfamily. Bilateral lower extremity spastic weakness often with urinary bladder disturbance. Atlastin Symptom onset may be earlier (less than 11 years). Typical features of AD HSP. Spastin Symptom onset may be later (after 20 years). Cognitive impairment in some cases. Non-imprinted Symptom onset may be later Prader-Willi syndrome 1 (after 20 years). Typical features of AD HSP. Unknown Symptom onset may be later (after 20 years). Typical features of AD HSP. Neuronal kinesin heavy Symptom onset may be earlier chain protein (less than 11 years). Typical features of AD HSP.

AR

HSP complicated

Unknown

SPG13: HSPD1 gene

60kDa heat shock protein

SPG19: 9q33-q34

Unknown

SPG5A: 8p12-q13

Unknown

SPG11: 15q13-q15

Unknown

SPG24: 13q14

Unknown

SPG9: 10q23.3-q24.1

Unknown

SPG17: (Silver Syndrome) BSCL2 gene

Seipin

SAX1: 12p13

Unknown

Symptom onset may be earlier (less than 11 years). Typical features of AD HSP. Symptom onset may be later (after 20 years). Typical features of AD HSP. Symptom onset may be later (after 20 years). Typical features of AD HSP. Childhood onset. Spasticity of lower limbs, abnormally active tendon reﬂexes, dysfunction of bladder spincter. Hereditary spastic paraparesis with thin corpus callosum. Hereditary spastic paraparesis with sensorineural deafness Mean age of onset, 3rd decade. Spastic paraplegia with cataracts, gastrointestinal reﬂux, motor neuronopathy. Onset juvenile or adult. Spastic paraplegia with amyotrophy – spasticity of lower limbs accompanied by weakness and wasting of small hand muscles. Spasticity, ataxia, dysarthria, dysphagia, and eye movement abnormalities.

130

131

132

133

134 135 136

117,137

138 697

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AD

SPG12: 19q13

Amyotrophic Lateral Sclerosis

HSP pure

(Continued)

698

Table 1b

Categories of Disorders of the Motor Neuron: Upper Motor Neuron Involvement Only (Continued )

Motor neuron disease

Inheritance

Type Gene/Loci

Protein

Clinical features of disease

References

AR

SPG7: SPG7 gene

Paraplegin

139

SPG15: 14q22-q24

Unknown

Troyer syndrome: SPG20 gene

Spartin

SPG21: SPG21 gene

Maspardin

ARSACS: SACS gene

Sacsin

Mean age of onset is 25 years. Progressive lower extremity weakness spasticity, hyperreﬂexia, dysarthria, dysphagia, optic disc pallor, optic atrophy, axonal neuropathy, evidence of vascular lesions on MRI. Spasticity with pigmented maculopathy, distal amyotrophy, dysarthria, and mental retardation. Onset is childhood. Spastic tetraplegia dysarthria, with distal muscle wasting, short stature, learning difﬁculties, delay in motor milestones, emotional lability. Young adult onset. Spasticity with cognitive decline. MRI shows cerebral, cerebellar, corpus callosum atrophy with white matter hyperintensity. Absent sensory nerve conduction, reduced motor-

HSP complicated

86

141

142

Siddique and Dellefave

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140

X-linked

Primary lateral sclerosis

SPG1: L1CAM gene

Neural cell adhesion molecule L1 protein

SPG2: PLP gene

Myelin proteolipid protein

SPG16: Xq11.2-q23

Unknown

Sporadic

Mutations in L1CAM can cause a variety of phenotypes: Spastic paraplegia with mental retardation and adducted thumbs; X-linked hydrocephalus; MASA syndrome; CRASH syndrome. Mutations in PLP can cause a variety of phenotypes ranging from Pelizaeus-Merzbacher to SPG2. SPG2 manifests as spastic paraparesis similar to uncomplicated AD-HSP. Phenotype similar to SPG2 however do not have mutations in PLP gene. Onset adulthood. Lower and upper extremity spasticity and weakness.

143

Amyotrophic Lateral Sclerosis

nerve velocity, hypermyelination of retinalnerve ﬁbers. High carrier frequency in descendents of Charlevoix-Saguenay-LacSaint-Jean region of Quebec.

144

145

146

a

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699

Can also present as UMN predominant ALS (Table 4). Abbreviation: AD, autosomal dominant; AR, autosomal recessive.

700

Siddique and Dellefave

Table 2 Overlapping Classiﬁcations Based on Topography of Involvement Condition

UMN

LMN

Sensory

Bulbar

Axial

UEs

LEs

PBP ALS PLS SP PMA Monomelic atrophy

þ þ þ þ –

– þ – – þ þ

– – – –

þ þ þ – – –

– – – –

– þ þ – þ þ (one extremity)

– þ þ þ þ þ (one extremity)

Abbreviation: UMN, upper motor neuron; LMN, lower motor neuron; PBP, progressive bulbar palsy; ALS, amyotrophic lateral sclerosis; PLS, primary lateral sclerosis, SP, spastic paraparesis; PMA, progressive muscular atrophy; LE, lower extremity; UE, upper extremity; (þ), present; (), absent.

Symptoms of ALS can occur initially as focal involvement of UMN and/or LMN dysfunction at several levels of the neuraxis without involvement of other systems. The diagnosis of ALS is one of exclusion, as there is no deﬁnitive test to diagnose it, and for purposes of research, the El Escorial Criteria is used as a standard for the diagnosis of ALS (Table 3) (3,4). Patients with ALS generally present with symptomatic weakness; the symptoms can vary based on the particular muscles involved. A history of fasciculations, muscle cramping, atrophy, and weakness can be usually elucidated. A diagnostic feature of ALS, unusual in other disorders, is the presence of hyper-reﬂexia in segmental regions of muscle atrophy, unaccompanied by sensory disturbance. At presentation, limb involvement occurs more often than bulbar involvement, with upper limbs more often affected than lower limbs in sporadic ALS (SALS) (5), whereas leg involvement is typical of familial ALS (FALS) (5,6). The pattern of involvement is almost always asymmetrical or focal. With UMN involvement, the symptoms are of clumsiness, stiffness, weakness and fatigue whereas LMN degeneration presents as weakness, atrophy, fasciculations, and cramps. Bulbar symptoms (as in progressive bulbar palsy) include slurring of speech, choking on liquids, difﬁculty initiating swallowing, drooling, weak cough, and sometimes hoarseness. Charcot ﬁrst deﬁned those symptoms as labio-pharyngeal paralysis. Paresthesias and sensory symptoms affect up to 25% of patients, but when present, are mild. There is wide variation in disease progression and duration, neither of which can be accurately predicted from age or site of onset, although older patients generally have a shorter survival. Bowel, bladder, and sexual functioning are usually spared, with about 4% of patients experiencing loss of sphincter control. ALS patients have diminished skin elasticity and rarely develop bedsores, possibly on account of the absence of sensory

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Amyotrophic Lateral Sclerosis

701

Table 3 Revised El Escorial Criteria for the Diagnosis of Amyotrophic Lateral Sclerosis The diagnosis of ALS requires the presence of symptoms of lower motor neuron (LMN) degeneration by clinical, electrophysiological or neuropathologic examination AND evidence of upper motor neuron (UMN) degeneration by clinical examination AND progressive spread of symptoms or signs within a region or to other regions, as determined by history or examination, together with the absence of electrophysiological or pathological evidence of other disease processes that might explain the signs of LMN and/or UMN degeneration and neuroimaging evidence of other disease processes that might explain the observed clinical and electrophysiological signs. The clinical diagnosis of ALS, without pathological conﬁrmation, may be categorized into various levels of certainty by clinical and laboratory assessment: Clinically deﬁnite ALS: The presence of UMN signs, as well as LMN signs, in three regions. Clinically deﬁnite familial-laboratory supported ALS: ALS presenting with progressive UMN and/or LMN signs in at least a single region (in the absence of another cause for the abnormal neurological signs) with an identiﬁed diseasecausing mutation in the SOD1 gene in the proband or a positive family history of an individual with an identiﬁed disease-causing mutation in the SOD1 gene. Clinically probably ALS: The presence of UMN and LMN signs in at least two regions with some UMN signs necessarily rostral to the LMN signs. Clinically probable-laboratory supported ALS: Clinical signs of UMN and LMN dysfunction are in only one region, or when UMN signs alone are present in one region, and LMN signs deﬁned by EMG criteria are present in at least two limbs. Clinically possible ALS: clinical symptoms of UMN and LMN dysfunction are found together in only one region or UMN signs are found alone in two or more regions; or LMN signs are found rostral to UMN signs and the diagnosis of clinically probable-laboratory supported ALS cannot be proven by evidence on clinical grounds in conjunction with electrodiagnostic , neurophysiologic, neuroimaging or clinical laboratory studies. Clinically suspected ALS: a pure LMN syndrome. Abbreviations: ALS, amyotrophic lateral sclerosis; SOD, superoxide dismutase; EMG, electromyography.

loss and possibly ongoing fasciculations. Accumulation of a basic protein in the skin and an alteration in collagen turnover has also been reported (7,8). The progressive paralysis in ALS usually affects respiratory function, resulting in ventilatory failure and death; 50% of patients die within three years of onset of symptoms and 90% within ﬁve years. Recently, there has been an increasing recognition of frontal lobe involvement in ALS, but obvious frontotemporal lobe dementia probably occurs in a very small minority of ALS patients. There is no known treatment to prevent, halt, or reverse the disease, although marginal delay in mortality has been noted with the drug Riluzole (9).

