Calcium and Bone Disorders in Children and Adolescents - Endocrine Development Vol 16

Calcium and Bone Disorders in Children and Adolescents

Endocrine Development Vol. 16

Series Editor

P.-E. Mullis

Bern

Calcium and Bone Disorders in Children and Adolescents Volume Editors

Jeremy Allgrove London Nick Shaw Birmingham 89 figures, 13 in colour, and 25 tables, 2009

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Shanghai · Singapore · Tokyo · Sydney

Endocrine Development Founded 1999 by Martin O. Savage, London

Jeremy Allgrove

Nick Shaw

Department of Paediatric Endocrinology Royal London Hospital London, UK

Clinical Lead for Endocrinology Birmingham Children's Hospital Birmingham, UK

Library of Congress Cataloging-in-Publication Data Calcium and bone disorders in children and adolescents / volume editors, Jeremy Allgrove, Nick Shaw. p. ; cm. -- (Endocrine development ; v. 16) Includes bibliographical references and indexes. ISBN 978-3-8055-9161-4 (hard cover : alk. paper) 1. Bone diseases in children--Case studies. 2. Calcium metabolism disorders in children--Case studies. I. Allgrove, Jeremy. II. Shaw, Nick. III. Series: Endocrine development ; v. 16. [DNLM: 1. Calcium Metabolism Disorders--physiopathology. 2. Adolescent. 3. Bone Diseases--physiopathology. 4. Child. W1 EN3635 v.16 2009 / WD 200.5.C2 C1433 2009] RJ482.B65C35 2009 618.92⬘71--dc22 2009018333

Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and Index Medicus Disclaimer. The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2009 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel ISSN 1421–7082 ISBN 978–3–8055–9161–4

Contents

VII Foreword Glorieux, F.G. (Montréal, Qué.) IX Preface Allgrove, J. (London); Shaw, N. (Birmingham) 1 8 32 49 58 73 93 115 133 157 170

Voyages of Discovery Allgrove, J. (London) Physiology of Calcium, Phosphate and Magnesium Allgrove, J. (London) Physiology of Bone Grabowski, P. (Sheffield) Bone Biopsy: Indications and Methods Rauch, F. (Montréal, Qué.) Bone Densitometry: Current Status and Future Perspectives Crabtree, N. (Birmingham); Ward, K. (Cambridge) A Practical Approach to Hypocalcaemia in Children Shaw, N. (Birmingham) A Practical Approach to Problems of Hypercalcaemia Davies, J.H. (Southampton) A Practical Approach to Rickets Allgrove, J. (London) Disorders of Phosphate Homeostasis and Tissue Mineralisation Bergwitz, C.; Jüppner, H. (Boston, Mass.) Primary Osteoporosis Bishop, N. (Sheffield) Secondary Osteoporosis Ahmed, S.F.; Elmantaser, M. (Glasgow)

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191 218 233 246

281

292 293

VI

Miscellaneous Bone Disorders Mughal, M.Z. (Manchester) Drugs Used in Paediatric Bone and Calcium Disorders Cheung, M. (London) Skeletal Aspects of Non-Accidental Injury Johnson, K. (Birmingham) Case Histories Howard, S. (London); Lyder, G. (Birmingham); Allgrove, J. (London); Shaw, N. (Birmingham) Appendix Author Index Subject Index

Contents

Foreword

The understanding of the biology and pathophysiology of the human skeleton has progressed at a remarkably fast pace in the past 35 years. Investigation and management of bone diseases were only chapters in textbooks on endocrinology or nephrology. Not anymore. Based on innovative diagnostic technologies, the development of new therapeutic modes, and the progress in medical genetics, the field of bone disease has established itself as a stand-alone specialty. As a consequence, a large number of books, thick and thin, have been and continue to be published on various aspects of skeletal diseases. In some, there are chapters on paediatric bone diseases, but it is only of late that entire books devoted to the study of the growing skeleton and its abnormalities have emerged, based on the impressive growth of knowledge in all aspects (cellular, organic, hormonal, structural) of bone metabolism and the diseases that interfere with the development, growth, remodelling and mineralisation of bone. The precursor opus was published in 1974 by Maroteaux [1]. It is a most extraordinary collection of radiographs, but with a timid approach at basic mechanisms. In 1980, there was also the book honouring Helen and Harold Harrison’s contribution to the understanding of paediatric bone diseases. It focused on biochemistry and clinical studies [2]. Then in 2003, an elaborate treatise was published as an attempt to organise the sum of current knowledge on paediatric bone diseases [3]. The editors of the present work have pursued the same goal in a concise and practical manner. They have recruited experts on various aspects of the biology of bone and the diseases affecting its structure and function. The field is well covered, but the true originality of the book lies within its last section where a series of case histories is presented. No doubt, the readers will appreciate this real-life approach to the problems discussed and, hopefully, connect them with their own experience. Francis H. Glorieux, OC, MD, PhD Shriners Hospital for Children, Montréal, Canada

VII

References 1 2 3

VIII

Maroteaux P: Maladies osseuses de l’ enfant. Paris, Flammarion, 1974. DeLuca HF, Anast CS (eds): Pediatric Diseases Related to Calcium. New York, Elsevier, 1980. Glorieux FH, Pettifor JM, Juppner H (eds): Pediatric Bone: Biology and Diseases. San Diego, Academic Press, 2003.

Glorieux

Preface

The idea for this book arose as a result of a number of initiatives. Several paediatricians in the UK with a clinical and research interest in calcium and bone disorders in children started to establish dedicated Metabolic Bone Disease clinics about 10 or more years ago. These clinics have attracted a wide variety of different conditions. Many of these are unusual but not really of sufficient rarity to warrant separate case reports. Others have already been published. There is, however, no publication that specifically brings together case histories related to bone and calcium disorders placed in the context of the background physiology and pathology, so, consequently, the idea arose of providing one. As a consequence of the rarity of some of the conditions we were seeing, a group of us started to meet twice a year to discuss challenging cases and share knowledge and expertise. Out of these initial meetings developed the British Paediatric and Adolescent Bone Group (BPABG), which is now affiliated to the Royal College of Paediatrics and Child Health as a speciality group with our own scientific session at the annual conference. The aim of this group is to promote knowledge and understanding of paediatric bone disease. As an additional development to fulfil this objective it has, for the past 3 years, run an annual postgraduate teaching course for senior trainees and consultants who are interested in learning more about the subject. An important aspect of these courses is that the delegates have been asked to bring case histories for presentation and discussion. Many of the cases described in the final chapter have been presented at these courses. Many of the chapters in this book are based on the lectures given at them and all of the founder members of BPABG have contributed. Additional contributions have come from other members of the BPABG together with some international authors.

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We are grateful to Karger for inviting us to edit this book, which is published as part of their Endocrine Development series under the overall editorship of Professor Primus Mullis. We are grateful to all of the authors for sending us their manuscripts in a timely manner. We wish to thank those clinicians whose cases are presented and who have allowed us to report them. Finally, we wish to thank our wives, Natalie and Vicki, for their long-suffering approach to our slaving over hot computers when more sociable activities beckoned. Jeremy Allgrove Nick Shaw

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Allgrove · Shaw

Chapter 1 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 1–7

Voyages of Discovery Jeremy Allgrove Royal London Hospital, and Great Ormond Street Hospital, London, UK

Abstract The metabolism of calcium and bone is controlled by five principal hormones: parathyroid hormone, 1,25-dihydroxyvitamin D, calcitonin, parathyroid hormone-related peptide and fibroblast growth factor 23, some of which have been known for several decades and some of which have only recently been identified. The stories of discovery of these hormones have constituted a series of complex journeys which have been undertaken over the past century or so and none of which has yet been completed. The complexities of bone and calcium metabolism have been and remain, to many people, somewhat mysterious and a daunting task to understand. This book is designed to try to unravel those mysteries and present them in an interesting and comprehensible manner. Copyright © 2009 S. Karger AG, Basel

The study of the diseases that affect bone and calcium has made huge strides over the past few decades. The initial realisation that rickets, which was rife in industrial cities, particularly in the UK, could be cured by exposing children to sunlight or supplementing them with foods such as cod liver oil was a major step in improving the health of children in the early part of the twentieth century. Subsequently, the discovery of other hormones that are involved in mineral metabolism, both calcium and phosphate, has enabled much wider understanding of the mechanisms of disease to be gained. This has led to the introduction of logical treatments based on this scientific understanding. There are five major hormones, vitamin D and its metabolites, parathyroid hormone (PTH), calcitonin (CT), parathyroid hormone-related peptide (PTHrP) and fibroblast growth factor 23 (FGF23), that are involved directly in the control of mineral metabolism in man. In addition, several other hormones, such as oestrogens and androgens, cortisol, growth hormone and thyroxine, have modifying effects. The story of the unravelling of these hormones is a long and complicated one that has gradually revealed itself over the past century or so. For each there has been a long voyage of discovery, some lasting longer than others, but each is still a journey in progress.

Vitamin D and Rickets

Rickets is an ancient disease. It was probably known in the ancient world, but is recorded in the UK since the 17th Century [1]. It became widespread with the increase in industrialisation during the 19th and 20th centuries. The first breakthrough in treatment came with the realisation, shortly after the end of the First World War, that most rickets could be cured either by exposure to sunlight or with supplements of cod liver oil [2]. Vitamin D was discovered to be the agent that effected the cure. As a consequence of this, rickets virtually disappeared in the UK until the first major wave of immigration, mainly from the old commonwealth countries. Most of this immigration was from either south Asia or the Caribbean and brought with it a greater predisposition to rickets than was present within the white population because of the need for greater sunlight exposure of more darkly pigmented skin in order to synthesise sufficient vitamin D [3]. This resulted in a second wave of rickets that again occurred mainly in the industrialised cities. Following a pilot study, it was demonstrated that the incidence of rickets in Glasgow could be effectively reduced by a campaign of supplementation [4]. Since then, the incentives to persist in such a campaign appear to have been lost and a third wave resurgence of rickets has been seen in many countries of the world [5]. In the meantime, during the 1960s it was discovered that vitamin D required metabolism in order to become effective and elevated it from the status of ‘vitamin’ to one of ‘hormone’. As a consequence, some forms of rickets that had previously been thought to be caused by vitamin D deficiency were now understood to result from inborn errors of metabolism and explained why some children had not previously responded to vitamin D treatment. Since then the metabolism of vitamin D has been well worked out and provides a logical basis for treatment. The third stage of investigation of vitamin D has been the demonstration that vitamin D deficiency may play an important part in contributing to the aetiology of a number of common diseases that previously had not been associated with vitamin D deficiency. These include certain cancers, especially of the breast and colon, diabetes, both type 1 and type 2, and coronary heart disease. These relationships remain to be worked out although increasing evidence is accumulating that vitamin D plays a part in the prevention of many of these diseases [6]. Whilst they are generally diseases of adulthood, it is conceivable that their origins lie in childhood. Vitamin D remains the preferred treatment of vitamin D deficiency but it seems extraordinary that, in modern societies, the ability to eliminate a fully preventable disease eludes us. It is arguable that effective vitamin D supplementation is the single most cost-effective treatment that could be given, at least to ‘at risk’ populations.

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Parathyroid Hormone

During the 1920s the role of the parathyroid glands in secreting a calcium-raising hormone was clarified [7]. The first description of hypoparathyroidism was made in 1929 [8] and this was followed in 1942 by the description of parathyroid hormone resistance in pseudohypoparathyroidism [9]. Fuller Albright was an early pioneer of parathyroid physiology and pathology who laid the basis of much of what we know about basic parathyroid actions. He was correct in describing pseudohypoparathyroidism as a hormone resistance syndrome. However, as it turns out, he was incorrect in referring to it as ‘an example of Seabright bantam syndrome’. The ‘Seabright bantam’, named after Sir John Sebright (sic), was misspelt in the original paper. It is characterised by the cock birds having a ‘hen-feathering’ appearance which led to the misapprehension that they were resistant to testosterone. In fact, they have excessive activity of aromatase P450 in extragonadal tissues that converts testosterone to oestrogen [10]. It is this that causes the characteristic feathering pattern. In most other aspects, he was correct and he made a huge contribution to our understanding of bone disease and several conditions bear his name eponymously. The first immunoassays for PTH were described in 1969 [11]. These had been developed in the wake of other immunoassays such as those for insulin and growth hormone. However, it rapidly became apparent that these were not straightforward since PTH, a large molecule containing 84 amino acids, circulates as a number of fragments [12]. These are particularly problematic in the presence of renal failure when the inactive fragments tend to circulate in higher quantities than normal. Since the original assays were developed, further refinements have been made that now allow measurement of physiological levels of intact hormone. The structure of PTH was difficult to establish and different structures were proposed initially. Once these were resolved, it became apparent that, although PTH contains 84 amino acids, only the first 34 are required for full biological activity [13]. The function of the remainder of the molecule remains unclear. Subsequent work revealed the mechanism of action of PTH via the Gsα second messenger which is common to a number of polypeptide hormones and which provides an explanation for the hormone resistance state known as pseudohypoparathyroidism that was originally described by Fuller Albright.