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702

Siddique and Dellefave

Pathology of ALS Neuronal loss of both UMNs and LMNs is characteristic of ALS. Other pathologic features can be present and can assist in the diagnosis but are not essential to diagnose ALS. Bunina bodies and ubiquinated skein-like inclusions are hallmark features of both SALS and FALS. The protein constituent of skein-like inclusions is unknown and does not stain for proteins typically seen in other neurodegenerative diseases, such as tau, alpha-synuclein, and cystatin C. The protein constituent of Bunina bodies has been identiﬁed as cystatin C (10). Cystatin C regulates extracellular cysteine protease activity, which results from release of lysosomal proteinases from dying or diseased cells; however, the function of the Bunina body is unknown. Hirano et al. (11) described neuronal Lewy-body-like hyaline inclusions (LBHIs) as a characteristic feature of FALS with posterior column involvement. In SOD1linked ALS these may contain SOD1 (12,13). Genetic Application to ALS Approximately 90% of ALS cases occur in individuals with no family history of ALS, and as such are said to have SALS (14) (Fig. 1). Approximately 10% of individuals with ALS have at least one other affected family member and are classiﬁed as having FALS. Because the majority of cases are sporadic, ALS was considered to be a non-genetic disease. However, as genetic technology and statistical tools became advanced, it became possible to address the genetic contribution in familial subgroups of largely sporadic disorders. Most FALS cases have a Mendelian pattern of inheritance, as has been shown in 20% of FALS families who have mutations in the Cu, Zn, Superoxide Dismutase (SOD1) gene as the cause of disease (15,16). Understanding

Figure 1 Distribution of familial versus sporadic ALS. Abbreviations: FALS, familial amyotrophic lateral sclerosis, SALS, sporadic amyotrophic lateral sclerosis.

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Amyotrophic Lateral Sclerosis

703

the basis of genetic locus heterogeneity in ALS and the etiopathogenesis in other motor neuron diseases (Table 1a and 1b) provides multiple starting points in search of possible common intersections in molecular pathways. APPROACHES TO MENDELIAN INHERITED ALS Single genes that cause Mendelian inherited disease are generally more penetrant than genes that cause disease through multifactorial inheritance and are more easily identiﬁed. The SOD1 locus for ALS was identiﬁed through the now commonly employed strategy of positional cloning (17). High consanguinity lends itself to homozygosity mapping (18) and was successfully used to identify loci for ALS2 (19) and ALS5 (17). These strategies use genotyping of highly polymorphic markers at average distances of 20, 10, or 5 centimorgans (cm). Recently, use of microsatellite markers and restriction fragment length polymorphisms (RFLPs) has been reported with dense arrays of single nucleotide polymorphisms (SNPs) placed on chips or beads for rapid genotyping. The application of DNA genotyping and statistical genetic technique was ﬁrst demonstrated in FALS in 1988 (14) and then used to identify genetic locus heterogeneity in FALS and linkage to the SOD1 locus on chromosome 21q22 (20). Tight linkage of FALS to markers on chromosome 21q22 just 2.5 megabases from the SOD1 gene led to the identiﬁcation of mutations in SOD1 and established it as the ﬁrst causative gene for ALS (15,16). Subsequently, six additional genetic loci for FALS and seven loci for related motor neuron degenerations were identiﬁed (Tables 4 and 5). Thus, locus genetic heterogeneity established the multi-etiologic basis of FALS, hence scleroses rather than sclerosis is a more accurate appellation, and is probably also true of SALS (ﬁrst publicly articulated by Stanley Appel). Once the causative gene for a disease is identiﬁed, the primary goal is to identify the mechanism by which disease is caused. This usually includes the development of genetically modiﬁed animal models. The gene mutation may result in the loss of a normal function (homozygous or dominant negative effect), a haploinsufﬁciency, or the gain of a completely novel and toxic function. The latter is especially true in many dominantly inherited disorders of neurodegeneration such as ALS (SOD1), Huntington’s disease (HD), Alzheimer’s disease (AD), Parkinson’s disease (PD), and Spinocerebellar ataxias (SCA). Mendelian Inherited ALS Mendelian inherited ALS accounts for a minority of ALS cases (10%) but provides an important resource to identify the etiopathogenesis of motor neuron degeneration. FALS can be transmitted as a dominant or a recessive

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704

Table 4 ALS Genes and Loci Percentage of patients with familial

Disease name

Gene

Locus

Inheritance

20%

ALS1

AD

FALS

SOD1

21q22.1

Rare

ALS2

AR

ALS2

2q33

Rare Rare

ALS3 ALS5

AD AR

One family Rare One family

ALS6 ALS7 XALS

AD AD X-linked dominant

Juvenile ALS Type 2 FALS Juvenile ALS Type 5 FALS FALS FALS

18q21 15q15.1q21.1 16q12 20ptel X

Product Superoxide Dismutase (Cu–Zn) Alsin

16

19,147

Unknown Unknown

148 17

Unknown Unknown Unknown

149,150,151 151 21

Abbreviations: ALS, amyotrophic lateral sclerosis; AD, autosomal dominant; AR, autosomal recessive.

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References

Siddique and Dellefave

Locus name

Percentage of patients with familial

Locus name

Inheritance

Disease name

Rare

ALS4a

AD

Rare

ALS8b

AD

FTDc/ALS FTD/ALS SPG17

AD AD AD AD

Heriditary motor neuropathy with pyramidal signs SMAIV, Finkel type ALS with FTD ALS with FTD Silver Syndrome IBMPFDd

SPG20

AR AR

Troyer syndrome Distal SMA5

Rare (<1%) Rare

Rare

Gene

Locus

Product

References

SETX

9q34

Senataxin

91,152

VAPB

20q13

VAPB

187

Unknown Unknown BSCL2 VCP

9q21-q22 17q 11q12-q14 9p21.1-p12

153 154 117,137 90

SPG20 Unknown

13q12.3 9p21.1-p12

Unknown Unknown Seipin Valosin containing protein Spartin Unknown

Amyotrophic Lateral Sclerosis

Table 5 ALS Related Motor Neuron Disorders Genes and Loci with both UMN and LMN Involvement

86 155

a

Never have bulbar involvement. Slow progression, long duration, distal wasting with pyramidal signs and sensory loss (previously called axonal CMT with pyramidal signs). Mutations in Senataxin have also been identiﬁed in a different disorder of ataxia and oculomotor apraxia (AOA2). b This is a disorder that appears to be proximal SMA 4 (Finkel type) with some UMN ﬁndings. c Frontotemporal dementia. d Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia. Mutations within the gene may also cause pure FTD. Abbreviations: ALS, amyotrophic lateral sclerosis; FTD, frontotemporal dementia; AD, autosomal dominant; AR, autosomal recessive; IBMPFD, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia; SMA, spiral muscular atrophy.

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trait, but it is usually inherited as an adult-onset disorder with an autosomal dominant (AD) inheritance pattern. Autosomal recessive (AR) inheritance is rare and limited to juvenile onset ALS or to those persons with a double dose of the D90A or N86S mutations in the SOD1 gene. Interestingly, an X-linked dominantly inherited ALS has also been described a rarely observed phenomenon in neurogenetics (21). In view of the genetic heterogeneity demonstrated in ALS, it is hoped that each additional disease-causing gene identiﬁed will contribute to the understanding of the multiple etiologies of ALS.

UNDERSTANDING ALS PATHOPHYSIOLOGY THROUGH MENDELIAN INHERITED ALS Genetically modiﬁed mouse models are an important resource to understand the biochemical and cellular pathology at selected stages of the disease, as such an approach is obviously not possible in humans. A mouse model over-expressing the G93A mutation in SOD1 was made following the discovery of SOD1 mutations in 1994 (22) and replicated with other SOD1 mutations (23–26). A large proportion of the literature (350 papers since 1994) on the etiopathogenesis of ALS use these models. Even though ALS is a disorder of people (or mice) and does not occur in cell culture, cell or organotypic models are used to understand the basic mechanism of disease at the cellular level. The mutant gene can be transfected into the cells and the interaction of the mutant protein with other proteins, its cellular localization, and predisposition to aggregate formation can be studied. For example, individual organelles of the cell containing the mutant protein can be separated and studied for the inimical effects of the mutant protein on the organelles such as damage to energy homeostasis due to the toxic effect of mutant protein inside mitochondria. SALS (the most common category of ALS) is not associated with mutations in SOD1, but is clinically and to a great extent pathologically, indistinguishable from SOD1-linked ALS; it is postulated that a common cellular or biochemical pathway of degeneration of motor neurons may be involved in the two forms of the disease. The ALS phenotype may therefore result from a variety of disruptions in the molecular pathways required for motor neuron maintenance. Known Pathophysiology of ALS Cu,Zn Superoxide Dismutase Gene The SOD1 gene is about 11 kb in size, containing ﬁve exons and four introns with several alternatively spliced forms (27). The SOD1 protein consists of 153 highly conserved amino acids with copper and zinc binding sites (15). Over 100 mutations, predominantly missense, have been found in 68 of

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the 153 codons, spread over all ﬁve exons; of these, seven were detected in exon 3 (28–33) (www.alsod.org.). Function of the normal SOD1 protein: The superoxide dismutases are a group of three isoenzymes that play a key role in reducing free-radicalinduced cellular damage. They scavenge the superoxide (O2) free radicals produced as a by-product of oxidative respiration and the cytochrome P450 system: O2 þ Enz Cu2þ þ Hþ !O2 þ Enz Cuþ

ð1Þ

O2 þ Enz Cuþ þ Hþ !H2 O2 þ Enz Cu2

ð2Þ

O2 þ Hþ !H2 O2 þ O2

ð3Þ

SOD1 is primarily a cytosolic enzyme (34), but is also present in small amounts in mitochondria and other organelles (35,36). Manganese SOD (SOD2) is present in the mitochondria and its gene is located on chromosome 6q27 (28). Extracellular SOD (SOD3), also contains copper and zinc and its gene is coded on chromosome 4p (28). No mutations have been described in SOD2 or SOD3 in ALS patients. SOD1 is a 320 kDa homodimeric protein. Each monomer has a b-barrel fold and binds to one copper and one zinc ion (37). The dimer interface is stabilized by hydrophobic interactions, and dimerization doubles the dismutase activity of SOD1. An electrostatic channel guides superoxide ions to the active site, which contains Cu2þ. The channel is lined by the amino acids Lys 122, Lys 134, and Arg 143, which provide positive charges (37). Four cysteine residues are present in human SOD1, two are free (C6, C111) and the other two are oxidized as a sulfydryl bridge (C57, C146). The zinc ion provides stability and increases its melting temperature, as does the cysteine bridge C57–C146. The rate of the dismutase reaction is very rapid, 2 109 M1 S1, which probably indicates that the rate of the reaction is limited only by substrate availability (38). Access to the active site is size and charge selective, and thus speciﬁcally allows in the negatively charged superoxide ion, while excluding larger and positively charged ions (37). The mechanisms by which mutations in SOD1 cause FALS are not fully understood. Transgenic mice or rats over-expressing ALS associated mutant SOD1 develop an ALS-like phenotype, while transgenic mice overexpressing wild-type SOD1 (wtSOD1) remain unaffected; SOD1 knockout mice show axonal damage but do not show motor neuron degeneration suggesting that the 100 plus SOD1 mutants associated with ALS have a toxic property that triggers motor neuron degeneration in ALS (22–26,39,40). Etiopathogenesis of SOD1 ALS: Human studies in SOD1 linked ALS. Certain phenotypic features may be typical of SOD1 linked FALS,

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but they are non-speciﬁc and therefore not distinct enough to make a clinical diagnosis of SOD1 FALS versus FALS caused by a different gene. Human ALS caused by SOD1 has a mean age of onset of 45.5 8.9 years, with lower extremity onset being more common than upper extremity, and much more common than bulbar onset; these symptoms occur without gender bias (41). The duration of disease may vary by SOD1 mutation, but not onset; the age of onset of symptoms for those with SOD1 mutations ranges from 15 to 81 years (41,42). The rate of progression of the disease has been correlated to speciﬁc SOD1 gene mutations. For example, the A4V mutation, present in approximately 50% of all North American families, has consistently been associated with a rapid disease course of 1.0 0.4 years (41). In sharp contrast, there are mutations that confer a signiﬁcantly longer mean duration of disease of at least 10 years, such as the G37R (18.7 11.4 years; n ¼ 8, one family), G41D (17.0 6.3 years; n ¼ 7, one family), H46R (17.4 6.4 years; n ¼ 5, a Japanese family; a second North America family had similar duration of disease), and G93C (10.1 6.2 years; n ¼ 7, one family) (41,43). Extensive variability in progression has been documented for patients with the I113T mutation (2.5–20 years) (44), G93R mutation (2–12 years) (44), and G85R mutation (16 months–13 years) (T. Siddique, unreported) versus less variability with the E100G mutation (5–8 years) (44). This variability is suggestive of additional genetic and/or environmental inﬂuences. Penetrance of SOD1 mutations is variable and is based on each speciﬁc mutation. The penetrance of some SOD1 mutations is reduced in comparison to other SOD1 mutations; most notable is the signiﬁcantly reduced penetrance of the I113T and D90A mutations (44,45). This is in contrast to a generally high penetrance of the A4V mutation. The dosage of certain SOD1 mutations appears to have an effect on the onset of ALS. An example of this is of an affected Pakistani girl of consanguineous parents with a homozygous N86S mutation of the SOD1 gene (42). Her disease onset was at the age of 13, while her uncle (a presumed heterozygote for the mutation) did not begin to manifest symptoms until the age of 32 (42). A second and more common example of dosage and SOD1 gene mutations is that of the homozygous D90A mutation identiﬁed in more than 80 cases belonging to 40 independent apparently sporadic and familial ALS pedigrees (46). Most of the cases are from Northern Scandinavia where the D90A allele frequency is 2.7% (47). D90A heterozygous individuals typically do not develop ALS when they are of Scandinavian origin, thus the D90A mutation has an AR inheritance pattern in those of Northern Scandinavian ethnicity. In contrast, a slowly progressive form of the disease has been described in affected individuals who are D90A homozygotes, originating from United States, France, United Kingdom, Belgium, and Byelorussia, and presents as apparent SALS. In addition, dominant pedigrees have also been identiﬁed (46–49). Thus, in ethnic backgrounds other than Northern

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Scandinavian, the D90A mutation could be inherited as AR or dominant. It is therefore prudent to test sporadic ALS cases of Northern Scandinavian or Swedish background with lower extremity onset and slow progression for the D90A mutation, and with presentation of a slowly progressive disorder for the I113T mutation. The relatively benign nature of the D90A mutation in individuals of Scandinavian origin is thought to be mediated by a genetic factor acting in cis close to the SOD1 locus (50). SOD1 enzyme activity level does not correlate with disease severity. For example, mutations with only marginally reduced enzyme activity, such as D90A, H46R, and G93D do produce the disease (47,51). Animal and Biochemical/Cellular Studies in SOD1 linked ALS SOD1 protein expression. SOD1 linked ALS animal models have shown that ALS from SOD1 mutations results from the gain of a new function that is toxic, and not due to decreased or abnormal enzymatic activity of the SOD1. Transgenic mice or rats over-expressing ALS associated mutant SOD1 develop an ALS-like phenotype (52), while transgenic mice overexpressing wild-type SOD1 (wtSOD1) remain unaffected (22). Muscles of SOD1 knockout mice do show ﬁber type grouping, indicative of denervation/reinervation, but apparently do not show motor neuron degeneration (22–26,39,40). Thus, contrary to conventional wisdom of genetics, the 100 plus SOD1 mutants associated with ALS trigger motor neuron degeneration in ALS from a toxic gain of function rather than from a loss of dismutase activity. In spite of the varied pathology described in animals with ALS overexpressing mutant SOD1 (22–25,53), the central lesson has been that onset of disease correlates with the levels of expression of protein, which indirectly relates to copy number of the transgene. This has been demonstrated by correlating RNA or protein expression to onset in transgenic mouse lines carrying the same mutation or by engineering homozygotes for the mutations (53). We (N. Cole and T. Siddique, unpublished) have shown that SOD1 protein levels are the highest in the spinal cord of the G93A animal models for ALS with levels diminishing in the brain and liver and the least amount in the kidneys; the accumulation of the toxic protein correlates with age. mRNA copy number of the transgene in the G93A mice was measured by using TaqmanÕ chemistry for detection of reversed transcribed mRNA from spinal cord, brain, liver, and kidney with real-time PCR. mRNA copy number did not signiﬁcantly differ with age. The mRNA turnover of the mutant transgene was not studied. These studies, taken together, suggest that the targeted region, the spinal cord and brainstem, are unable to effectively deal with the mutant protein load leading to the region speciﬁc pathology and dysfunction noted in ALS mice, and probably in humans. Because SOD1 mutations confer novel toxic properties, an attractive therapeutic approach is to target the cause of the disease by reducing levels

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of mutant SOD1 expression in the spinal cord. RNA interference (RNAi) is a post-transcriptional mechanism of gene silencing mediated by small interfering RNA (siRNA). The siRNA promotes speciﬁc endonucleolytic cleavage of mRNA targets through an RNA-induced silencing complex (54). Two simultaneous publications support the possibility of using siRNA as a mechanism to silence SOD1 and consequently substantially slowing disease onset. One study used lentiviral based expression systems to drive the production of short hairpin RNA species (shRNAs) to provide long lasting RNAi based gene knockdown (55). This method showed decreases in SOD1G93A levels of 70.3 2.4% and 74.6 9.6% in motoneurons and glial cells, respectively, compared to non-infected cells at three days after injection (55). They also demonstrated a substantial reduction in SOD1 levels after interspinal delivery of the viral vectors in the ventral horn cells of SOD1G93A mice, prior to disease onset as well as an improvement in neuromuscular function of the ALS mice by 40% (55). A second study also used lentiviral based expression systems to drive production of siRNAs (56). In human cell lines, the SOD1 vector used to express the target sequences (EIAV-SOD1HP1) resulted in a signiﬁcant reduction in SOD1 mRNA compared with control-transduced samples (P < 0.05, n ¼ 3). In SOD1G93A derived neurons, SOD1 expression showed a reduction in protein levels at day 4 (50%) and day 7 (70%) compared with controls (56). In vivo models using the SOD1G93A mice, prior to the onset of symptoms, showed that intramuscular injections of the EIAV-SOD1HP1 vectors had a high transduction efﬁciency and the mice injected with the EIAV-SOD1HP1 vector had a highly signiﬁcant delay in the onset of symptoms compared with controls (202.1 7.5 d; P < 0.001), representing an increase in age of onset by 115% (56). However, a disappointing ﬁnding was that once the symptoms began, the progression of deterioration was similar to the controls, suggesting that onset and progression may be mediated by different mechanisms. Survival of the EIAV-SOD1HP1 injected mice increased by 77% (56). Neuropathology of the spinal cord of mice injected with the EIAV-SOD1HP1 vector showed increased survival of motor neurons, indicating these motor neurons were protected from degeneration (56). Based on the results of the two independent studies, RNAi as a method of treatment needs to be further investigated as the results seem promising at this initial stage of development. Reactive oxygen species and metal binding. Etiopathogenesis related to reactive oxygen species due to the presence of metal binding properties of SOD1 has been well studied. Mutant SOD1 exhibits enhanced oxidative activity by acting as a peroxidase (57–61), superoxide reductase (62), or by actually producing O2 rather than scavenging it, thus leading to production of peroxynitrite and/or hydroxyl radical. These reactive nitrogen and oxygen species cause toxicity by cumulative damage to proteins, nucleic acids, and lipids in affected cells (62–66). Structural mapping studies of the mutations on the protein structure of mutant SOD1 suggest that

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increased ﬂexibility in structure of the mutant SOD1 allows the catalytic site to become shallower and more exposed. As a result, molecules normally excluded may gain access to the catalytic reactive site (15,67–69). Several studies have proposed that cell injury may occur when the mutant SOD1 reacts with peroxynitrite (ONOO) formed from superoxide and nitric oxide with resultant nitration of tyrosine residues of critical cytosolic protein (63). The access of peroxynitrite would be in keeping with the model of promiscuous accessibility to the copper site. These studies have received wide dissemination, but the role of copper essential to these studies has been challenged. Breeding mutant SOD1 mice on a background knocked out of copper chaperone of SOD1 (CCS) does not alter the course of disease (70). CCS is the main transporter of copper to SOD1 and when copper binding sites of SOD1 are eliminated, the transgenic mice still develop the ALS phenotype (71). Protein aggregation and the ubiquitin proteosome system. Aggregates of SOD1, like mutant protein aggregation in other neurodegenerative disorders, have been proposed as mechanisms of toxicity for ALS. Transgenic mouse models have identiﬁed mutant SOD1 protein aggregates in the brain and spinal cord. In spite of intense effort, the role of these aggregates, of both SOD1 and other aggregated proteins within the brain and spinal cord, is not fully understood. It is debated that the aggregates could be a mechanism of detoxiﬁcation or of toxicity, depending on their cellular location. As the ubiquitin–proteosome is a major protein disposal system, protein aggregation suggests problems in the pathway for this system. However, very little mutant SOD1 is ubiquitinated in the transgenic mouse models of ALS (72). In order to explain the aggregation, it has been reasoned that a component of the ubiquitin–proteosome system such as an E3 enzyme may be impaired. Niwa et al. (73) identiﬁed Dorﬁn, a RING ﬁnger E3 ubiquitin ligase localized to the inclusion bodies of motor neurons of human FALS patients with SOD1 mutations, SALS patients as well as mutant SOD1 transgenic mice. Dorﬁn is involved in the ubiquitination and targeted degradation of mutant SOD1; however, wtSOD1 does not appear to be the substrate for Dorﬁn (73). Recently, valosin-containing protein (VCP) has been shown to interfere with E3 ligase activity of Dorﬁn (74). Role of wild-type SOD1; conversion to ALS phenotype. In animal models, transgenic mice over-expressing wild-type SOD1 (wtSOD1) do not develop ALS, but do show ultrastructural vacuolar pathology in the spinal cord and motor axons. When transgenic mice over-expressing wtSOD1 are crossbred with transgenic mice over-expressing mutant SOD1, the ALS phenotype and pathology appear earlier. This has been noted with G93A and L126Z transgenic mice (52). In addition, deposits of wtSOD1 can be identiﬁed in spinal cords of clinically asymptomatic L126Z mice. wtSOD1 can also convert over-expressing A4V transgenic mice to an ALS phenotype. A4V transgenic mice, on their own, do not develop ALS. In addition, G93A mice crossbred with SOD1 knockout demonstrate