Calcitonin

This hormone was first described in 1963 [14] and its structure elucidated in 1968 [15]. It is a 32 amino acid protein and, unlike PTH, has a disulphide bond between the cystine residues at positions one and seven. It has an action that is largely opposite to that of parathyroid hormone, i.e. it has a calcium-lowering effect. There are considerable interspecies differences in structure [16] and, interestingly, the salmon

Voyages of Discovery

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hormone has considerably greater activity than its human counterpart in humans. For this reason, it has been used as a therapeutic agent to lower calcium in certain hypercalcaemic conditions although its use in this respect has been largely superseded by the introduction of the bisphosphonates. CT is now known to be one product of the α-CT/CT gene-related peptide (CALCA) which, as a result of alternative splicing, gives rise to at least two products, α-CT and CT gene-related peptide (CGRP) [17]. Each is produced mainly by different tissues, CT by the C cells of the thyroid and CGRP by the hypothalamus. The physiological role of these proteins, together with those of two other closely related proteins, amylin and adrenomedullin, have yet to be identified precisely, but CT probably makes a contribution to bone formation and CGRP is mainly a neuropeptide which plays a part in vascular tone. It may have a role in the pathogenesis of migraine. Nevertheless, it is also thought that all four proteins may play some part in bone formation [18, 19] possibly via a network of neurones that exists in bone. However, pathological states in man in which CT is produced in excess, such as medullary carcinoma of the thyroid (MCT), do not result in hypocalcaemia and the principal significance of CT is as a marker of MCT and as a therapeutic agent.

Parathyroid Hormone-Related Peptide

The first indication that there is a substance that has PTH-like activity but is not PTH came with a publication in 1985 demonstrating that human umbilical cord blood contained a compound which had PTH-like bioactivity and yet could not be identified as PTH on immunoassay [20]. The calcium concentration in foetal cord blood is unusual in being one of the few substances which is present at higher levels than in the mother, i.e. there is a positive gradient across the placenta. Whilst it had previously been suggested that foetal PTH levels are suppressed because of these relatively high levels of calcium, the question had never been asked as to what maintains the gradient. It seems that it is PTHrP that is responsible. Subsequently, a humoral factor was identified and purified from malignant tissue that was found to be responsible for some instances of humoral hypercalcaemia of malignancy [21]. This was a PTH-like factor that shared some properties with PTH, including its ability to stimulate cyclic-AMP, but was sufficiently dissimilar as to be undetectable on standard PTH immunoassays. It is a considerably larger molecule than PTH itself and has some limited homology with PTH such that it binds to the PTH1 receptor. The role of PTHrP in man seems to be principally in the foetus to maintain the calcium gradient across the placenta and to have a paracrine function in promoting cartilage development. In postnatal life, it seems not to have a classical endocrine role but is important as a mediator of humoral hypercalcaemia of malignancy [22].

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Fibroblast Growth Factor 23

The factors controlling phosphate metabolism have, until relatively recently, not been well understood. The discovery of FGF23 in 2000 [23] led to an explosion of discoveries relating to phosphate. The relationship of this to PHEX, DMP1, GALNT3, FGFR1, Klotho and the sodium/phosphate co-transporter in renal tubular cells has widened our understanding considerably and led to a much greater knowledge of the pathological processes that go towards explaining the mechanisms of disorders of phosphate metabolism. Further details of all these hormones are given in the relevant chapters. The discovery of DNA in the early 1950s and its role as a genetic blueprint has allowed the identification of a whole host of diseases that are genetically based. The diseases related to bone and calcium are no exception and, if one excludes vitamin D deficiency and secondary osteoporosis, it is the case that virtually all other causes of bone and calcium diseases have a genetic origin. Indeed, they encompass the full gamut of genetic conditions including autosomal and X-linked dominant and recessive, mitochondrial and imprinting disorders. It is therefore necessary to have at least a modicum of understanding of genetics in order to be able fully to understand their mechanisms. Fortunately, modern technology allows the rapid advances in genetics to be recorded electronically without having to ‘go back to the books’ all the time. It also ensures that updates to discoveries can be made available to a wide audience more rapidly than previously. The most useful tool is the creation of the Online Mendelian Inheritance in Man (OMIM) website which was the brainchild of the late Victor McKusick [24] when his original paper version became too unwieldy and difficult to update. The website is accessible at http://www.ncbi.nlm.nih.gov/sites/entrez?db = omim and gives details of all disorders that are or are thought to be genetically based, together with the genes involved. Because of the diversity of the disorders of bone and calcium metabolism, each of the clinical chapters in this book is accompanied by at least one table that gives the OMIM reference numbers for these disorders and their genes. Each entry is accompanied by a detailed bibliography which is regularly updated. Hopefully, this will prove useful to readers. An explanation of any abbreviations appearing in the text and which are not defined at the time can be found in the Appendix together with relevant conversion factors for readers who are not familiar with either SI or ‘conventional’ units of measurement. The final chapter in this book is a series of case histories. These are intended to illustrate some of the problems that are discussed in the previous chapters. It is not a comprehensive coverage of all the conditions mentioned but, since this book will be available on line, it will be possible in the future to add further cases. When the text describes a case that is included in the case history section, the number of that case is shown in the text. References to these cases may appear in more than one chapter.

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‘Dr. Donne’s verses are like the peace of God; they pass all understanding’. With these words, King James I of England and VI of Scotland is said to have replied to Archdeacon Plume when asked to comment on the poetry of John Donne [25]. There are many, even in the world of paediatric endocrinology, for whom the same is true of the study of bone and calcium disorders in children and for whom they remain a mystery. This was recognised by the late Graham Chapman, of Monty Python fame, comedian, bon viveur and erstwhile medical student, who, in a book of collected sketches, letters and essays, wrote a brief essay entitled ‘Calcium Made Interesting’ [26]. This book is designed not only to enable mineral metabolism to be understood, but to ‘make calcium interesting’.

References 1 O’Riordan JL: Rickets in the 17th century. J Bone Miner Res 2006;21:1506–1510. 2 DeLuca HF: The vitamin D story: a collaborative effort of basic science and clinical medicine. FASEB J 1988;2:224–236. 3 Parra EJ: Human pigmentation variation: evolution, genetic basis, and implications for public health. Am J Phys Anthropol 2007;(suppl 45):85–105. 4 Dunnigan MG, Glekin BM, Henderson JB, McIntosh WB, Sumner D, Sutherland GR: Prevention of rickets in Asian children: assessment of the Glasgow campaign. Br Med J (Clin Res Ed) 1985;291:239– 242. 5 Chesney RW: Rickets: the third wave. Clin Pediatr (Phila) 2002;41:137–139. 6 Bouillon R, Eelen G, Verlinden L, Mathieu C, Carmeliet G, Verstuyf A: Vitamin D and cancer. J Steroid Biochem Mol Biol 2006;102:156–162. 7 Collip JB: The internal secretion of the parathyroid glands. Proc Natl Acad Sci USA 1925;11:484–485. 8 Albright F, Ellsworth R: Studies on the physiology of the parathyroid glands. I. Calcium and phosphorus studies on a case of idiopathic hypoparathyroidism. J Clin Invest 1929;7:183–201. 9 Albright FBC, Smith PH, Parson W: Pseudohypoparathyroidism: an example of ‘Seabright-Bantam syndrome’. Endocrinology 1942;30:922–932. 10 Matsumine H, Wilson JD, McPhaul MJ: Sebright and Campine chickens express aromatase P-450 messenger RNA inappropriately in extraglandular tissues and in skin fibroblasts. Mol Endocrinol 1990; 4:905–911. 11 Berson SA, Yalow RS, Aurbach GD, Potts JT: Immunoassay of Bovine and Human Parathyroid Hormone. Proc Natl Acad Sci USA 1963;49:613– 617.

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12 Berson SA, Yalow RS: Immunochemical heterogeneity of parathyroid hormone in plasma. J Clin Endocrinol Metab 1968;28:1037–1047. 13 Rosenblatt M, Segre GV, Tregear GW, Shepard GL, Tyler GA, Potts JT Jr: Human parathyroid hormone: synthesis and chemical, biological, and immunological evaluation of the carboxyl-terminal region. Endocrinology 1978;103:978–984. 14 Copp DH: Calcitonin: a new hormone from the parathyroid which lowers blood calcium. Oral Surg Oral Med Oral Pathol 1963;16:872–877. 15 Neher R, Riniker B, Rittel W, Zuber H: Human calcitonin: structure of calcitonin M and D. Helv Chim Acta 1968;51:1900–1905. 16 Niall HD, Keutmann HT, Copp DH, Potts JT Jr: Amino acid sequence of salmon ultimobranchial calcitonin. Proc Natl Acad Sci USA 1969;64:771– 778. 17 Rosenfeld MG, Lin CR, Amara SG, Stolarsky L, Roos BA, Ong ES, et al: Calcitonin mRNA polymorphism: peptide switching associated with alternative RNA splicing events. Proc Natl Acad Sci USA 1982; 79:1717–1721. 18 Huebner AK, Keller J, Catala-Lehnen P, et al: The role of calcitonin and alpha-calcitonin gene-related peptide in bone formation. Arch Biochem Biophys 2008;473:210–217. 19 Naot D, Cornish J: The role of peptides and receptors of the calcitonin family in the regulation of bone metabolism. Bone 2008;43:813–818. 20 Allgrove J, Adami S, Manning RM, O’Riordan JL: Cytochemical bioassay of parathyroid hormone in maternal and cord blood. Arch Dis Child 1985;60: 110–115.

Allgrove

21 Burtis WJ, Wu T, Bunch C, et al: Identification of a novel 17,000-dalton parathyroid hormone-like adenylate cyclase-stimulating protein from a tumor associated with humoral hypercalcemia of malignancy. J Biol Chem 1987;262:7151–7156. 22 Kaiser SM, Goltzman D: Parathyroid hormone-related peptide. Clin Invest Med 1993;16:395–406. 23 Yamashita T, Yoshioka M, Itoh N: Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain. Biochem Biophys Res Commun 2000; 277:494–498.

24 Obituary: Professor Victor McKusick: advocate of the Human Genome Project. The Times 2008 Aug 1. 25 Oxford Dictionary of Quotations, ed 2. London, Oxford University Press, 1953. 26 Chapman G: Calcium Made Interesting; in Yoakum J (ed): Calcium Made Interesting: Sketches, Letters Essays and Gondolas. London, Sidgwick & Jackson, 2005, pp 88–89.

Jeremy Allgrove, MD Department of Paediatric Endocrinology, David Hughes Building, First Floor Royal London Hospital, Whitechapel London E1 1BB (UK) Tel. +44 20 7377 7468, Fax +44 20 7943 1353, E-Mail [email protected]

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Chapter 2 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 8–31

Physiology of Calcium, Phosphate and Magnesium Jeremy Allgrove Royal London Hospital, and Great Ormond Street Hospital, London, UK

Abstract The physiology of calcium and the other minerals involved in its metabolism is complex and intimately tied in with the physiology of bone. Five principal humoral factors are involved in maintaining plasma levels of calcium, magnesium and phosphate and coordinating the balance between these and their content in bone. The transmembrane transport of these elements is dependent on a series of complex mechanisms that are controlled by these hormones. The plasma concentration of calcium is initially sensed by a calcium-sensing receptor which then sets up a cascade of events that initially determines parathyroid hormone secretion and eventually results in a specific action within the target organs, mainly bone and kidney. This chapter describes the physiology of these humoral factors and relates them to the pathological processes that give rise to disorders of calcium and bone metabolism. It details the stages in the calcium cascade and describes the effects on the various target organs. The pathology of disorders of bone and calcium metabolism is described in detail Copyright © 2009 S. Karger AG, Basel in the relevant chapters.