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a small but measurable delay in onset of disease (HX Deng and T. Siddique, Unpublished Observation). Possible explanations for these observations include increased load in the mutant SOD1 clearing system, or conversion of wtSOD1 to a toxic variety. It should be pointed out that mutant SOD1 may itself have to undergo a conformational change to be toxic. Thus both wild-type and SOD1 may exist as both non-toxic and toxic forms. It may be energetically economical to convert the mutant form to a highly stable toxic variety than the wild-type form, but the mutant could possibly chaperone such a change in wtSOD1. Alternatively, an increase in SOD1 load may relate to an overburdened RNA turnover system (HX. Deng, Y. Shi, and T. Siddique, Unpublished Observation, 2004). Lately, aggregation studies have focused on suborganelle damage due to mislocalization of SOD1 aggregates. Mitochondria have been implicated because initial studies of the G93A showed vacuolar enlargement associated with the smooth endoplasmic reticulum (ER) and mitochondria in the spinal cord of ALS mice (22,75). Protein aggregates in mitochondria were considered a possible cause of disease due to toxicity related to energy failure or apoptosis. Studies in our laboratory used the wtSOD1 transgenic mouse crossbred with the G93A, L126Z and A4V transgenic mouse lines and showed that the wtSOD1 protein may be involved in the pathogenesis of ALS by targeting the mitochondria. Biochemical and Immunogold electron microscopy studies show the detergent resistant form of SOD1 wild-type protein co-localizes with the mitochondria and speciﬁcally targets both the outer and inner membranes of the mitochondria (HX. Deng, Y. Shi, E. Liu, and T. Siddique, Unpublished Observation 2004). Recent experiments suggest that the pathway to neurodegeneration may involve conversion of the dimeric holoenzyme of SOD1 to apoenzyme and reduction of its cysteine bridges to produce a population of molecules that would melt at body temperature (76). Such monomers would then be able to bind to membranes or other hydrophobic entities and enter organelles to form toxic complexes that damage normal functioning (76). ‘‘Bad neighborhood’’ or non-motor neuron autonomous degeneration. Deposition of SOD1 in glia of the G85R mice was reported to occur before the appearance of deposits in motor neurons, and the role of glia were then explored by using chimeric mice. Chimeric mice expressing mutant or wild-type SOD1 in mutant or wild-type background were studied (77) and reported to show that the mechanism of motor neuron dysfunction resides primarily in the non-motor neuron cells of the spinal cord. As expression levels of the SOD1 in individual motor neurons could not be measured, it is unclear if the experiments, as reported, conﬁrm the hypothesis of ‘‘a bad neighborhood,’’ or non-cell autonomous degeneration. Previous studies using promotors that targeted mutant SOD1 to motor neurons (78) or glial cells (79) failed to produce ALS. A drawback of the latter studies is that cDNA constructs were used rather than standard genomic constructs

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previously used in ALS transgenic mice. In contrast to these studies, neuronal culture studies have demonstrated cell death or over-expression of SOD1 without a need for glial input. In addition, SOD1 aggregates do not overlap with GFAP in glial cells in the G93A and L126Z mouse models of ALS. Excitotoxicity. Glutamate excitotoxicity caused by decreased glutamate transport from the synaptic clefts of the motor neurons has been hypothesized as a cause of ALS. This interesting and physiologically relevant hypothesis rests on the observed absence of EAAT2 immunoreactivity in the cortex of sporadic ALS patients (80) and anterior gray matter of the spinal cords of G93A transgenic mice and rats (81). These observations were augmented by the detoxiﬁcation of EAAT2 splice variants, some of which were noted only in ALS tissue. Unfortunately, this theory has not found support in genetic studies. For example, convincing mutations have not been found in familial cases of ALS (82). Moreover, over-expression of the EAAT2 transporter in double transgenic mice does not ameliorate the disease onset (70) and knockout of EAAT2 had a very subtle effect (83). Recent work by Rothstein’s group using the antibiotic ceftriaxone in G93A mice showed a marginal increase in survival of 10 days (83). This effect of ceftriaxone was attributed to an increase in the expression of the glutamate transporter, once again showing that glutamate excitotoxicity is not the major pathway in ALS pathogenesis. Thus glutamate excitotoxicity may be a contributing factor in the pathogenesis of ALS or the loss of the EAAT2 receptors may reﬂect the loss of the motor neurons or their connection, as an epiphenomenon in motor neuron degeneration. Non-glutamate excitotoxicity. Whereas glutamate toxicity may be a minor contributor to motor neuron degeneration, other mechanisms of excitotoxicity may also be contributive. Preliminary studies with cultured embryonic motor neurons from G93A mice demonstrate an increase in the persistent sodium channel that may lower the threshold for motor neuron excitotoxicity (84,85). Etiopathogenesis of the ALSIN Gene Mutations in ALSIN are a rare experiment of nature that results in severe and progressive dysfunction of the corticospinal tract. The ALSIN gene encodes the protein product alsin and is associated with juvenile onset PLS and UMN-predominant ALS (19). The ALSIN gene makes two transcripts of the protein by alternate splicing, a short and a long form. It is hypothesized that the homozygous loss of the long form results in the phenotype of juvenile onset PLS, whereas a homozygous loss of both short and long forms results in the phenotype of juvenile onset ALS (19). The speciﬁc function of alsin is not fully known, but it is believed to be a guanine nucleotide exchange factor involved in membrane transport events in the neuron. The vacuolar protein sorting (VPS) or other motifs associated

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with endosomal trafﬁcking have been implicated in other motor neuron disorders besides ALS2: Troyer syndrome (SPG20 gene) (86), ALS8 (Finkel type SMA/SMA4) (VAPB gene) (87); as well as in other neurodegenerative diseases: chorea-acanthocytosis (CAC gene) (88) and Niemann–Pick type C1 (NPC1) (89). Knockout models of the ALSIN gene do not exhibit a robust phenotype of motor neuron degeneration, but special copper–silver staining methods show distal axonal degeneration in the corticospinal tracts of knockout mice (HX Deng and T. Siddique, Unpublished Data). The corticospinal tracts in rodents are small and lie behind the central cord, raising the concern about rodents as appropriate models of UMN disease and spinal cord injury. Etiopathogenesis of Other Genes Identified in ALS Related Motor Neuron Disorders with Both UMN and LMN Involvement (Table 5) Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) features adult onset proximal and distal muscle weakness, early onset Paget disease of bone and premature frontotemporal dementia (FTD). IBMPFD is caused by the gene encoding vasolin containing protein (VCP), which is associated with cellular activities including cell cycle control, membrane fusion and the ubiquitin–proteosome degradation pathway (90). Finkel type of spinal muscular atrophy type 4 (SMA4) is an adult-onset AD SMA caused by mutations in the VAPB gene, which has been shown to be involved in ER-Golgi transport and secretion (87). Troyer syndrome, characterized by spastic paraparesis with dysarthria, distal amyotrophy, mild developmental delay, and short stature, is caused by mutations in the SPG20 gene encoding the protein spartin, which may be involved in endosomal trafﬁcking (86). ALS4, also known as distal hereditary motor neuropathy with pyramidal features, is a rare childhood or adolescent onset form of ALS characterized by slow disease progression, limb weakness, severe muscle wasting, and pyramidal signs. ALS4 is caused by mutations in the SETX gene encoding the protein Senataxin, which contains a DNA/RNA helicase domain with a possible role in RNA processing (91). Curiously, mutations in SETX have been known to cause a recessively inherited disorder of oculomotor apraxia and cerebellar ataxia (AOA2) (91). APPROACHES TO SALS AS A GENETICALLY COMPLEX OR MULTIFACTORIAL DISORDER Approximately 90% of ALS patients belong to the SALS subgroup (14). SALS is believed to be a multifactorial disease (multiple genes interacting with environmental factors). SALS and its multiple etiologies remain to be elucidated. Complex genetic diseases characteristically do not show a Mendelian pattern of inheritance but a genetic component contributes to

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disease susceptibility. Identiﬁcation of susceptibility genes provides clues to the pathogenesis and may point to intersecting environmental factors. The human genome project and advances in large-scale genotyping, statistical genetics, and bioinformatics have made it possible to investigate genes and DNA variations that confer disease susceptibility in disorders such as SALS. Some polymorphisms may not inﬂuence susceptibility but may affect onset, severity, and duration, thus inﬂuencing the phenotype. Susceptibility genes can be analyzed for their association between a speciﬁc allele and a disease in a large population. Successful gene mapping in complex diseases depends on many factors which include study design, adequate statistical power of a large sample size, extent of genetic heterogeneity, and appropriate mechanisms for veriﬁcation of the susceptibility genes. Association Studies in SALS Association studies focus on whether a speciﬁc allele of a genetic marker is found with increased frequency in individuals with the disease as compared to the frequency of the marker in individuals without the disease. Association studies can be conducted with population based case-control samples or family-based samples. Population based association studies involve recruitment of individual subjects, both affected cases and controls. Population based studies are more efﬁcient in terms of time, resources, and logistics; however, the limitation is that cases and controls may not be well matched, resulting in confounding data. As a rule of thumb, the cases and controls should be matched by ethnicity, age and gender and some investigators advocate a ratio of 1:2, cases versus controls. Family-based association studies involve recruitment of the affected individual in addition to the unaffected parents (called a trio) or an unaffected sibling (called a sib-pair). An advantage of family-based studies is that the affected samples and control samples have the same genetic background and are therefore well matched. The limitation of family-based studies is the difﬁculty of acquiring enough parents and siblings for the necessary statistical power. This can especially be the case in late-onset disorders, such as ALS, where collecting samples from both parents may not be possible because one or both parents are frequently deceased and as such, the ascertained population is skewed towards younger individuals. Association studies have been utilized in identifying potential susceptibility genes in ALS. APOE The Apolipoprotein E (APOE) gene was initially shown to be associated with late-onset Alzheimer’s disease (LOAD) and subsequently shown to

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have an effect on onset of LOAD (92). The effect of APOE polymorphisms on onset, rather than causation of disease, has lead to it being tested in many neurological disorders, including ALS. The APOE gene polymorphisms comprising alleles 2, 3, and 4 has been the focus of at least ﬁve association studies with ALS (93–97). A recent study identiﬁed the E2 allele as protective against an early onset of ALS (98); this is the ﬁrst study to subclassify the role of the APOE polymorphism in ALS. SMN The survival motor neuron gene (SMN gene) was screened in association studies of ALS because both SMA and ALS are caused by motor neuron degeneration. LMN-dominant ALS is an especially enticing subgroup of SALS to study, because it resembles adult-onset SMA, even though SMA is usually proximal in onset. The SMN region contains two, almost identical, copies of SMN, called SMN1 (or SMNt for telomeric) and SMN2 (or SMNc for centromeric); SMN1 and SMN2 are highly homologous. The SMN2 copy number is known to be a modifying gene to SMN1. Several studies have been performed in SALS patients attempting to correlate the SMN2 copy number or deletions within the SMN1 gene to ALS disease (99–101). Some studies showed a modest difference in SMN2 copy number in SALS patients; the data have not been reproduced to conﬁrm a genetic susceptibility of SMN to be associated with ALS. VEGF The vascular endothelial growth factor (VEGF) gene is another potential susceptibility gene because it has been shown to promote growth of blood vessels, and is a neuronal growth factor. In addition, when the promoter region of VEGF was knocked out in genetically modiﬁed mice, a subgroup of mice survived and developed motor neuron-like disease. An association study between SNP polymorphisms in the VEGF promoter and ALS showed ALS to be associated with susceptibility haplotypes of the VEGF promoter responsible for decreased VEGF product (102). Additional studies in SALS patients are necessary to conﬁrm these results. Intermediate Filaments Peripherin (103) is a type III intermediate neuroﬁlament protein expressed predominantly in the peripheral nervous system. Neuroﬁlaments have been implicated in the pathogenesis of motor neuron diseases based on mouse models (104), their presence in inclusions within motor neurons (105–107), and reported association of polymorphisms in the neuroﬁlament heavy chain (NFH) (108–110). A frameshift mutation was identiﬁed in peripherin in one individual with ALS; additional studies of ALS patients are therefore necessary to conﬁrm these results (103).

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Other Studies Examining the Mechanisms Behind SALS A recent study identiﬁed the presence of a retroviral marker (reverse transcriptase, RT, activity) in the serum of sporadic ALS patients (111). The serum RT activity was detected in 47% of ALS patients (n ¼ 14 of 30) compared with 18% of non-blood related controls (n ¼ 8 of 44), but unexpectedly a similar number of blood relatives (43%) to ALS patients had RT activity (111). This suggests that endogenous (inherited) retroviral activity may be involved, but also that additional factors may be necessary for the disease to manifest itself if unaffected blood relatives have similar RT activity. The detection of increased prevalence of RT activity in the serum of SALS patients raises questions about the potential role of retroviruses in disease.

CLINICAL MANAGEMENT Drug Development from Pathophysiology The in vitro experiments demonstrating that excitotoxicity caused by glutamate can kill neurons in cell culture and the absence of EAAT2 glutamate transporter in the motor cortex of ALS patients and in spinal cords of G93A mice led to clinical testing of Riluzole in ALS. Human clinical trials showed Riluzole slowed progression of disease by an average of three months. Currently, Riluzole is the only FDA approved medication indicated to slow the progression of ALS. Drug Screening Drugs and compounds are screened for their impact on ALS disease models. Typically, drugs and compounds are screened on cellular models of disease ﬁrst, such as cells containing the mutant SOD1 gene or for the effect on apoptosis or expression of glutamate receptors (EAAT2). Cell growth is recorded and those drugs or compounds that protect the cells from apoptosis or from developing aggregates of mutant SOD1 are predicted to have an effect in ALS. Those drugs or compounds that provide encouraging results in the cellular models are then tested in the mutant SOD1 transgenic mouse lines. Those drugs or compounds showing prolonged survival in the mutant SOD1 transgenic mouse lines are considered as candidates for testing in Phase I human clinical trials. Unsuccessful Clinical Trials A large number of drugs have been tested in human clinical trials for ALS (Table 6). However, as more drugs and compounds are tested and identiﬁed as potential drugs for ALS based on cellular and transgenic mouse models, a wider range of drugs will be available for human trials in the future.

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Table 6 Clinical Trials for ALS Completed clinical trials with unfavorable results Recombinant human ciliary neurotrophic factor Nimodipine Gabapentin Recombinant insulin-like growth factor Superoxide dismutase Xaliproden Glial cell-like derived neurotrophic factor Topiramate TCH346 Completed clinical trials with FDA approval to use for ALS Riluzole Combination of Dextromethorphan and Quinidine for pseudobulbar affect

REMAINING QUESTIONS TO BE ANSWERED Obviously there is much to be learned about the pathophysiology of ALS. Identiﬁcation of additional FALS genetic loci and genes will provide the means to develop additional cellular and animal models to study the disease process. The genetic models of familial disease will be the mainstay of research until the cause(s) of sporadic disease is/are identiﬁed and replicated; therefore, the current task of identifying causative genes requires urgent attention. ALS is a multietiologic disease and many genes are involved in causing ALS (both FALS and SALS); a major priority will be to elucidate the pathways by which these genes cause disease. A wider understanding will be obtained when the points of intersection of these pathways and the disease processes are identiﬁed. These nodal points will be important queries for environmental factors that may trigger interactions between genes and environment in causing sporadic ALS.

ACKNOWLEDGMENTS Acknowledgements of this work to: The National Institute of Neurological Disorders and Stroke (NS050641, NS046535), Les Turner ALS Foundation, Vena E. Schaff ALS Research Fund, Harold Post Research Professorship, Herbert and Florence C. Wenske Foundation, the Muscular Dystrophy Association of America, Ralph and Marian Falk Medical Research Trust, Abbott Labs Duane and Susan Burnham Professorship, and The David C. Asselin MD Memorial Fund.

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131. Hansen JJ, Durr A, et al. Hereditary spastic paraplegia SPG13 is associated with a mutation in the gene encoding the mitochondrial chaperonin Hsp60. Am J Hum Genet 2002; 70(5):1328–1332. 132. Valente EM, Brancati F, et al. Novel locus for autosomal dominant pure hereditary spastic paraplegia (SPG19) maps to chromosome 9q33-q34. Ann Neurol 2002; 51(6):681–685. 133. Hentati A, Pericak-Vance MA, et al. Linkage of ‘pure’ autosomal recessive familial spastic paraplegia to chromosome 8 markers and evidence of genetic locus heterogeneity. Hum Mol Genet 1994; 3(8):1263–1267. 134. Winner B, Uyanik G, et al. Clinical progression and genetic analysis in hereditary spastic paraplegia with thin corpus callosum in spastic gait gene 11 (SPG11). Arch Neurol 2004; 61(1):117–121. 135. Hodgkinson CA, Bohlega S, et al. A novel form of autosomal recessive pure hereditary spastic paraplegia maps to chromosome 13q14. Neurology 2002; 59(12):1905–1909. 136. Seri M, Cusano R, Forabosco P, et al. Genetic mapping to 10q23.3-q24.2, in a large Italian pedigree, of a new syndrome showing bilateral cataracts, gastroesophageal reﬂux, and spastic paraparesis with amyotrophy. Am J Hum Genet 1999; 64(2):586–593. 137. Windpassinger C, Wagner K, Petek E, Fischer R, Auer-Grumbach M. Reﬁnement of the Silver syndrome locus on chromosome 11q12-q14 in four families and exclusion of eight candidate genes. Hum Genet 2003; 114(1): 99–109. 138. Meijer IA, Hand CK, Grewal KK, Stefanelli MG, Ives EJ, Rouleau GA. A locus for autosomal dominant hereditary spastic ataxia, SAX1, maps to chromosome 12p13. Am J Hum Genet 2002; 70(3):763–769. 139. Casari G, De Fusco M, et al. Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease. Cell 1998; 93(6):973–983. 140. Hughes CA, Byrne PC, et al. SPG15, a new locus for autosomal recessive complicated HSP on chromosome 14q. Neurology 2001; 56(9):1230–1233. 141. Simpson MA, Cross H, et al. Maspardin is mutated in mast syndrome, a complicated form of hereditary spastic paraplegia associated with dementia. Am J Hum Genet 2003; 73(5):1147–1156. 142. Engert JC, Berube P, et al. ARSACS, a spastic ataxia common in northeastern Quebec, is caused by mutations in a new gene encoding an 11.5-kb ORF. Nat Genet 2000; 24(2):120–125. 143. Jouet M, Rosenthal A, et al. X-linked spastic paraplegia (SPG1), MASA syndrome and X-linked hydrocephalus result from mutations in the L1 gene. Nat Genet 1994; 7(3):402–407. 144. Willard HF, Riordan JR. Assignment of the gene for myelin proteolipid protein to the X chromosome: implications for X-linked myelin disorders. Science 1985; 230(4728):940–942. 145. Starling A, Rocco P, Cambi F, Hobson GM, Passos Bueno MR, Zatz M. Further evidence for a fourth gene causing X-linked pure spastic paraplegia. Am J Med Genet 2002; 111(2):152–156.