Calcium, phosphate and magnesium metabolism is intimately bound and it is necessary to discuss all three together. Furthermore, this metabolism is, in many ways, different from that of most other substances by virtue of the fact that the majority of each is contained within bone which acts as a structural material, as well as a reservoir, whilst also acting as an important physiological regulator. Thus, calcium is required to be kept within narrow limits within plasma in order to maintain optimum neuromuscular function and phosphate is involved in virtually all metabolic processes whilst magnesium is required to ensure optimum parathyroid hormone secretion. The mechanisms required to maintain these levels are complex and dependent on a number of factors. It is the purpose of this chapter to describe these factors and to indicate how disorders of function give rise to clinical problems.

Calcium Physiology

A fully grown adult contains approximately 1,200 g of calcium. In foetal and neonatal life, the total calcium content is related to body weight and a very close relationship exists between the two under normal circumstances. This relationship is expressed by the formula: Ca = 0.00075*BWt1.3093,

where Ca and BWt are both expressed in grams [1]. The relationship has been observed during the foetal and neonatal period and probably largely holds true throughout the period during which bone accretion is occurring. About 99% of calcium is normally contained within bone, the remainder being present either as an intracellular cation or circulating in plasma. Within plasma, there are three main fractions: ionised, protein bound and complexed, mainly to citrate or sulphate. The ionised fraction constitutes approximately 50% of the total. Most blood gas machines found within critical or intensive care units can measure ionised calcium directly. Of the remainder, most circulates bound to albumin and plasma albumin levels affect the total concentration of calcium. Various formulae are used to ‘correct’ total calcium to allow for this and many laboratories automatically provide a value for ‘corrected’ calcium (see chapter 6). The concentration of ionised calcium is normally kept within very narrow limits (1.1–1.3 mmol/l), a level which is necessary to maintain normal neuromuscular activity. Complex mechanisms are involved in maintaining this level. These involve altering calcium absorption in the gut, changing excretion within the renal tubules and balancing the rate of deposition into or removal from bone. If calcium levels vary significantly from this, either upwards or downwards, symptoms may develop. These are discussed in more detail in the relevant chapters.

Control of Plasma Calcium

Five principal humoral factors are involved in the maintenance of normal levels of calcium and phosphate in plasma. Plasma calcium is mainly influenced by parathyroid hormone (PTH) and the active form of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)2D). In addition, calcitonin (CT) and parathyroid hormone-related peptide (PTHrP) play a more minor role, at least in postnatal life, but attain greater significance in a number of pathological situations. Plasma phosphate is also influenced by PTH and 1,25(OH)2D, but another factor, fibroblast growth factor 23 (FGF23) also plays an important part in its metabolism. Magnesium is influenced, though to a lesser degree, by the same factors that control calcium but, in addition, itself influences calcium indirectly by altering the PTH secretion in response to hypocalcaemia.

Calcium Physiology

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Calcium TRPV5

TRPV6 Claudin 16

Mainly GI

Mainly renal

Na+

Paracellular

CB28k CB9k

Ca2+ PMCA1b

NCX1 Ca2+

Fig. 1. Schematic representation of the mechanisms of calcium transport in the gut and renal tubules. Similar mechanisms are present in both tissues although the importance of each differs between them. The principal mechanisms in the gut are shown on the right hand side and the more important ones in the renal tubules are shown on the left. Abbreviations are explained in the Appendix.

Transmembrane Calcium Transport

Calcium balance is controlled principally by transport across membranes in the gastrointestinal tract and in renal tubules. The mechanisms for both are similar but with differences of emphasis depending on which organ is affected. Transport occurs by both paracellular and transcellular mechanisms. They are summarised in figure 1. Paracellular transport occurs through the tight junctions between cells and is facilitated by a number of proteins including, amongst others, the claudins. The most important of these is claudin 16 (*603959) (also known as paracellin 1). It is coded for by a gene on chromosome 3q27 and has its action mainly in renal tubules where it also facilitates passive transport of magnesium. Mutations in this gene cause the hypomagnesaemia, hypercalciuria and nephrocalcinosis (HOMG3) syndrome (#248250) in which both calcium and magnesium are poorly reabsorbed (see chapter 6). The most important mechanism for calcium absorption in the gut is via active transport. Three steps are involved in this process [2]. There is initial absorption of calcium from the lumen followed by transcellular transport and lastly extrusion of

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calcium across the basolateral membrane. A similar process involving related proteins is present in renal tubules (see below). Two recently discovered proteins, TRPV5 (*606679) and TRPV6 (*606680), which are members of the transient receptor potential (TRP) channel protein family, are thought to play an important role in promoting active calcium transport [2, 3]. In the gut the most important of these is TRPV6 whilst TRPV5 plays a larger role in renal tubules. They are inwardly rectifying calcium channels whose affinity is greater for calcium than for magnesium. Once calcium reaches the intracellular compartment, cytosolic diffusion across the cell is facilitated by two further proteins, calbindin28K (*114050) and calbindin9K (*302020). These bind calcium and transport it across the cytoplasm. At the basolateral surface, extrusion of calcium is facilitated by both an ATP-dependent Ca+-transporting ATPase (PMCA1b) (*108731) and a Na+/ Ca+ exchanger (NCX1) (*182305). The former of these is more important in the gut. There are vitamin D receptors in the small intestinal cells and both this and TRPV6 are stimulated by it. If these receptors are defective, as in hereditary 1α,25(OH)2Dresistant rickets (HVDRR) (#277440), calcium cannot be absorbed properly and rickets results (see chapter 8 for further details). Calcium absorption is also influenced by a number of other factors. In particular, absorption can be reduced in the presence of large quantities of calcium-binding agents such as phytate or oxalate [4]. Bisphosphonates also bind to calcium in the gut and, if used orally for therapeutic purposes, should be taken as far away from meals as possible. Alternatively, a high calcium intake helps to protect from the effects of vitamin D deficiency [5], presumably by increasing passive absorption. Calcium reabsorption in renal tubules is largely passive. It is influenced by a number of dietary factors including a high sodium, protein or acid load, all of which increase calcium excretion. About 70% of filtered load is reabsorbed passively in the proximal tubule in conjunction with sodium. A further 20% is reabsorbed in the loop of Henle by paracellular processes under the influence of claudin 16 (paracellin 1). Paracellular reabsorption does not occur unless claudin 16 is present. The remaining 5–10% is reabsorbed in the distal tubule. Similar mechanisms to those in the gut are present although TRPV5 is thought to be the major influence. Transcellular transport is facilitated by calbindins. At the basolateral surface NCX1 is the more important mechanism. This is under hormonal influence, mainly PTH, and, in the presence of hypoparathyroidism, treatment with active vitamin D analogues must be monitored carefully to prevent hypercalciuria.

Magnesium Metabolism

Magnesium is, like calcium, a divalent cation that is important for bone and calcium metabolism. It is normally present in plasma at a concentration of between 0.7 and 1.2 mmol/l. Adequate plasma magnesium is required for normal secretion of PTH

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Magnesium TRPM6

TRPM7

Claudin 16 Claudin 19

Paracellular

+

Na+ Pro-EGF ? EGFR

? ␣ ␤ Y

Mg2+ EGF

Na-K ATPase Mg2+

Fig. 2. Schematic representation of the mechanisms of magnesium transport in the gut and renal tubules. Similar mechanisms are present in both tissues although the importance of each differs between them. Abbreviations are explained in the Appendix.

which occurs in response to a magnesium-dependent adenylate cyclase system. In the presence of hypomagnesaemia, PTH secretion is inadequate and correction of hypocalcaemia cannot occur normally [6]. Magnesium absorption occurs in the small intestine by mechanisms that are very similar to those of calcium although these mechanisms are not so well understood [3]. They are summarised in figure 2. Two proteins, TRPM6 and TRPM7, which are related to the corresponding proteins involved in calcium absorption, are present in renal tubules and intestinal cells. Mutations in TRPM6 cause hypomagnesaemia with secondary hypocalciuria (HOMG1) (#602014) as a result of impaired magnesium absorption in the gut (see chapter 6). Transcellular transport of magnesium probably involves similar calbindin proteins to those of calcium. Renal tubular reabsorption of magnesium mostly occurs by passive reabsorption in the ascending loop of Henle along with calcium. The tight junction protein claudin 16 (Paracellin 1) (*603959) is principally responsible for this and mutations in its gene cause the HOMG3 syndrome (#248250). Because calcium reabsorption is also impaired, this is accompanied by hypercalciuria and nephrocalcinosis (see chapter 6).

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Further along the renal tubule, active reabsorption takes place in the DCT where TRPM6 is situated. It is under the influence of epidermal growth factor (EGF) which is present on the basolateral membrane of the renal cells where it is processed from Pro-EGF [7]. Following cleavage from Pro-EGF, it interacts with its receptor, the EGFR (*131550), which, amongst its other actions, stimulates magnesium absorption via TRPM6 on the luminal surface. It has recently been shown that mutations in the EGF gene disrupt the basolateral sorting of Pro-EGF resulting in understimulation of TRPM6 and impaired magnesium reabsorption [8]. The resulting condition is known as isolated recessive renal hypomagnesaemia (IRH) (see chapter 6). Transcellular transport of magnesium is probably effected by proteins similar to the calbindins involved in calcium transport but these mechanisms are not well understood. At the basolateral membrane, magnesium is transported partly by a mechanism that involves Na+/K+ ATPase. This has three subunits, α, β and γ, the latter of which is coded for by the FXYD2 (*601814) gene. Mutations in this gene result in defective magnesium reabsorption in the autosomal-dominant renal hypomagnesaemia associated with hypocalciuria (HOMG2) (#154020) syndrome (see chapter 6). The thiazide-sensitive sodium chloride co-transporter (NCC) is also involved in magnesium transport and mutations in the coding gene, SLC12A3 (*600968), cause Gitelman’s syndrome (#263800) in which hypermagnesuria is a feature. Raised urinary magnesium excretion is also present in some cases of Bartter’s syndrome which is caused by a variety of mutations affecting chloride and sodium reabsorption in the loop of Henle. In the last part of the renal tubules, the collecting ducts, both calcium and magnesium are again reabsorbed passively via the tight junction protein, claudin 19 (*610036). Mutations in this gene cause renal hypomagnesaemia with ocular involvement (#248190) (see chapter 6). Renal tubular transport of magnesium can also be increased by several non-genetic causes including diuretics, diabetic ketoacidosis, gentamicin, mercury-containing laxatives, transplanted kidney, urinary tract obstruction, the diuretic phase of acute renal failure and cisplatin.

Phosphate Metabolism

A fully grown adult contains approximately 700 g phosphate. As with calcium, the total body content of phosphate is closely related to body weight and is expressed by the formula: PO4 = 0.00037*BWt1.2409.

Approximately 80% of phosphate is contained in bone. Of the remainder, 45% (9% of the total) is present in skeletal muscle, 54.5% in the viscera and only 0.5% in extracellular fluid. Most of the phosphate is present in inorganic form but plays a crucial

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part in many intracellular processes. In plasma, phosphate circulates in the form of phospholipids, phosphate esters, and free inorganic phosphate (Pi). Plasma Pi concentrations are not as tightly controlled as those of calcium and reflect the fluxes of phosphate entering and leaving the extracellular pool. In contrast to calcium, phosphate concentrations in plasma vary considerably during life, being highest during phases of rapid growth. Thus, phosphate concentrations in premature infants are normally above 2.0 mmol/l (6.4 mg/dl), falling to 1.3–2.0 mmol/l (4.2–6.4 mg/dl) during infancy and childhood and to 0.7–1.3 mmol/l (2.2–4.3 mg/dl) in young adults. Phosphate transport across membranes is controlled by a series of sodium-dependent active transport mechanisms (Na/Pi co-transporters). Three classes are known to exist. Type 1 is present in renal tubular brush borders but is not thought to have a major role in renal tubular reabsorption of phosphate. Type 2, which has three subtypes, 2a, 2b and 2c, are probably the most important in regulating phosphate absorption and reabsorption. Type 3 is present in many tissues but is thought to have more of a ‘gatekeeping’ role. Phosphate is readily absorbed throughout the small bowel by both passive and active mechanisms. Approximately 70% is absorbed via type 2b Na/Pi co-transporter, the remainder being by passive absorption. This active transport is stimulated directly by 1,25(OH)2D and therefore indirectly by hypocalcaemia and PTH [9]. Since hypophosphataemia is a powerful stimulant of 25-hydroxyvitamin D-1-alphahydroxylase (1α-hydroxylase), phosphate deficiency itself stimulates increased absorption. However, the total amount absorbed is dependent on the dietary phosphate load and may be inhibited by phosphate-binding agents such as calcium acetate (Phosex®), calcium carbonate (Tetralac®) or sevelamer (Renagel®). These are of value in hyperphosphataemic states such as chronic renal failure when phosphate absorption needs to be limited. The metabolism of phosphate has, until recently, been relatively poorly understood. However, in 2000, a new member of the fibroblast growth factor family, FGF23 was discovered [10]. This was subsequently shown to be mutated in cases of autosomaldominant hypophosphataemic rickets (ADHR) (#193100) [11] and it is now thought to play a key role in phosphate metabolism. FGF23 is derived from bone cells, particularly osteocytes, circulates in plasma, and is subject to a variety of feedback mechanisms. As a result, it is now considered to be a classic hormone. Its synthesis and secretion are modified by several factors, especially PHEX and DMP1 (see below), and it undergoes post-translational modification, which results in either inactivation (cleavage) or activation (O-glycosylation) before becoming active. Once activated, its principal target organ is the renal tubule where it stimulates renal phosphate excretion and inhibits 1α-hydroxylase activity to reduce levels of 1,25(OH)2D. Hypophosphataemia also inhibits FGF23 secretion. These actions are summarised in figure 3. FGF23 (*605380) is a 251 amino acid protein which includes a 24 amino acid signal sequence. It is coded for by a gene on chromosome 12p13.3. It has a crucial cleavage site between residues arginine179 and serine180 where it is cleaved by a subtilisin/ furin-like enzyme that renders it inactive [12]. A second arginine residue is present at

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DMP1

BMP1 etc

Gs␣

GALNT3 Endopeptidase

FGFR1c

FGF23

Klotho

Renal tubule Subtilisin/furin Na/Pi PHEX

N-terminal

C-terminal

f1␣-hydroxylase Degradation products

DPhosphaturia DPhosphate absorption

fPlasma phosphate

f1,25(OH)2D

Fig. 3. Schematic representation of the control of phosphate metabolism. Fibroblast growth factor 23 (FGF23) sits at the centre. Its secretion is influenced by several other factors and it has to undergo modification before becoming active. Its receptor on the renal tubule enables it to promote phosphate excretion. Solid lines represent stimulatory effects and interrupted lines inhibitory actions. Abbreviations are explained in the Appendix.

position 176 and mutations in either of these arginine residues renders FGF23 resistant to cleavage without affecting its intrinsic activity. As a consequence, circulating FGF23 levels remain high and result in the excessive renal phosphate loss in ADHR (#193100) (see chapter 9 for further details). Activation of FGF23 occurs by O-glycosylation. This occurs under the influence of UDP-nacetyl-alpha-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) (*601756). This is a 633 amino-acid protein that is coded for by a gene on chromosome 2q24-q31 [13]. The gene has ten exons and GALNT3 itself has a single transmembrane spanning region. It catalyses the O-glycosylation of serine and threonine residues on the native protein. Its crucial role in phosphate metabolism is demonstrated by the fact that inactivating mutations in this gene result in hyperphosphataemic familial tumoral calcinosis type 1 (HFTC1) (#211900) [14] in which, although FGF23 levels are high, these are inactive and hyperphosphataemia and soft tissue calcinosis occurs (see chapter 9). The principal target organ of FGF23 is the renal tubule. Once it has been activated, it acts on a receptor on the surface of the tubules. This receptor is part of the fibroblast growth factor receptor family, FGFR1(IIIc) (*136350), which is coded for by

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a gene on chromosome 8p11.2-p11.1. The FGF receptors, of which four classes are described, are involved in a wide variety of functions, and mutations in FGFR1 give rise to several conditions including Kallmann syndrome, hypogonadotrophic hypogonadism and a number of skeletal abnormalities [15]. FGFR1 is therefore not specific for FGF23. However, in 2006 another factor, α-Klotho (KL) (+604824) was found to act as a cofactor which confers specificity of FGFR1(IIIc) for FGF23 [16]. Klotho (named after the Greek Fate who spins the thread of life) is coded for by a gene on chromosome 13q12. Patients with chronic renal failure (CRF) have low renal expression of KL and it has been suggested that this may accelerate the degenerative processes, such as atherosclerosis, osteoporosis and skin ageing, seen in CRF [17]. KL is not capable of acting as an FGF23 receptor on its own, but requires FGFR1. Similarly, FGFR1 is not active as an FGF23 receptor if KL is inhibited or mutated [16]. Rare patients have been described who have inactivating mutations of KL which cause a form of tumoral calcinosis and activating mutations have been associated with hypophosphataemic rickets that is associated with hyperparathyroidism (see chapter 9). Once the FGFR1-KL complex has been activated by FGF23, it increases renal tubular phosphate excretion by means of the NaPi-IIc/SLC34A3 (*609826) exchanger at the luminal surface of the cells. Mutations in the NaPi-IIc exchanger result in hereditary hypophosphataemic rickets with hypercalciuria (HHRH) (#241530). However, unlike those conditions which are associated with high FGF23 levels, low levels of active FGF23 are present. 1α-Hydroxylase activity is therefore not inhibited. The resulting raised levels of 1,25(OH)2D not only stimulate calcium and phosphate absorption in the gut but also increase calcium excretion in renal tubules, as a consequence of which hypercalciuria and renal stones are present. The secretion and initial processing of FGF23 is under the influence of several other factors. The most important of these is the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) (*300550). Several studies in the hyp-mouse, an animal model of X-linked dominant hypophosphataemic rickets (XLH), have demonstrated that PHEX is somehow involved in the regulation of FGF23 despite the fact that it is not present in renal tubules. The precise mechanisms by which this occurs are not fully understood but may involve either modification of the activity of the subtilisin/furin enzyme activity that cleaves FGF23, or by modifying dentin matrix protein 1 (DMP1) that also affects FGF23 (see below). Whatever the precise mechanism, mutations in PHEX result in failure of cleavage of FGF23 which therefore causes hyperphosphaturia and hypophosphataemia. DMP1 (*600980) is another protein synthesised by osteocytes. It is one of a number of small integrin-binding ligand, N-linked glycoproteins (SIBLING) that are involved in bone mineralisation. It may act as a mechanostat that responds to changes in stresses within bone transmitted via the fluid filled canaliculi within bone in which the osteocytes lie. It is cleaved into two fragments of 35 and 57 kDa, respectively, possibly by the action of bone morphogenic protein 1 (BMP1). The latter of these is an active inhibitor of FGF23 secretion. Thus, DMP1 has a controlling effect on the

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action of FGF23. Homozygous or compound heterozygous mutations in DMP1 result in excess FGF23 secretion which results in autosomal-recessive hypophosphataemic rickets (ARHR) (#241520) (see chapter 9). Other SIBLING proteins include bone sialoprotein (BSP) (*166490), osteopontin (OPN) (*166490), dentin sialophosphoprotein (DSPP) (*125485) and matrix extracellular phosphoglycoprotein (MEPE) (*605912). Some of these are upregulated in certain forms of cancer and may be responsible for alterations in FGF23 secretion that causes TIO. Some individuals with the McCune-Albright polyostotic fibrous dysplasia, caused by somatic mutations in the alfa subunit of the stimulatory G-protein (Gsα), have an associated excess phosphate excretion secondary to increased FGF23 by an, as yet, ill understood mechanism. Hypophosphataemia and rickets are also seen in several primary renal tubular abnormalities, such as the Fanconi syndrome (whatever the cause), in which a generalised proximal renal tubular defect, which results in bicarbonaturia, glycosuria and amino aciduria as well as a phosphate leak, is present. The most common inherited cause of Fanconi syndrome is cystinosis (#219800) and rickets may be the presenting feature of this condition. Hyperparathyroidism also causes a mild form of Fanconi syndrome and patients with parathyroid tumours may have a mild metabolic acidosis and aminoaciduria in addition to hypercalcaemia. These features resolve when the tumour is removed.

The Calcium Cascade

The concentration of calcium in plasma is normally maintained within very narrow limits. The initial stage of this process is binding of calcium to a specific calciumsensing receptor. This then initiates a cascade of events that terminates in the action of PTH on its target organs (fig. 4).

The Calcium Sensing Receptor

The calcium-sensing receptor (CaSR) (+601199) is a large molecule consisting of 1078 amino acids. It is coded for by a gene on chromosome 3q13-q21. It has a large extracellular calcium-binding domain consisting of approximately the first 610 residues followed by a seven-transmembrane domain consisting of the next 250 residues with a further 210 residues making up the intracellular component. The receptor is present in many tissues, especially the parathyroid glands and renal tubules, but also in bone and cartilage as well as other tissues [18]. When calcium binds to the extracellular domain, it alters PTH secretion via both phospholipase Cb and G-protein second messengers. As a consequence, PTH secretion changes in a sigmoidal fashion in response to acute changes in plasma calcium (fig. 5), and there is a continuous tonic secretion of PTH, which maintains plasma-ionised calcium at whatever level is ‘set’

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Ca2+ CaSR

Parathyroid Glands PTH

PTH1R

Gs␣,␤␥

Target Organs – Kidney Bone (Gut)

Fig. 4. The calcium cascade. Plasma calcium levels are controlled by a series of events that begin with the effect of calcium on the calcium sensing receptor and end with the response of the target organs.

by the CaSR [19]. Magnesium also binds to the CaSR and influences PTH secretion in a similar, but less potent, manner to that of calcium. However, severe magnesium deficiency inhibits PTH secretion, probably because the adenylate cyclase coupled to the G-protein is itself magnesium dependent [6]. Mutations within the CaSR gene result in either inactivation or activation of the receptor, which result in hyper- and hypocalcaemia respectively. Inactivating mutations cause insensitivity to calcium, which shifts the curve of PTH secretion in response to plasma calcium to the right (fig. 5). As a consequence, PTH secretion is switched off at a higher concentration than normal, and hypercalcaemia results [18]. The receptors are also present in the renal tubule, and renal calcium excretion is thereby reduced. The resulting condition is known as familial benign hypercalcaemia (FBH) or familial hypocalciuric hypercalcaemia (FHH) (#145980) (see chapter 7). In contrast, activating mutations of the receptor shift the PTH secretion curve to the left (fig. 5) causing chronic hypocalcaemia and hypercalciuria, a condition known as autosomal-dominant hypocalcaemia (ADH) (#146200). One particular mutation causes a constitutive activation of the receptor which remains constantly active whatever the calcium concentration so that, rather than shifting the curve to the left, PTH secretion remains permanently switched off [20] (see chapter 6).

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80 70 Inactivating mutations 60 50 40

Intact PTH

30 20

Activating mutations

10 Ca2+ 0 0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

1.40

1.45

1.50

Fig. 5. Schematic representation of the relationship between plasma ionised calcium and PTH secretion as determined by the calcium sensing receptor. Inactivating mutations generally shift the curve to the right whilst activating mutations do so to the left.

Many of the mutations found in FBH are clustered around the aspartate- and glutamate-rich regions of the extracellular domain of the molecule, and it has been postulated that this region contains low-affinity binding sites for calcium. Many of the FBH kindreds have been found to have unique mutations. Mutations have also been detected within the transmembrane domain but only rarely within the intracellular domain. Mutations within this latter domain may have a greater effect on the CaSR in the parathyroid glands than in the renal tubules and patients are described in whom inactivating mutations are associated with hypercalciuria and PT gland hyperplasia necessitating parathyroidectomy [21]. Similarly, most activating mutations that cause ADH are present within the extracellular calcium-binding domain. One hundred and twenty-eight mutations, three of which are polymorphisms, have been described so far, and an online database has been established to keep track of them (http://www. casrdb.mcgill.ca). Two other loci, located on chromosomes 19p and 19q13, respectively, have been identified by family linkage studies. The precise nature of the gene products of these loci remains uncertain, but mutations within them result in clinical syndromes that are very similar to those resulting from inactivating mutations of the CaSR itself. Not all families with FBH have mutations within the CaSR gene, and it has been suggested that there may be abnormalities either within the CaSR gene promoter or within one

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of these two loci found on chromosome 19. The three variants of FBH linked to chromosome 3q, 19p and 19q, have therefore been referred to as FBH types 1–3.