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146. Pringle CE, Hudson AJ, Munoz DG, Kiernan JA, Brown WF, Ebers GC. Primary lateral sclerosis. Clinical features, neuropathology and diagnostic criteria. Brain 1992; 115(Pt 2):495–520. 147. Hentati A, Bejaoui K, et al. Linkage of recessive familial amyotrophic lateral sclerosis to chromosome 2q33-q35. Nat Genet 1994; 7(3):425–428. 148. Hand CK, Khoris J, et al. A novel locus for familial amyotrophic lateral sclerosis, on chromosome 18q. Am J Hum Genet 2002; 70(1):251–256. 149. Ruddy DM, Parton MJ, et al. Two families with familial amyotrophic lateral sclerosis are linked to a novel locus on chromosome 16q. Am J Hum Genet 2003; 73(2):390–396. 150. Abalkhail H, Mitchell J, Habgood J, Orrell R, de Belleroche J. A new familial amyotrophic lateral sclerosis locus on chromosome 16q12.1–16q12.2. Am J Hum Genet 2003; 73(2):383–389. 151. Sapp PC, Hosler BA, et al. Identiﬁcation of two novel loci for dominantly inherited familial amyotrophic lateral sclerosis. Am J Hum Genet 2003; 73(2):397–403. 152. Chance PF, Rabin BA, et al. Linkage of the gene for an autosomal dominant form of juvenile amyotrophic lateral sclerosis to chromosome 9q34. Am J Hum Genet 1998; 62(3):633–640. 153. Hosler BA, Siddique T, et al. Linkage of familial amyotrophic lateral sclerosis with frontotemporal dementia to chromosome 9q21-q22. Jama 2000; 284(13):1664–1669. 154. Wilhelmsen KC, Forman MS, et al. 17q-linked frontotemporal dementiaamyotrophic lateral sclerosis without tau mutations with tau and alpha-synuclein inclusions. Arch Neurol 2004; 61(3):398–406. 155. Christodoulou K, Zamba E, et al. A novel form of distal hereditary motor neuronopathy maps to chromosome 9p21.1-p12. Ann Neurol 2000; 48(6): 877–884.

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28 Prion Diseases William S. Baek and James A. Mastrianni Department of Neurology, University of Chicago Hospitals, Chicago, Illinois, U.S.A.

INTRODUCTION Also known as transmissible spongiform encephalopathies (TSEs), and previously considered to result from a ‘‘slow virus,’’ this unique family of transmissible neurodegenerative disorders results from the accumulation within the brain of misfolded prion protein (PrP). Prion disease (PrD) is typiﬁed by its most common phenotype, Creutzfeldt–Jakob disease (CJD), which presents as a rapidly progressive dementia associated with ataxia and myoclonus; however, at least four other phenotypes are recognized, namely Gerstmann–Stra¨ussler–Scheinker disease (GSS), fatal insomnia (FI), new variant (v) CJD, and kuru. While the majority of cases occur on a sporadic basis, approximately 15% are associated with an autosomal dominant mutation within the PrP gene (PRNP), and a susceptibility polymorphism at codon 129 within PRNP appears to play a role in both. This chapter will review PrD, with special attention to the genetic aspects of disease. HISTORICAL BACKGROUND Human PrD was ﬁrst described by Creutzfeldt and then Jakob in 1929 (1–3), as a progressive dementia with associated gait abnormalities and with histopathology consisting of extensive vacuolation and astrocytic gliosis of the brain. Later, in the mid-1950s, Gajdusek described kuru, a disease endemic to the natives living in the highlands of New Guinea. Kuru, translated as

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‘‘abnormal gait,’’ is characterized as a progressive gait ataxia in conjunction with behavioral abnormalities, myoclonus, and a progressive course to death. The pathologic features of kuru and CJD overlap with scrapie, a natural disease of sheep, also known to be transmissible. Each produced extensive vacuolation of the neuropil, also referred to as ‘‘spongiform change’’ (4). This eventually prompted experiments leading to the successful transmission of kuru to chimpanzees, followed shortly thereafter by the transmission of CJD (5,6). The source of kuru was eventually traced to the brain of affected individuals honored during cannibalistic rituals. Women and children, who had the greatest contact with the brain, developed kuru with the greatest frequency. Carleton Gadjusek was awarded a Nobel Prize for his work on kuru. A second Nobel Prize was awarded years later to Stanley Prusiner, for his discovery of the etiologic agent of these diseases; he designated this novel infectious agent, composed largely if not entirely of a protein, a prion (7). PRION PROTEIN (PrP) AND THE PRION CONCEPT PrP is a 253 amino acid (209 amino acid in its mature form) host-encoded cell surface glycoprotein highly expressed in nervous tissues, and especially within neuronal synapses (8). The protein is present in all vertebrates, and although there are some variations, the protein is generally well conserved. The normal function of PrP is unknown, although its roles in synaptic function (9–11), copper binding and delivery (12–14), and cell signaling (15) have all been supported. In the original line of mice in which the PrP gene was ablated (Prnp%), no obvious phenotype or alteration of life span was observed, suggesting a noncritical or redundant function of PrP (16). The central feature of PrD is the accumulation of a pathogenic ‘‘scrapie-associated’’ isoform (PrPSc) of PrP, a conformational deviant of the normal cellular isoform (PrPC). PrPSc is distinguished from PrPC both structurally and biochemically. Whereas PrPSc is high (40%) in b-pleated sheet content, insoluble in nonionic detergents, and partially resistant to proteases, PrPC is predominantly a-helical, detergent soluble, and completely sensitive to Proteinase-K (PK). PrPSc accumulates primarily within nervous tissue of patients with PrD but can be recovered from lymphoreticular tissue in experimental rodent models of disease and in humans with variant CJD (vCJD) (17–21). The accumulation of PrPSc generally correlates with the progression of disease; however, infectivity in the absence of PK-resistant PrP has been noted experimentally (22). PrD in humans occurs as a result of three possible etiologies: (i) spontaneous conversion of endogenous PrPC to the PrPSc conformation, (ii) exposure to prions, or (iii) a genetic mutation within the coding segment of the PrP gene (PRNP). It is presumed that the sequence variations associated with genetic PrD predispose the protein to misfold into the

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pathogenic PrPSc conformation. Regardless of the mechanism, once generated, PrPSc triggers the conversion of normal PrPC in a template-assisted process of protein–protein interaction of the two isoforms. Propagation of PrPSc may occur by association–dissociation of the PrPSc–PrPC complex, or by extension of PrPSc amyloid via the addition of PrPC to the ﬁbril ends, ultimately leading to the accumulation of PrPSc throughout the brain. Early studies (23) suggested that the conversion to PrPSc requires the trafﬁcking of PrPC to the plasma membrane, where the association with PrPSc occurs. More recent evidence from Vey et al. (24) supports this concept and pinpoints the site of conversion to early endocytic vesicles, or caveoli-like membranes. While this may hold true for the propagation of PrPSc, the mechanism for the de novo generation of prions, especially those associated with PRNP gene defects, is still unanswered. Prion generation could result from impairment in function of the proteasome, a cytosolic multisubunit structure that hydrolyzes misfolded proteins within the cytosol (25). Evidence suggests that PrP is misfolded early after translation and translocation into the ER lumen, where ER quality control mechanisms may either refold the protein or eject it to the cytosol. This process of ER-associated degradation (ERAD) is thought to explain the spontaneous generation of PrPSc from PrPC and its enhanced production from mutated PrP as a result of gene defects that promote its misfolding in the ER. Proteasomal degradation has been demonstrated in cell culture for a handful of PrPs carrying pathogenic mutations, in addition to the wild-type PrP sequence (26,27). Transgenic mice that over-express PrP directly in the cytosol of neurons develop ataxia and focal atrophy of the cerebellar granule cell layer, yet PrPSc was not detectable in their brains (28). Other studies have questioned the role of ERAD in PrPSc generation. More studies are needed to clarify the details of the spontaneous generation of prions, and the speciﬁc role of genetic mutations in promoting the production of PrPSc. PATHOGENESIS OF PRIONS It is not known precisely how prions induce neuronal death, although the intracellular accumulation of PrPSc is assumed to be detrimental in some way. Support for such a gain in toxic function of PrPSc comes from several lines of evidence: (i) apoptosis has been linked to the accumulation of PrPSc in animal models (29) and humans (30); (ii) the protease-resistant core of PrPSc, PrP27–30, is toxic when applied to cell cultures (31); (iii) PrP knockout mice do not develop neurodegeneration (32); and (iv) the so-called ‘‘toxic fragment’’ of PrP, a ﬁbrillogenic peptide composed of the human sequence PrP106–126, is toxic to hippocampal cultures (33), mixed cerebellar cultures (34), and primary neuronal cultures from PrP-expressing (but not PrP knockout) mice (35). As mentioned above, the expression of misfolded PrP in the cytosol of neurons of transgenic mice also appears to be toxic,

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yet whether this is a true model of PrD has not yet been proven. A loss of function of PrPC as the cause of disease is supported by in vitro studies that show PrPC is protective against Bax-induced apoptosis (36). In addition, cells from PrP knockout mice are hypersensitive to oxidative stress (37). A third model recognizes that some genetic forms of PrP are associated with a transmembrane topology, rather than the typical anchoring to the external leaﬂet of the cell membrane. This topology correlates with the progression of disease in animal models (38). While this was ﬁrst associated with a substitution of valine for alanine at position 117 (39), a mutation that causes GSS in humans, this has not been universally observed with other mutations that cause PrD (27). Interestingly, residue 117 lies within the putative transmembrane region of PrP, composed of a short stretch of hydrophobic amino acids. How the transmembrane form of PrP induces cell death has not yet been explained.