The Parathyroid Glands

The parathyroid (PT) glands, usually four in number but sometimes as many as seven, are derived embryologically from the third (lower glands) and fourth (upper glands) branchial arches. Several transcription factors are involved in their development [22]. Some, such as Hoxa3 (thyroid and thymus, chromosome 7p15-p14.2) (*142954), GATA3 (hearing sensation and kidney, chromosome 10p13–14) (*131320), Tbx1 (thymus, cardiac outflow tract and the face, chromosome 22q11) (*602054) and UDF1L are involved in the development of other structures. The latter two genes are located on the long arm of chromosome 22. Mutations within or deletion of the genes responsible for these factors result in congenital hypoparathyroidism that is associated with other conditions such as the hypoparathyroidism, deafness, renal anomalies (HDR) (#146255) syndrome and the 22q deletion complex, of which the DiGeorge syndrome (DGS) (#188400) is part. The homologue of drosophila glial cells missing 2 (GCM2) (*603716) is a highly conserved gene that is necessary for PT gland development. It has no other known function in man. Mutations in this gene cause autosomal recessive familial isolated hypoparathyroidism (FIH) (#146200). It is also thought that the SRY-related HMGbox gene 3 (SOX3) (*313430), located on the X-chromosome, is involved in PT gland development and mutations in this gene may be responsible for X-linked recessive familial isolated hypoparathyroidism (%307700). Apart from these autosomal- and X-linked syndromes, there are several mitochondrial genes that are involved in PTG development. Mutations in these genes give rise to a variety of syndromes in which hypoparathyroidism is a feature. Because the genes are mitochondrial, these syndromes are maternally inherited (for full details of these conditions, see chapter 6). In addition to these genetic causes, destruction of the glands may occur as a result of surgery (e.g. following thyroidectomy), infiltration (e.g. with iron in β-thalassaemia) or antibodies. These may either be isolated or associated with autoantibodies to other organs as in the polyendocrinopathy type 1 syndrome (APS1), also known as the APECED syndrome (#240300) (see chapter 6 for further details).

Parathyroid Hormone

PTH (*168450) is a single-chain polypeptide hormone containing 84 amino acids. It is encoded by a gene on chromosome 11. It is synthesised by the parathyroid glands from prepro-PTH, which has an additional 31 N-terminal amino acids. Synthesis occurs in

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the ribosomes, where the initial 25-amino-acid ‘pre’ sequence acts as a signal peptide to aid transport through the rough endoplasmic reticulum [23, 24]. The ‘pre’ sequence is cleaved, and pro-PTH then travels to the Golgi apparatus where the 6-amino-acid ‘pro’ sequence is cleaved to yield the mature hormone, which is stored in secretory vesicles that fuse with the plasma membrane prior to secretion of the hormone [25]. Very little PTH is stored within the glands, and most of the secreted hormone is newly synthesised. Mutations in the PTH gene, involving the pre-pro- sequence have been described that result in inherited hypoparathyroidism (see chapter 6). Only the first 34 N-terminal amino acids are required for full activity, and the function of the remainder of the molecule is not understood. The half-life of PTH in the circulation is 1–2 min [25]. The molecule is cleaved at various sites, which results in a number of fragments that can be identified in the circulation. The best modern assays of PTH measure ‘intact’ PTH, are able to measure physiological concentrations of PTH, correlate well with bioactivity and ignore the inactive fragments. This is particularly important in conditions such as chronic renal failure where inactive fragments are cleared less rapidly than normal. Normal levels of PTH in the circulation are about 1–6 pmol/l (10–60 pg/ml) but vary depending on the assay used.

The PTH Receptors

PTH acts via two receptors. The first and principal one is PTH1R (also called PTH/ PTHrP) receptor (*168468), which has equal affinity for both PTH and PTHrP. It consists of 593 amino acids coded by a gene on the long arm of chromosome 3 [26]. It has an extracellular binding domain of 190 residues, a seven-transmembrane domain, and a cytosolic component of 134 residues. Both inactivating and activating mutations of the PTH1R have been described. These result in the very rare conditions of Blomstrand lethal chondrodysplasia (#215045 ) and Jansen disease (#156400) respectively. A second PTH2 receptor (PTH2R) is present in the central nervous system. PTHrP is not a ligand for it.

Intracellular Signalling

Intracellular signalling occurs principally by coupling of the cytosolic component of the PTH1R to G-protein second messengers, Gs and Gq [27]. These are heterotrimeric, consisting of α, β, and γ subunits. In the resting state, they are associated, and the Gsα subunit is bound to GDP. Binding of the ligand with the receptor results in GDP being exchanged for GTP and dissociation of the Gsα subunit from the β,γ complex. The Gsα is then free to stimulate adenylate cyclase, which results in an increase in intracellular cAMP, which activates the various actions of PTH via specific protein kinases. Intrinsic GTPase activity associated with the Gsα subunit hydrolyses GTP to GDP, which causes

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reassociation of the components of the G-protein. At the same time, phosphodiesterases inactivate the cAMP to AMP, and the cell reverts to its resting state. This mechanism is common to several hormones, including thyroid-stimulating hormone (TSH), gonadotropins, and growth hormone-releasing hormone (GHRH) [27]. The Gsα subunit is coded by a gene, GNAS1, (+139320) located on chromosome 20q13.3. This complex gene contains 13 exons that code for the Gsα subunit itself plus several other exons, known as A/B, XL, NESPAS (*610540) (which is an antisense transcript) and NESP55 which is only expressed in renal tubules. Alternative promoter use and splicing results in several different mRNA transcripts. In most tissues, these show biallelic expression, but the transcripts arising from the A/B, XL and NESPAS exons are paternally derived whilst those arising from the NESP55 exon are maternally expressed. This results from methylation of these uniparental alleles that either switches on or switches off the activity of those alleles in an epigenetic manner (fig. 6). In addition, there is a further gene, Syntaxin (STX16) (*603666), upstream of the GNAS complex which appears to influence the methylation of the A/B exon. Mutations within the biallelic coding region (exons 2–13) of the gene result in resistance to the action of PTH which clinically causes pseudohypoparathyroidism type 1a (PHP1A) (#103580) if they are associated with the maternally derived transcripts but cause pseudopseudohypoparathyroidism (PPHP) (#612463) and/or progressive osseous heteroplasia (POH) (#166350) if derived from paternal sources [28]. These patients frequently have resistance to other hormones whose action is mediated via the Gsα second-messenger mechanism and many display features of Albright’s hereditary osteodystrophy (AHO). Somatic activating mutations in the GNAS complex are responsible for the McCune-Albright syndrome (MAS) (#174800) (see chapter 12 for more details). Alterations in the methylation patterns of the monallelic exons, particularly A/B, cause pseudohypoparathyroidism type 1b (PHP1B) (#603233) when they are on the maternally derived alleles. Under these circumstances there are no mutations found in the coding regions of the GNAS gene and the patients do not usually have evidence of AHO. Mutations in STX16 have also been associated with some forms of pseudohypoparathyroidism type 1b probably by influencing the methylation of the A/B exon (see chapter 6).

The Target Organs

The principal target organs of PTH are bone and kidney. In bone, PTH has two main effects. Under physiological conditions, it promotes bone formation via receptors on the osteoblasts. Under circumstances of hypocalcaemia, PTH stimulates bone resorption in order to retrieve calcium from the large reservoir within bone so that normocalcaemia can be restored. There are very few receptors for PTH in osteoclasts and bone resorption occurs as a result of changes within the relationship between osteoblasts and osteoclasts. Both RANKL and osteoprotegerin (OPG) are produced by osteoblasts. RANKL stimulates osteoclast differentiation whilst OPG acts as a decoy ligand

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STX1 M P

Nesp55 – +

Nespas + –

XL + –

A/B + –

1

2

3 3N

4 5

6

7

13

Allele-specific methylation

Bi-allelic (most tissues)

Exons 2–13 Exon1

Gsα

Exons 2–13 A/B Paternal

Exons 2–13 XL Exons 2–13 Nespas

Maternal

Exons 2–13 Nesp55

Fig. 6. Diagrammatic representation of the GNAS gene showing the different products that result from alternative splicing. Native Gsα is expressed biallelically. The A/B, XL and Nespas transcripts are principally expressed in the paternal allele whilst the Nesp55 transcript is mainly expressed in the maternal allele. Since the latter is present in renal tubules and the others only in other tissues, mutations in exons 2–13 result in AHO and, if derived from the maternal allele, are associated with pseudohypoparathyroidism. If the paternal allele is the origin, pseudopseudohypoparathyroidism is the result. Alterations to the methylation pattern of the various alternative splicing products without mutations in exons 2–13 result in pseudohypoparathyroidism type 1b if they are of maternal origin. (Adapted and reprinted from Bastepe M, Jűppner H, Thakker RV: Parathyroid disorders; in Glorieux FH, Pettifor JM, Jűppner H (eds): Pediatric Bone: Biology and Diseases. Academic Press, San Diego, 2003, p 493, with permission from Elsevier.)

for RANKL and inhibits its action. PTH alters the balance between the two in such a way as temporarily to change the balance in favour of bone resorption (see chapter 3 for further details). In the absence of PTH over long periods, such as in unrecognised hypoparathyroidism, bone becomes undermineralised (see chapter 15, case 13).

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In the nephron, PTH has three main actions. In the convoluted and straight parts of the proximal tubule, it stimulates the conversion of 25OHD to 1,25(OH)2D. In the distal tubules, it promotes the reabsorption of both calcium and magnesium. It also promotes the excretion of phosphate which allows excess phosphate that is resorbed from bone by PTH to be excreted. There is also an effect of PTH on bicarbonate and amino acid reabsorption in the proximal tubule that results in a mild form of Fanconi syndrome in hyperparathyroidism. This resolves when the hyperparathyroidism is reversed.

Parathyroid Hormone-Related Peptide

The presence of a PTH-like substance with similar biological activity but different immunological properties was originally suggested in 1985 [29]. These studies showed that neonatal cord blood contained high PTH-like activity although N-terminal immunoreactivity was absent. The bioactivity was related to the positive gradient of calcium across the placenta and the authors suggested that it was PTHrP that maintained this gradient. It had also been recognised for some time that some patients with malignancy developed hypercalcaemia with undetectable levels of PTH. Subsequently, a protein was purified from lung cancer cells that had similar biological properties to PTH but which was clearly different from PTH itself [30]. This protein was subsequently identified as PTHrP (+168470). PTHrP is a 141 amino acid polypeptide that is coded for by a gene on chromosome 12p12.1-p11.2. It has some homology with PTH in its N-terminal end but diverges from PTH after residue 13. PTHrP cannot normally be measured in the circulation and has no significant classical hormone action in post natal life but does have an important paracrine role in chondrocyte proliferation and maturation. PTHrP has equal activity with PTH on the PTH1R, and some of the changes seen in Jansen’s metaphyseal chondrodysplasia (#156400) are thought to be related to overactivity of these receptors. PTHrP is not a ligand for the PTH2R which is mainly present in brain. However, PTHrP is secreted by the lactating breast and women with hypoparathyroidism who are breast feeding may need to reduce their dose of vitamin D analogues. The principal pathological importance of PTHrP in postnatal life is as a cause of hypercalcaemia of malignancy (see chapter 6).

Calcitonin

CT (*114130) is a 31 amino acid protein that is synthesised by the C cells of the thyroid gland. It is coded for by a gene on chromosome 11p15.2-p15.1 which, by alternative splicing, also results in another protein, calcitonin gene-related peptide (CGRP). CT is mainly active in the thyroid gland whilst CGRP plays more of a role in the hypothalamus.

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It is secreted in response to hypercalcaemia and acts via a specific receptor that is coded for by a gene on chromosome 7q21.3 (*114131). The principal action of CT is to lower plasma calcium in a manner opposite to that of PTH. It may have a role in promoting skeletal mineralisation in the foetus but has little physiological role in postnatal life. It is sometimes used therapeutically to reduce plasma calcium in symptomatic hypercalcaemia, although bisphosphonates are now used more frequently for this purpose, but its principal value is as a marker of malignancy in familial medullary carcinoma of the thyroid (MTC) (#155240).

Alkaline Phosphatase

This enzyme is present in several tissues and exists in three main isoforms, intestinal (IAP) (*171740), placental (PLAP) (*171810), and liver (tissue non-specific) (LALP, TNSAP) (*171760). A gene on chromosome 2q34–37 codes for the first two, and a gene on chromosome 1p36.1-p34 codes for the last [31]. Different post-translational modifications of TNSAP enzyme result in three tissue-specific forms found in bone, liver, and kidney that can be distinguished by their different isoelectric points and heat lability, the bone-specific form (bTNAP) being the least stable. It has been suggested that there are three codominant alleles (HN, HC and HI) of this enzyme and that the presence or absence of hypophosphatasia and its severity depends on which alleles are present. The HN allele is by far the commonest and is homozygous in most individuals. The HI allele results in the most serious reduction in activity whilst the HC allele is intermediate. Homozygous HI alleles result in the perinatal lethal or infantile forms of hypophosphatasia (#241500), whilst heterozygous HN/HC or HN/HI cause the adult form (#146300). The intermediate childhood form (#241510) results from HC/HC or HC/HI combination [32]. For a clinical description of these conditions, see chapter 12. bTNSAP is secreted by osteoblasts and promotes bone mineralisation. Circulating TNSAP is largely derived from liver and bone. Levels in plasma during childhood reflect growth rate [33] and are also raised in the presence of rickets (see chapter 8), in juvenile Paget’s disease (#239000) and in fibrous dysplasia (see chapter 12). Low levels are seen in hypophosphatasia, which results from mutations in the TNSAP gene. A database that keeps track of these mutations (currently 194) has been established and can be accessed at http://www.sesep.uvsq.fr/Database.html.

Vitamin D Metabolism

Although referred to as a vitamin, vitamin D is mainly available not from dietary sources but as a result of the action of sunlight on 7-dehydrocholesterol. Ultraviolet light of wavelength 270–300 nm breaks the B-ring of the steroid molecule creating a

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25

secosteroid. Further rearrangement of the molecule occurs by the action of body heat to create cholecalciferol (vitamin D3). Vitamin D is also available from plant sources as ergocalciferol (vitamin D2), which is synthesised from ergosterol and which differs structurally from cholecalciferol only in the presence of an additional double bond in the side chain. Both compounds are then metabolised in a similar manner and are thought to be equipotent. Collectively, they are referred to as vitamin D or calciferol. Under normal circumstances, approximately 80% of vitamin D requirements are obtained from this action of sunlight but synthesis is dependent on the amount of sunlight exposure, the strength of the UV light in that sunlight and skin colour. Cultural practices that necessitate substantial covering of the skin limit sunlight exposure. In addition, in temperate climates there is insufficient UV light available in sunlight during winter months even if skin exposure is possible. Melanin absorbs UV light of the appropriate wavelength and, since the melanophores that determine skin colour are situated in the skin above the keratinocytes that synthesise vitamin D, darker skinned individuals require a greater degree of sunlight exposure to achieve the same effect as light-skinned people [34]. There may be as much as a sixfold difference in requirement to overcome this barrier. If this is achieved, darker-skinned individuals are equally capable of synthesising vitamin D. There is generally little vitamin D in food although some oily fish have a relatively high content and it is a common misconception that, because a child is taking a ‘healthy diet’, they are not at risk of vitamin D deficiency. If sunlight exposure is halved, vitamin D intake must be trebled to compensate for this and the only realistic way of achieving this is by giving adequate dietary supplementation. Vitamin D is stored in liver and adipose tissue. Obese subjects have lower circulating levels of vitamin D than non-obese subjects, possibly because they sequester more vitamin D into their fat stores [35]. Following synthesis, vitamin D is bound to a specific vitamin D binding protein (DBP) and passes to the liver. Native vitamin D has little biological activity and requires metabolism via two hydroxylation steps, firstly at the 25- and subsequently at the 1- position in order to become fully active [36]. All of the steps in vitamin D metabolism are catalyzed by cytochrome P450 enzymes (fig. 7). The first step is catalysed by vitamin D 25-hydroxylases. There are at least four different enzymes that have an influence on 25-hydroxylase activity. They are distinguishable by their different affinities and capacities and by their intracellular localisation. The first to be cloned, a low-affinity, high-capacity enzyme (CYP27A1) (*606530) is located in mitochondria. However, there are no reports of rickets resulting from mutations in this gene, but they do cause cerebrotendinous xanthomatosis (#213700). A second high-affinity, low-capacity enzyme (CYP2R1) (*608713), which is probably of greater physiological significance, is located within hepatic microsomes. It contains 501 amino acids and is coded for by a gene on chromosome 11p15.2 [37]. Rare cases are described of rickets associated with mutations in this gene (#600081) [38]. Two other enzymes, CYP3A4 (*124010) and CYP2J2 (*601258) probably also have some effect on 25-hydroxylase but are mainly involved in drug metabolism.

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7-Dehydrocholesterol

Diet

Sunlight PrevitaminD Body heat

1␣-hydroxycholecalciferol (alfacalcidol)

Cholecalciferol/Ergocalciferol (Vitamin D) Vitamin D 25-hydroxylase

25-OH vitamin D 25-hydroxyvitamin D 1␣-hydroxylase

Vitamin D 25-hydroxylase

1,25(OH)2 vitamin D Vitamin D receptor

Vitamin D 24-hydroxylase

Peripheral action

24,25-Dihydroxyvitamin D 1,24,25-Trihydroxyvitamin D

Fig. 7. Diagrammatic representation of vitamin D metabolism.

The resulting product, 25-hydroxyvitamin D (25OHD), circulates in plasma bound to the DBP in nanomolar concentrations. Assay of 25OHD gives a measure of vitamin D status. Its level varies depending on the supply of vitamin D and shows a considerable annual variation with a peak about 6 weeks after maximal exposure to sunlight. It is now generally agreed that vitamin D sufficiency is defined by a plasma concentration above 50 nmol/l [39]. It has some weak activity, which is not normally of clinical significance, but may become so in the presence of vitamin D excess. Vitamin D 25-hydroxylase also catalyses the conversion of the synthetic vitamin D analogues, 1α-hydroxy-cholecalciferol (alfacalcidol) and 1α-hydroxy-ergocalciferol (doxercalciferol), to 1,25(OH)2D3 and 1,25(OH)2D2, respectively. 25OHD is metabolised to its active hormone 1,25(OH)2D by 25-hydroxyvitamin D 1α-hydroxylase, which is active only against metabolites that are already hydroxylated at position 25 [40]. A single enzyme has been identified located in convoluted and straight portions of the proximal renal tubule. Activity is also present in osteoblasts, keratinocytes, and lymphohaematopoietic cells, where 1,25(OH)2D may have an autocrine or paracrine role. During foetal life, 1α-hydroxylase activity is found in the placenta. In pathological states, it is present in the macrophages of sarcoid tissue and subcutaneous fat necrosis (see chapter 7). It is a mitochondrial enzyme

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(CYP27B1) (*609506) consisting of 508 amino acids with considerable homology to other P450 enzymes. It is encoded by a single gene on chromosome 12q13.1-q13.3. Mutations in this gene are responsible for the condition known variously as pseudovitamin D deficiency rickets (PDDR), vitamin D-dependent rickets type I (VDDR-I), Prader rickets or 1α-hydroxylase deficiency (#264700). Activity of 1α-hydroxylase is stimulated by PTH via its cAMP/protein kinase actions. Hypocalcaemia stimulates 1α-hydroxylase activity, but this effect is not a direct effect but mediated via PTH. Plasma phosphate has a direct effect on 1α-hydroxylase activity, although there is some evidence to suggest that this may be modulated by growth hormone (GH); calcitonin may also regulate the enzyme. Its activity is inhibited by FGF23. 1,25(OH)2D is a highly potent compound that circulates in picomolar concentrations. However, measurement of 1,25(OH)2D in plasma gives no measure of vitamin D status. Its synthesis is tightly controlled by the plasma calcium concentration. In order to enable changes in 1,25(OH)2D to occur rapidly, a second enzyme, 25-hydroxyvitamin D 24-hydroxylase (25OHD 24-OHase) (*126065), exists. This is yet another cytochrome P450 enzyme that can use both 25OHD and 1,25(OH)2D as substrates to form 24,25-dihydroxyvitamin D (24,25(OH)2D) and 1α,24,25-trihydroxyvitamin D (1,24,25(OH)3D) respectively. The role of this enzyme is probably to divert metabolism of 25OHD away from 1,25(OH)2D synthesis when this is not needed and to participate in the degradation of existing 1,25(OH)2D. It is inhibited by PTH and stimulated by 1,25(OH)2D and FGF23. 1,24,25(OH)3D has limited potency (about 10% of 1,25(OH)2D) and is probably an intermediate degradation metabolite of 1,25(OH)2D. The role, if any, of 24,25(OH)2D is uncertain. Some authors have argued that it has no role to play whereas others have suggested that it may influence bone mineralisation. In addition, people of South Asian origin possess higher 25OHD 24-OHase activity than those of European origin [41] and this seems to contribute to their susceptibility to vitamin D deficiency rickets. 1,25(OH)2D acts via a specific vitamin D receptor [42] (*601769). It is a member of the steroid-thyroid-retinoid superfamily of nuclear receptors and, in many respects, is typical of this group with ligand binding, DNA binding, dimerisation, and transcriptional activation domains. It is encoded by a gene on chromosome 12 near the 1α-hydroxylase gene. The receptors are widely distributed in gut, parathyroid glands, chondrocytes, osteoblasts, and osteoclast precursors. 1,25(OH)2D plays a critical role in promoting calcium absorption in the small intestine, suppresses PTH secretion from the parathyroids, influences growth plate mineralisation, and stimulates differentiation of osteoclasts. In addition, there are receptors present in many tissues that are not directly related to calcium homeostasis such as skin, breast, prostate, colon, etc., and it has been postulated that 1,25(OH)2D may play a part in preventing cancers of these tissues [43]. Mutations in the vitamin D receptor occur throughout the molecule but particularly in either the ligand-binding (ligand-binding-negative) or the DNA-binding

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(ligand-binding-positive) domains [42]. These mutations cause severe rickets, and many individuals, especially those with defects in DNA binding, also have alopecia. Originally referred to as vitamin D-dependent rickets type II (VDRR-II), it is now more properly called hereditary 1,25(OH)2D-resistant rickets (HVDRR) (#277440). In another form of HVDRR, no mutations of the receptor have been identified, but is thought to be caused by overexpression of a nuclear ribonucleoprotein that binds with the hormone receptor complex to attenuate its action (%600785) (see chapter 8 for details).

Conclusions

The mechanisms that are involved in maintaining normal calcium, magnesium and phosphate are complex and involve several different hormonal mechanisms that influence both calcium, magnesium and phosphate in an independent but linked manner. Normal calcium and phosphate physiology demands that these mechanisms all function satisfactorily in order to maintain good bone health and a suitable milieu in which muscle and nerve function can be optimised. Disruptions to these mechanisms may be either environmental, principally due to vitamin D deficiency, or, in many instances, genetic. A thorough understanding of the physiology is required before a correct diagnosis can be made.

References 1 Allgrove J: Practical management of disorders of calcium metabolism; in Aynsley-Green A (ed): Paediatric Endocrinology in Clinical Practice. Lancaster, MTP Press, 1984, pp 241–263. 2 Hoenderop JG, Nilius B, Bindels RJ: Calcium absorption across epithelia. Physiol Rev 2005;85: 373–422. 3 Hoenderop JG, Bindels RJ: Epithelial Ca2+ and Mg2+ channels in health and disease. J Am Soc Nephrol 2005;16:15–26. 4 Heaney RP: Nutrition and risk of osteoporosis; in Marcus R, Feldman D, Kelsey J (eds): Osteoporosis. San Diego, Academic Press, 2001. 5 Khadilkar A, Das G, Sayyad M, et al: Low calcium intake and hypovitaminosis D in adolescent girls. Arch Dis Child 2007;92:1045. 6 Allgrove J, Adami S, Fraher L, Reuben A, O’Riordan JL: Hypomagnesaemia: studies of parathyroid hormone secretion and function. Clin Endocrinol (Oxf) 1984;21:435–449. 7 Muallem S, Moe OW: When EGF is offside, magnesium is wasted. J Clin Invest 2007;117:2086–2089.