TRANSMISSIBLE PROPERTIES OF PRIONS Two key features of prion transmission that proved difﬁcult to explain on the basis of a ‘‘protein-only’’ hypothesis are (i) the species barrier of prion transmission and (ii) prion strains. Transmission of prions across species does occur, although it is unpredictable at ﬁrst passage but improves with subsequent passage within the species. Differences in the amino acid sequence at speciﬁc regions of PrP, presumably at compatible exposed surfaces of the protein, are now thought to partially explain the species barrier (40). Prion strains manifest as differences in incubation period and clinicopathologic phenotype (41). One of the most distinctive human PrD, FI, has provided important insights into the determinants of prion strains. The characteristic clinical and pathologic proﬁle of FI is quite distinct from any other PrD. This unique phenotype is invariably linked to a protease-resistant fragment of PrPSc with a characteristic molecular weight that differs from that derived from typical sporadic CJD (42,43). This difference in molecular weight is explained by a difference in the conformation of PrPSc that causes FI. This is true regardless of whether the FI-related PrPSc comes from sporadic or familial forms of FI. When FI prions are transmitted to susceptible transgenic mice, the PrPSc recovered from their brains is similar in conformation to PrPSc from patients with FI rather than CJD, indicating that the distinctive conformation of PrPSc is transferred from FI prions to PrPC within the mouse brain. This conformational transfer is associated with a speciﬁc and reproducible pathologic picture in the mice, indicating that the strain of disease is carried within the conformation of PrPSc. Earlier studies of transmissible mink encephalopathy in hamsters laid the foundation for these concepts (44–46). While other forms of PrD are not as well deﬁned as FI, there are clearly

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characteristic phenotypes that are either tightly or loosely associated with speciﬁc mutations of PRNP. EPIDEMIOLOGY The overall incidence of PrD from all causes is low, averaging one case per million per year worldwide, without relation to geographical location (47). Sporadic CJD constitutes the overwhelming majority of PrD cases, while familial PrD is much less common, accounting for approximately one in 10 cases. There is no obvious gender preference nor is there a predilection for a particular ethnic group, although certain PRNP mutations that cause familial disease may be more restricted to certain regions of the world, depending on the origin of the mutation (48). Age at disease onset differs depending on the occurrence, with sporadic PrD typically presenting between 60 and 70 years of age; familial PrD commonly presents prior to age 55. Acquired PrD is the least common form, although the prominent press received by the outbreak of mad cow disease and its human counterpoint, vCJD, gives the perception that it is quite common. In addition to vCJD, acquired PrD has also occurred from the use of contaminated biologicals (growth hormone, gonadotrophic hormone), transplantation of infected tissues (dura mater grafts, corneal transplants), and the use of improperly sterilized surgical or neuro-diagnostic tools. MUTATIONS OF PRNP CAUSE FAMILIAL PrD The PRNP gene is located on the short arm of chromosome 20 in humans. The entire coding segment of the gene is contained within a single exon. Over 25 base pair alterations and insertions of PRNP have been found to associate with various phenotypes of PrD (Fig. 1). All disease-associated mutations are in-frame and heterozygous, following autosomal dominant inheritance. In one example, ﬁve patients were found to be homozygous, although no difference in onset and disease phenotype was recognized, conﬁrming a true autosomal dominant inheritance (49). Three major types of pathogenic mutations have been described: (i) nucleotide substitutions that affect the primary structure of the protein; (ii) duplications of one or more of eight amino acid stretches (known as the octarepeat region), normally repeated ﬁve times within the N-terminal of the protein from amino acid 51 to 90, thereby producing an extended PrP; and (iii) nonsense mutations, producing an early stop signal and a C-terminally truncated PrP. Table 1 lists the currently known disease-associated mutations of PRNP. While the insertion mutations are strictly conﬁned to within the N-terminal octarepeat region, base-pair substitutions occur anywhere C-terminal to the octarepeat region, and two occurrences of an early stop signal have been reported at position 145 and 160.

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Figure 1 Known mutations and polymorphisms of PRNP. The relative positions of polymorphic changes (top) and disease-associated mutations (bottom) are schematically represented along the length of the PRNP gene. Reported polymorphisms include single base pair changes resulting in amino acid substitutions or deletion of an octarepeat segment, whereas disease-associated mutations result from missense mutations, insertions of a variable number of octarepeat segements, or nonsense mutations (-). The letter preceding the residue number indicates the normal sequence and the amino change is represented by the letter following the residue number. Abbreviations: A, alanine; D, aspartate; E, glutamate; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; (-), nonsense (stop) signal.

The most common mutations are substitutions at codons 178 and 200. A glutamate (E) to lysine (K) substitution at codon 200 (E200K) appears to be the most common mutation worldwide, the largest foci of which are found in the Middle East (Lybian Jews) and western Europe (Slovakia). An aspartate (D) to asparagine (N) substitution (D178N) is nearly as common and is found throughout the world. While both mutations are associated with familial CJD (fCJD), others are characteristically associated with different presentations. Despite the lack of clear experimental evidence, it has been hypothesized that alterations in the normal PrP sequence destabilize the protein and predispose it to misfold into the PrPSc conformation. This idea is supported by the existence of genotype– phenotype correlations that are predicted to result from differences in the speciﬁc conformation of PrPSc, based on the location and nature of the particular mutation.

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Table 1 Mutations of PRNP Predict the Phenotype of PrD Phenotype CJD

CJD/GSS CJDa or FFIb GSS

GSS/HD-like

Amino acid change V180I T183A H187R E196K V210I E211Q 24 bp octarepeat insertion 48 bp octarepeat insertion 96 bp octarepeat insertion 120 bp octarepeat insertion 144 bp octarepeat insertion 168 bp octarepeat insertion M232R D178N P102L P105L A117V G131V Y145Stop Q160Stop F198S D202N V203I Q212P Q217R 216 bp octarepeat insertion 192 bp octarepeat insertion

Octarepeat segment is [Pro-(His/Gln)-Gly-Gly-Gly-(-/Gly)-Trp-Gly-Gln]5. a When codon 129 is Val. b When codon 129 is Met. Abbreviations: P, Proline; L, Leucine; A, Alanine; V, Valine; Q, Glutamine; Y, Tyrosine; D, Aspartate; N, Asparagine; I, Isoleucine; T, Threonine; E, Glutamate; R, Arginine; K, Lysine; F, Phenylalanine; S, Serine; H, Histdine; M, Methionine; HD, Huntington’s disease.

PRNP POLYMORPHISMS Several polymorphic sites exist within PRNP, some of which modify the risk to, or the phenotype of PrD. The most common of these is codon 129, where either methionine (Met) or valine (Val) may be encoded. Homozygosity for either Met or Val occurs in roughly 50% of Caucasians and is found in at least 85–95% of patients with CJD, suggesting that homozygosity increases the risk to PrD (50–54). The epidemiology of this polymorphism displays cultural differences. For instance, in Japan the vast majority of the

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population is homozygous for Met at codon 129, thereby masking any associated risk to PrD (55). The polymorphic codon 129 also inﬂuences the clinical phenotype of disease; Met homozygotes (129MM) more commonly display progressive dementia at onset, while heterozygotes (129MV) or Val homozygotes (129VV) tend to present with ataxia at onset, and the course of disease is more protracted (56). Homozygosity at codon 129 has also been associated with an earlier onset and more rapid progression of familial PrD (57). The importance of codon 129 is also demonstrated by the ﬁnding that all patients thus far diagnosed with vCJD are homozygous for Met, with the exception of one patient who was infected by transfusion with contaminated blood, suggesting greater susceptibility of such individuals to BSE. The underlying basis for the greater susceptibility of homozygotes to PrD is presumed to reﬂect the greater efﬁciency of interaction of PrPSc with a homologous PrPC molecule. Another polymorphism at codon 219, in which glutamate is substituted by a lysine (K) (E219K) has been reported in roughly 6% of the healthy Japanese population, yet it was absent in a small cohort of CJD patients, raising speculation that the lysine substitution is protective (58). Experimental evidence for this was documented in cell culture and transgenic mouse models. When expressed in mouse neuroblastoma N2a cells chronically infected with prions (ScN2a), normal sequence PrP, and several other mutated PrPs are converted to protease-resistant PrPSc, but not when these PrPs carry the mouse equivalent of the E219K polymorphism (59). In addition, transgenic mice expressing PrP with the E219K mutation, either alone or in the presence of normal PrP, displayed a dominant negative effect by delaying the onset of disease (60). It is postulated that glutamate at position 219 is necessary for binding to Protein X, a chaperone protein proposed to assist in the conversion of PrPC to PrPSc (61). However, familial disease has been reported with the P102L mutation allelic to the E219K polymorphism, although an inﬂuence on the typical phenotype of disease was described (62). At codon 171, a polymorphism results in the substitution of asparagine (N) for serine (S) (63). Initially identiﬁed incidentally in a healthy control, this was later reported in several members of a family with a high prevalence of schizophrenia (64), and in subjects with a high risk of mesial temporal lobe epilepsy and cortical malformation (65), suggesting a potential role for PrP in such conditions. These associations are, however, quite preliminary. PRION-LIKE PROTEIN GENE (Prnd ) The prion-like protein gene (Prnd) encodes doppel (Dpl). This gene was recognized when additional lines of PrP knockout mice were generated and, in contrast to the original knockout line, developed an ataxic phenotype (66). Initially thought to result directly from the lack of PrP expression,

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the ataxic phenotype was found to be related to the nature of the disruption of the Prnp gene, which led to an increase in expression of the downstream Prnd gene, and Dpl accumulation within cerebellar Purkinje cells (67). Directed expression of Dpl in PrP knockout mice produces ataxia and loss of cerebellar Purkinje cells at 6–12 months of age, yet no PK-resistant PrP or pathology characteristic of PrD was detected (67). When mice co-expressing Dpl and PrP were challenged with scrapie, there was no alteration in the properties of scrapie transmission (68,69). The role of Dpl in prion disease is yet unclear, and while Dpl may be directly toxic to neurons (70), no mutations of the doppel gene in humans (PRND) have yet been directly linked to familial PrD (71), nor is there evidence for elevated expression during the course of prion disease in humans (72). Croes et al. (73) have suggested that a polymorphism within PRND (T174M) may increase the risk of developing CJD independent of the effect of the M129V polymorphism of PRNP, resulting in an almost three-fold increased risk for homozygotes. Conﬁrmatory studies are pending. THE PHENOTYPES OF PrD PrD is associated with a spectrum of phenotypes. These are broadly classiﬁed into ﬁve major categories, including kuru, CJD, GSS, FI, and vCJD. The salient features of each disease subtype are outlined in Table 2 and in the subsequent sections. Of these, only GSS is caused solely by a genetic mutation of PRNP, whereas CJD and FI occur either sporadically or genetically. The major clinical features that distinguish these diseases include the rapidly progressive dementia of CJD, the slowly progressive ataxia of GSS, the onset of intractable insomnia and autonomic disturbances of FI, and the psychiatric symptoms (especially depression and apathy) and peripheral pain syndrome of vCJD. Pathologic distinctions include the diffuse vacuolation and gliosis of CJD, the PrP-amyloid plaque deposition of GSS, the focal thalamic gliosis of FI, and the intense spongiform change surrounding PrP-amyloid deposits, also known as ﬂorid plaques, of vCJD. In general, the genetic forms of disease present at a younger age, and progress more slowly than the sporadic forms of disease. In FI, however, the age at onset and disease course is similar, regardless of whether it occurs on a genetic or sporadic basis. Creutzfeldt–Jakob Disease The clinical features of CJD include the classic ‘‘triad’’ of progressive dementia, ataxia, and myoclonus. Onset is typically in the seventh to eighth decade, and confusion and memory disturbance are the most common presenting features (70% of cases) with or without ataxia, while the remaining cases may present either with ataxia or less commonly, cortical blindness. The latter has been labeled the Heidenhain variant of CJD (74). Other atypical presentations include focal or generalized weakness, peripheral neuropathy,