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8 Groenestege WM, Thebault S, van der WJ, et al: Impaired basolateral sorting of pro-EGF causes isolated recessive renal hypomagnesemia. J Clin Invest 2007;117:2260–2267. 9 Wilz DR, Gray RW, Dominguez JH, Lemann J Jr: Plasma 1,25-(OH)2-vitamin D concentrations and net intestinal calcium, phosphate, and magnesium absorption in humans. Am J Clin Nutr 1979;32: 2052–2060. 10 Yamashita T, Yoshioka M, Itoh N: Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain. Biochem Biophys Res Commun 2000; 277:494–498. 11 ADHR Consortium: Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet 2000;26:345–348. 12 Fukumoto S: Post-translational modification of fibroblast growth factor 23. Ther Apher Dial 2005;9: 319–322.

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13 Bennett EP, Hassan H, Clausen H: cDNA cloning and expression of a novel human UDP-N-acetylalpha-d-galactosamine: polypeptide N-acetylgalactosaminyltransferase, GalNAc-t3. J Biol Chem 1996; 271:17006–17012. 14 Topaz O, Indelman M, Chefetz I, et al: A deleterious mutation in SAMD9 causes normophosphatemic familial tumoral calcinosis. Am J Hum Genet 2006; 79:759–764. 15 Passos-Bueno MR, Wilcox WR, Jabs EW, Sertie AL, Alonso LG, Kitoh H: Clinical spectrum of fibroblast growth factor receptor mutations. Hum Mutat 1999; 14:115–125. 16 Urakawa I, Yamazaki Y, Shimada T, et al: Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature 2006;444:770–774. 17 Koh N, Fujimori T, Nishiguchi S, et al: Severely reduced production of Klotho in human chronic renal failure kidney. Biochem Biophys Res Commun 2001;280:1015–1020. 18 Brown EM, MacLeod RJ: Extracellular calcium sensing and extracellular calcium signalling. Physiol Rev 2001;81:239–297. 19 Conlin PR, Fajtova VT, Mortensen RM, LeBoff MS, Brown EM: Hysteresis in the relationship between serum ionized calcium and intact parathyroid hormone during recovery from induced hyper- and hypocalcemia in normal humans. J Clin Endocrinol Metab 1989;69:593–599. 20 Zhao XM, Hauache O, Goldsmith PK, Collins R, Spiegel AM: A missense mutation in the seventh transmembrane domain constitutively activates the human Ca2+ receptor. FEBS Lett 1999;448:180–184. 21 Carling T, Szabo E, Bai M, et al: Familial hypercalcemia and hypercalciuria caused by a novel mutation in the cytoplasmic tail of the calcium receptor. J Clin Endocrinol Metab 2000;85:2042–2047. 22 Parfitt AM: Parathyroid growth: Normal and abnormal; in Bilezikian JP, Marcus R, Levine MA (eds): The Parathyroids: Basic and Clinical Concepts. San Diego, Academic Press, 2001. 23 Habener JF, Potts JT Jr: Biosynthesis of parathyroid hormone (second of two parts). N Engl J Med 1978; 299:635–644. 24 Habener JF, Potts JT Jr: Biosynthesis of parathyroid hormone (first of two parts). N Engl J Med 1978; 299:580–585. 25 Kronenberg HM, Bringhurst FR, Segre GV, Potts JT: Parathyroid hormone biosynthesis and metabolism; in Bilezikian JP, Marcus R, Levine MA (eds): The Parathyroids: Basic and Clinical Concepts. San Diego, Academic Press, 2001.

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26 Nissensen RA: Receptors for parathyroid hormone and parathyroid hormone-related protein: signaling and regulation; in Bilezikian JP, Marcus R, Levine MA (eds): The Parathyroids: Basic and Clinical Concepts. San Diego, Academic Press, 2001. 27 Farfel Z, Bourne HR, Iiri T: The expanding spectrum of G protein diseases. N Engl J Med 1999; 340:1012–1020. 28 Bastepe M: The GNAS locus and pseudohypoparathyroidism. Adv Exp Med Biol 2008;626:27–40. 29 Allgrove J, Adami S, Manning RM, O’Riordan JL: Cytochemical bioassay of parathyroid hormone in maternal and cord blood. Arch Dis Child 1985;60: 110–115. 30 Moseley JM, Kubota M, Diefenbach-Jagger H, et al: Parathyroid hormone-related protein purified from a human lung cancer cell line. Proc Natl Acad Sci USA 1987;84:5048–5052 31 Smith M, Weiss MJ, Griffin CA, et al: Regional assignment of the gene for human liver/bone/kidney alkaline phosphatase to chromosome 1p36.1-p34. Genomics 1988;2:139–143. 32 Igbokwe EC: Inheritance of hypophosphatasia. Med Hypotheses 1985;18:1–5. 33 Round JM: Changes in plasma urate, creatinine, alkaline phosphatase and the 24 hours excretion of hydroxyproline during sexual maturation in adolescents. Ann Hum Biol 1980;7:83–88. 34 Lo CW, Paris PW, Holick MF: Indian and Pakistani immigrants have the same capacity as Caucasians to produce vitamin D in response to ultraviolet irradiation. Am J Clin Nutr 1986;44:683–685. 35 Wortsman J, Matsuoka LY, Chen TC, Lu Z, Holick MF: Decreased bioavailability of vitamin D in obesity. Am J Clin Nutr 2000;72:690–693. 36 Okuda K, Usui E, Ohyama Y: Recent progress in enzymology and molecular biology of enzymes involved in vitamin D metabolism. J Lipid Res 1995; 36:1641–1652. 37 Cheng JB, Motola DL, Mangelsdorf DJ, Russell DW: De-orphanization of cytochrome P450 2R1: a microsomal vitamin D 25-hydroxilase. J Biol Chem 2003;278:38084–38093. 38 Cheng JB, Levine MA, Bell NH, Mangelsdorf DJ, Russell DW: Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase. Proc Natl Acad Sci USA 2004;101:7711–7715. 39 Holick MF: Vitamin D: a D-lightful health perspective. Nutr Rev 2008;66:S182–S194. 40 St Arnaud R, Messerlian S, Moir JM, Omdahl JL, Glorieux FH: The 25-hydroxyvitamin D 1-alphahydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus. J Bone Miner Res 1997;12:1552–1559.

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41 Awumey EM, Mitra DA, Hollis BW, Kumar R, Bell NH: Vitamin D metabolism is altered in Asian Indians in the southern United States: a clinical research center study. J Clin Endocrinol Metab 1998;83:169–173.

42 Haussler MR, Haussler CA, Jurutka PW, et al: The vitamin D hormone and its nuclear receptor: molecular actions and disease states. J Endocrinol 1997; 154(Suppl):S57–S73. 43 Holick MF: Vitamin D and sunlight: strategies for cancer prevention and other health benefits. Clin J Am Soc Nephrol 2008;3:1548–1554.

Jeremy Allgrove, MD Department of Paediatric Endocrinology, David Hughes Building, First Floor Royal London Hospital, Whitechapel London E1 1BB (UK) Tel. +44 20 7377 7468, Fax +44 20 7943 1353, E-Mail [email protected]

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Chapter 3 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 32–48

Physiology of Bone Peter Grabowski Faculty of Medicine, Dentistry and Health, Academic Unit of Child Health, University of Sheffield, Sheffield, UK

Abstract Bone serves three main physiological functions. Its mechanical nature provides support for locomotion and offers protection to vulnerable internal organs, it forms a reservoir for storage of calcium and phosphate in the body, and it provides an environment for bone marrow and for the development of haematopoietic cells. The traditional view of a passive tissue responding to hormonal and dietary influences has changed over the past half century to one of a dynamic adaptive tissue responding to mechanical demands. This chapter gathers together some recent advances in bone physiology and molecular cell biology and discusses the potential application of the bone’s functional adaptation to loading in enhancing bone strength during childhood and adolescence. Copyright © 2009 S. Karger AG, Basel

Bone is a dynamic mineralised connective tissue with multiple physiological functions. At the organ level, bones provide mechanical support for load bearing and locomotion, offer physical protection to vulnerable internal organs, form a mobilisable reservoir of calcium and phosphate ions, and provide an environmental niche for bone marrow and the development of haematopoietic cells. At the tissue level, the coordinated activities of bone formation and resorption provide mechanisms for bone modelling – i.e. the formation of new bone during growth and development – and remodelling – i.e. the coordinated process by which old bone is firstly removed then replaced during skeletal maintenance, and for responding rapidly to the body’s immediate calcium, phosphate and acid-base homeostatic requirements. At the cellular level, bone matrix formation and mineralisation are mediated by osteoblasts and bone resorption is mediated by osteoclasts, while at the molecular level, a range of systemic and local factors regulate cellular and tissue level processes in bone. Bones are highly dependent upon other organs for their growth and development, in particular the intestine and kidney, through which mineral and nutritional factors are absorbed, reabsorbed and excreted, as well as the hypothalamus, pituitary, gonads, parathyroid glands, liver and skin that produce hormonal factors regulating bone

growth and mineral homeostasis. Whereas in adults bone physiology is concerned with skeletal maintenance, in children the context is one of bones that are growing in size, mass and mineral density, while at the same time being modelled into their final adult shape and form. Much of what we know of basic skeletal biology and physiology derives from the study of adults or animals, but the study of children’s bone is an active and expanding area of research. Macroscopically, bone tissue is classified as either cortical or trabecular. Cortical bone is found most commonly in the shafts of long bones and consists of a dense compact tissue penetrated by blood vessels and canaliculi which surround osteocytes and their connecting cellular processes. Trabecular or cancellous bone is found at the ends of long bones, in vertebrae and near joint surfaces and consists of a network of thin plates and connecting struts surrounded by bone marrow. Cortical and trabecular bone are very similar in their cellular and molecular composition but differ significantly in their function and mechanical properties. For much of the 20th century, bone physiology largely centred on understanding the hormonal regulation of osteoblasts and osteoclasts in skeletal maintenance and to a lesser extent in bone growth. Since the mid-1960s bone physiology has seen a change of focus largely due to the efforts of Frost, Jee and others (reviewed in [1–3]), giving a vision of bone as a dynamic tissue that responds at the tissue level to the mechanical demands placed upon it, developing the concept of the mechanostat [2], and leading to an increased interest in the role of cells within the bone matrix and marrow as sensors of local mechanical stimuli and regulators of local bone turnover [4]. Advances in tissue and cell culture techniques have contributed to our understanding of the development, regulation and function of osteoblasts and osteoclasts and, more recently, the development of molecular genetics and the ability to generate targeted transgenic mice have revolutionised the study of individual gene product functions in bone.

Skeletal Development

The evolutionary landmarks giving rise to the vertebrate skeleton are reflected in the developmental biology of bone tissue. Axial skeletal patterning, segmentation, growth and condensation are regulated by homeobox (hox) genes (reviewed in [5]), bone morphogenic proteins (BMPs) and other members of the transforming growth factor-β (TGF-β) superfamily (reviewed in [6]), fibroblast growth factors (FGFs) [7], hedgehog [8] and Wnt proteins [9]. Many of these factors act not only in skeletal patterning and development but also in the recruitment and differentiation of osteoblasts during bone modelling and remodelling throughout life. The axial skeleton is laid down initially as a cartilaginous matrix model by chondrocytic cells of mesenchymal origin (fig. 1). The chondrocytes mineralise the matrix and, through

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GP

GP a

b

c

Fig. 1. Early stages of long bone development by endochondral ossification. a A condensation of mesenchymal cells leads to the formation of a cartilage model of the bone. b Chondrocytes beneath the perichondrium (black) differentiate, become hypertrophic and eventually undergo apoptosis, resulting in cartilage mineralisation (shaded) and release of metalloproteinases. c Angiogenic factors released by chondrocytes encourage vascular invasion, recruiting osteoclast and osteoblast precursors that differentiate and convert calcified cartilage into true bone. Growth plates (GP) establish from chondrocytes in the epiphyseal regions.

a process of hypertrophy, eventually die by apoptosis. Metalloproteinases released by the chondrocytes dissolve some of the matrix and generate angiogenic signals promoting vascularisation and the influx of osteoclasts which begin to resorb the mineralised cartilage. Along with the osteoclasts, osteoblast precursors enter the primitive bone and begin to form true bone behind the advancing osteoclasts, giving rise to the primary spongiosa under the growth plates. In the growth plate, gradients of Indian hedgehog (IHH) and parathyroid hormone-related peptide (PTHrP) regulate the directional proliferation and differentiation of chondrocytes to a hypertrophic phase characterised by mineralisation and metalloproteinase secretion, leading to longitudinal bone growth. In contrast, bones of the cranial vault form through a process of intramembranous ossification in membranes of mesenchymal condensations which progress to bone formation without chondrocyte involvement. Bone formed through both mechanisms undergoes remodelling, a process initiated by activation of osteoclasts, followed by a period of resorption, a reversal phase in which osteoclasts die and osteoblasts are activated, a period of matrix formation followed by a mineralisation phase and a return to the resting state. Communication between osteoblasts and osteoclasts coordinates this series of events, known as the bone remodelling cycle, within a basic multicellular unit (BMU). For a more detailed description of skeletal development see Karaplis [10]. Hormonal influences on bone development are described elsewhere in this book.