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Table 2 Human PrD Phenotypes Disease

Occurrence

Kuru

Cannibalism

CJD

Sporadic (sCJD) Iatrogenic (iCJD)

Genetic (fCJD)

Age at onset (range)

Time to death (months)

40 years (29–60) 60 years (17–83) Depends on age at ‘‘infection’’ <55 (20s–80sa)

3–12 <12 (typically 4–6) <12

>12 and <72

Genetic

<60 years (20s–80s)

12–60

FI

Sporadic (sFI)

45 years

12–16

Genetic (FFI)

Same as above

Same as above

Beef consumption?

30 years (18–53)

12–18

vCJD

a

Gait ataxia, myoclonus, dementia Early dementia, then gait ataxia and myoclonus Variable, often ataxia, then dementia

Presence of kuru-type plaques Extensive vacuolation (spongiosis) and gliosis Extensive vacuolation (spongiosis) and gliosis

Early dementia, then gait Extensive vacuolation ataxia, and myoclonus (spongiosis) and gliosis Early ataxia and dysarthria, PrP-amyloid plaques, late dementia gliosis, less vacuolation than CJD Thalamic and olivary Refractory insomnia, nucleus neuronal loss and autonomic dysfunction, gliosis, minimal then ataxia, and late spongiform pathology dementia Same as above Same as above Psychiatric or pain Presence of ‘‘ﬂorid’’-type PrP-amyloid plaques syndrome, then gait dysfunction, late dementia

Typically less than 55 years, although some mutations may present later in life (e.g., E200K, V210I, V180I).

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Clinical phenotype

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choreiform movements, hallucinations, primary language disturbance, supranuclear ophthalmoplegia, and alien hand syndrome (75–77). Essentially all patients will develop dementia at some point during the course of disease. Myoclonus, the third key feature, develops in over 75% of patients in the middle to late stages of the disease. This occurs as either spontaneous or stimulus-induced (hence the classic description of ‘‘startle myoclonus’’) asynchronous jerks of large muscle groups. The course of CJD averages from four to ﬁve months, with 70% of patients expiring within six months, often as a result of aspiration pneumonia, malnutrition, or sepsis. Extensions in the course of disease to greater than one year are known. Clinical and pathologic features of familial CJD are similar to sporadic CJD, although the age at onset for fCJD is younger, typically less than 55 years, and disease progression is slower, extending from one to ﬁve years. In addition, diagnostic studies, such as EEG and CSF 14-3-3 testing are more likely to be positive in sCJD rather than fCJD. Gerstmann–Stra¨ussler–Scheinker Disease First described by Gerstmann in 1928, GSS is a hereditary progressive ataxia associated with dysarthria, pyramidal and extrapyramidal signs, and late development of dementia (78). Myoclonus may or may not be present. The onset is usually before age 50, and course of disease ranges from two to seven years. Aspiration pneumonia is a major cause of death due to early onset ataxia and dyscoordination of swallowing. PrP amyloid deposits (GSS plaques) are observed primarily within the cerebellum. GSS plaques have also been associated with atypical phenotypes that include spastic paraparesis (79) and a telencephalic variant with dementia rather than ataxia (80). In addition to amyloid deposits, neuroﬁbrillary tangles and congophilic angiopathy have also been reported in association with speciﬁc mutations. Molecular analysis of GSS plaques has revealed that they are composed predominantly of PrP peptide segments encompassing amino acids 88–145, suggesting that both amino and carboxy terminal processing play a role in plaque generation. At least 10 mutations of the PRNP gene are associated with the GSS pathology, two of these resulting in a foreshortened PrP with an early stop codon at positions 145 and 160. Interestingly, two to seven insertional repeats are typically associated with fCJD, while eight or nine inserts result in GSS. Fatal Insomnia This PrD was originally described as fatal familial insomnia (FFI), an unusual disorder manifesting with insomnia at onset (81). A sporadic form of FI (sFI) has since been described in several patients (82,83). The average age of onset is 48 years, and most patients will die within 1–16 months from the start

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of symptoms (84–86). Additional features include autonomic dysfunctions such as blood pressure, heart rate, and respiration rate abnormalities. Analysis of the PRNP gene of affected family members later revealed that the disease co-segregated with a PrP—Asp178/Met129 genotype (87). Transmission studies in transgenic mice expressing a chimeric mouse–human PrP that are receptive to human prions show that FFI and sFI are the same ‘‘strain’’ of PrD, likely because they carry similar conformations of PrPSc. Neuropathology reveals neuronal loss and astrogliosis primarily in the thalamus and inferior olives, with minimal spongiform change and little PK-resistant PrP present, which posed a challenge for the identiﬁcation of this PrD. Variant CJD Between 1995 and January 2005, 153 cases of vCJD have been documented, primarily in the United Kingdom. Compared with CJD, vCJD is distinguished by several features: (i) a younger onset (median age ¼ 28, compared with 65 years for sCJD), (ii) the presence of psychiatric symptoms (especially depression and apathy) and, in many cases, peripheral pain syndromes, likely due to thalamic involvement of the disease, and (iii) the presence of PrP-amyloid deposits surrounded by intense vacuolation, labeled ‘‘ﬂorid plaques’’ (88,89). Although not a genetic form of prion disease, the codon 129 polymorphism appears to play a signiﬁcant role in susceptibility (for further details see previous sections of this chapter). DIAGNOSIS OF PRION DISEASE Difﬁculties in diagnosis of PrD stem from its broad range of clinical presentations and the lack of a speciﬁc diagnostic test. As this is both a neurodegenerative and infectious disease, the work-up is often extensive, necessitating evaluation for CNS infection while considering various entities associated with dementia, ataxia, or a movement disorder. Since the familial forms of prion disease classically follow a more protracted course, these are often misdiagnosed as one of the more common neurodegenerative diseases, including Alzheimer’s disease, dementia with Lewy bodies, frontotemporal dementia, Huntington’s disease, Parkinson’s disease, spinocerebellar ataxia, or progressive supranuclear palsy. Other conditions such as ceroid lipofuscinosis (Kufs disease), Wilson’s disease, and Whipple’s disease, may also be considered in the evaluation. Some familial forms of CJD, and in particular sCJD, progress, very rapidly, bringing into consideration toxic–metabolic, infectious, or inﬂammatory conditions, including viral, bacterial, and parasitic-related encephalopathies/encephalitides, CNS or systemic vasculitides, heavy metal poisoning (especially bismuth ingestion), autoimmune diseases such as Hashimoto’s thyroiditis with related encephalopathy, multiple sclerosis, and paraneoplastic syndromes. Complete PRNP gene analysis is also performed on a research basis at a handful of laboratories.

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DIAGNOSTIC STUDIES Magnetic resonance imaging (MRI) of the brain of patients with PrD may show dramatic and diffuse atrophy early in the course of disease but this is rare. In recent years, advances in MRI have yielded better diagnostic tools for CJD. Diffusion-weighted imaging (DWI) shows hyperintensity of the basal ganglia or cortical ribbon in up to 90% of patients with CJD (90–93). In vCJD, hyperintensity of the pulvinar detected by proton-weighted MRI or DWI is a characteristic. Other forms of neuroimaging, such as PET or SPECT scanning, are of limited use in CJD patients (94,95). However, in early stages of FI, the PET scan often shows focal hypometabolism within the thalamus (96). In sCJD and some cases of fCJD, the EEG shows 0.5–2 per second generalized bursts of periodic sharp wave complexes (PSWCs) (97–99). They may be unilateral at onset and disappear with disease progression. CSF analysis typically shows mildly elevated protein (about 10% over normal), and no immunologic response. The 14-3-3 protein is often elevated in the CSF (98,100), especially in typical CJD, but less often in atypical presentations of CJD (101) or in other PrD phenotypes. Variability of sensitivity ranges from <60% of autopsy-proven cases (102–105) to well over 90%. False positive results are seen with herpes encephalitis, hypoxic brain damage due to stroke, Hashimoto’s encephalopathy, Alzheimer’s disease, and even multiple sclerosis. The diagnostic criteria for CJD by the World Health Organization includes a positive 14-3-3 test as one criterion that may substitute for a positive EEG in making a diagnosis of probable CJD. TREATMENT The most recent promising drugs that were ‘‘curative’’ in cell culture include quinacrine and chlorpromazine (106). Clinical trials with quinacrine have begun; this agent does not have proven efﬁcacy in rodents inoculated with prions. The timing of drug administration may be an important issue in this case. A variety of agents are effective in cell culture models, but not in rodent models, suggesting that cell culture is not an ideal test ground for prion therapeutics. This is a rich area for future development. REFERENCES ¨ ber eine eigenartige herdfo¨rmige Erkrankung des Zentral1. Creutzfeldt HG. U nervensystems. Z Gesamte Neurol Psychiatrie 1920; 57:1–18. ¨ ber eigenartige Erkrankungen des Zentralnervensystems mit bemer2. Jakob A. U kenswertem anatomischen Befunde (spastische Pseudoscklerose-Encephalomyelopathiemit disseminierten Degenerationsherden) Preliminary communication. Dtsch Z Nervenheilk 1921; 70:132–146. 3. Jakob A. Spastische pseudosklerose. Die Extrapyramidalen Erkrankungen 1923; 215–245.

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