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Mesenchymal stem cell

Osteochondroprogenitor

Osteoblasts Osteocytes Lining cells

Sox9

Runx2

Osterix

Fig. 2. Osteoblast differentiation. Osteoblasts arise from a multipotent precursor cell of mesenchymal origin (mesenchymal stem cell). An osteochondrogenitor cell capable of forming both chondrocytes and osteoblasts arises under the control of the transcription factor Sox9. Runx2 is the key regulator of osteoblast differentiation and is constitutively expressed in osteoblasts at all stages of differentiation. A second transcription factor, Osterix, acts downstream of Runx2 in osteoblast differentiation. Osteoblasts can further differentiate into osteocytes that become embedded in the bone matrix or into lining cells on bone surfaces.

Osteoblast Differentiation and Function

Osteoblasts, bone-lining cells and osteocytes all arise from a multipotent precursor of mesenchymal origin that also gives rise to chondrocytes, adipocytes, myocytes and fibroblasts, most commonly called a mesenchymal stem cell [11] (fig. 2). The early differentiation process leading to osteochondroprogenitor cells involves Sox9, the key transcriptional regulator of chondrogenesis. Significant advances in the last 10 years have defined Runx2 [12, 13] and Osterix [14] as the two critical transcription factors determining osteoblast lineage differentiation. Runx2 (also called CBFA1) is a member of the Runx transcription factor family that is characterised by a DNA binding domain homologous with the Drosophila gene runt. It was identified as a causative gene for cleidocranial dysplasia (table 1) [12, 13]. It is expressed in mouse embryonic tissues in cells destined to become osteoblasts or chondrocytes in the developing embryo, and in all osteoblasts regardless of their differentiation stage [15]. Runx2–/– mice are unable to produce either endochondral or intramembranous bone [13, 16] but can produce adipocytes and chondrocytes [17]. Osterix is a zinc finger containing transcription factor of the SP transcription factor family. Osterix–/– mice are deficient in osteoblasts and do not form intramembranous bone [14]. They do express Runx2, but Runx2–/– mice do not express Osterix, indicating that Osterix acts downstream of Runx2. The Wnt signalling pathway in osteoblasts contains a number of molecules that are now viewed as amongst the most important regulators of bone formation during growth and development, mediating some of the regulatory dialogue between osteoblasts and osteoclasts [18]. Wnts are glycoproteins that in osteoblasts act on receptors composed of a Frizzled (Fz, a G protein coupled receptor-like protein) and one of the low density lipoprotein receptor related proteins LRP5 or LRP6. Activation

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Table 1. Regulatory, structural and processing genes in bone and cartilage with known skeletal disorders in children Gene/protein

OMIM

Disorder

OMIM

Inheritance

*604539 Ehlers-Danlos syndrome, type VIIC #225410 AR ADAMTS2 Procollagen I N-proteinase ADAMTS10 A disintegrin-like metalloproteinase with thrombospondin type 1 motif, 10

*608990 Weill-Marchesani syndrome

#277600 AR

ALPL (TNSALP)* Alkaline phosphatase, liver/bone/kidney

*171760 hypophosphatasia, perinatal and infantile hypophosphatasia, childhood hypophosphatasia, adult

#241500 ?AR

CA2* Carbonic anhydrase II

*611492 osteopetrosis, autosomal recessive type III

#259730 AR

CASR* Calcium sensing receptor

+601199 severe neonatal hyperparathyroidism familial hypocalciuric hypercalcaemia with hyperparathyroidism

#239200 AR

*602727 osteopetrosis, autosomal recessive type IV osteopetrosis, autosomal dominant type II

#611490 AR

COL1A1* Pro-α1 collagen type I

+120150 osteogenesis imperfecta type IA osteogenesis imperfecta type IIA osteogenesis imperfecta type III osteogenesis imperfecta type IV Caffey disease

#166200 #166210 #259420 #166220 #114000

AD AD AD AD AD

COL1A2* Pro-α2 collagen type I

*120160 osteogenesis imperfecta type IB osteogenesis imperfecta type II osteogenesis imperfecta type III osteogenesis imperfecta type III osteogenesis imperfecta type IV

166240 #166210 #259420 203760 #166220

AD AD AD AR AD

CLCN7* Chloride channel 7

36

#241510 AD #146300 AD

#145980 AD

#166600 AD

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Table 1. Continued Gene/protein

OMIM

COL2A1 Pro-α1 collagen type II

+120140 achondrogenesis type 2 (Langer-Saldino) platyspondylic dysplasia (Torrance) hypochondrogenesis spondyloepiphyseal dysplasia, congenital spondyloepimetaphyseal dysplasia (Strudwick) Kniest dysplasia spondyloperipheral dysplasia Stickler syndrome type 1 otospondylomegaepiphyseal dysplasia

#200610 AD

COL5A1 Pro-α1 collagen type V

*120215 Ehlers-Danlos syndrome type I Ehlers-Danlos syndrome type II

#130000 AD #130010 AD

COL5A2 Pro-α2 collagen type V

*120190 Ehlers-Danlos syndrome type I Ehlers-Danlos syndrome type II

#130000 AD #130010 AD

COL9A1 Pro-α1 collagen type IX

+120210 multiple epiphyseal dysplasia type 6 Stickler syndrome, autosomal recessive, Col9a1-related

#120210 AD

COL9A2 Pro-α2 collagen type IX

*120260 multiple epiphyseal dysplasia type 2

#600204 AD

COL9A3 Pro-α3 collagen type IX

*120270 multiple epiphyseal dysplasia type 3

#600969 AD

COL10A1 Pro-α1 collagen type X

*120110 metaphyseal chondrodysplasia (Schmid)

#156500 AD

COL11A1 Pro-α1 collagen type XI

*120280 Stickler syndrome type 2 Marshall syndrome

#604841 AD #154780 AD

COL11A2 Pro-α1 collagen type XI

*120290 otospondylomegaepiphyseal dysplasia

#215150 ?AR

CRTAP* Cartilage-associated protein

*605497 osteogenesis imperfecta type IIB osteogenesis imperfecta type VII

#610854 AR #610682 AR

CTSK* Cathepsin K

*601105 pycnodysostosis

#265800 AR

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Disorder

OMIM

Inheritance

#151210 ? usually lethal #200610 AD #183900 AD #184250 AD #156550 #271700 #108300 #215150

AD AD AD AD

#108300 AR

37

Table 1. Continued Gene/protein

OMIM

Disorder

OMIM

Inheritance

FGF23* *605380 hypophosphataemic rickets, Fibroblast growth factor 23 ADHR familial hyperphosphataemic tumoral calcinosis

#193100 AD

FGFR3 Fibroblast growth factor receptor 3

*134934 achondroplasia thanatophoric dysplasia type I thanatophoric dysplasia type II hypochondroplasia

#100800 #187600 #187601 #146000

LEPRE1* Collagen prolyl 3-hydroxylase 1

*610339 osteogenesis imperfecta type VIII

#610915 AR

LRP5* Low-density lipoprotein receptor-related protein 5

*603506 osteoporosis-pseudoglioma syndrome high bone mass osteopetrosis, autosomal dominant type I endosteal hyperostosis, autosomal dominant

#259770 AR

*120360 Torg-Winchester syndrome MMP2 multicentric osteolysis, nodulosis Matrix metalloproteinase 2 and arthropathy

#211900 AD AD AD AD AD

#601884 AD #607634 AD #144750 AD #259600 AR #605156 AR

MMP13 Matrix metalloproteinase 13

*600108 spondyloepimetaphyseal dysplasia type II metaphyseal anadysplasia

#602111 AD

OSTM1* Osteopetrosis-associated transmembrane protein 1

*607649 osteopetrosis, autosomal recessive type V

#259720 AR

PHEX* Phosphate-regulating endopeptidase homologue, X-linked

*300550 X-linked hypophosphataemia

#307800 XLD

PLEKHM1* *611466 osteopetrosis, autosomal Pleckstrin homology recessive type VI domain-containing, family M (with RUN domain) member 1 PLOD1 Procollagen lysyl hydroxylase 1

38

*153454 Ehlers-Danlos syndrome type VIA Nevo syndrome

309645

AD

#611497 AR

#225400 ?AR #601451 ?AR

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Table 1. Continued Gene/protein

OMIM

Disorder

OMIM

Inheritance

PLOD2* Procollagen lysyl hydroxylase 2

*601865 Bruck syndrome type 2

#609220 AR

PLOD3 Procollagen lysyl hydroxylase 3

*603066 bone fragility with contractures, arterial rupture and deafness

#612394 AR

PTHR1* Parathyroid hormone receptor 1

*168468 metaphyseal chondrodysplasia (Jansen) chondrodysplasia (Blomstrand) endochondromatosis (Ollier) Eiken syndrome

#156400 AD

RUNX2* Runt-related transcription factor 2

*600211 cleidocranial dysplasia

#119600 AD

SOST* Sclerostin

*605740 sclerosteosis endosteal hyperostosis (Van Buchem)

#269500 AR #239100 AR

SOX9 Sex-determining region Y-related homeobox gene 9

*608160 campomelic dysplasia

#114290 XLR

TCIRG1 Vacuolar proton pump α-subunit 3

*604592 osteopetrosis, autosomal recessive type I

#259700 AR

TGFβ1* Transforming growth factor-β1

*190180 Camurati-Engelmann disease

#131300 AD

TNFRSF11A* Receptor activator of NF-κB (RANK)

*603499 familial expansile osteolysis Paget disease of bone osteopetrosis, autosomal recessive type VII

#174810 AD #602080 AR #612301 AR

TNFRSF11B* Osteoprotegerin (OPG)

*602643 juvenile Paget disease

#239000 AR

TNFSF11* Receptor activator of NF-κB ligand (RANKL)

*602642 osteopetrosis, autosomal recessive type II

#259710 AR

#215045 ?AR #166000 ?AD #600002 ?AR

The genes are listed in alphabetical order and those genes associated with conditions that are described in more detail in the book are indicated by (*). The genes involved and the conditions arising from mutations in those genes are shown with their appropriate OMIM numbers. See also Superti-Furga and Unger [55] for a comprehensive classification of genetic skeletal disorders.

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a

b

c

Fig. 3. Wnt signalling in osteoblasts. a Osteoblasts express canonical Wnt pathway coreceptor molecules including LRPs and Frizzled family members. Wnts interact with LRP5/6 and Frizzled proteins to form a complex that leads to the inhibition of β-catenin destruction mediated by Dishevelled. As a result, β-catenin accumulates and is translocated to the nucleus where it interacts with transcription factors to modulate gene expression. b Dickkopf (Dkk) inhibits Wnt signalling by binding LRPs to Kremen, enhancing their internalisation and destruction. c Sclerostin (Sost) competes with Wnt for binding to LRPs, preventing the interaction of LRPs with Frizzled proteins.

of the Wnt receptor results in dephosphorylation and accumulation of intracellular β-catenin and its translocation into the nucleus, where it interacts with transcription factors to control osteoblast gene expression (fig. 3). The Dickkopf family of proteins act as negative regulators of Wnt signalling by binding to LRP5/6 and another cell surface co-receptor Kremen, causing internalisation and destruction of the resulting complex and reducing the density of Wnt receptors at the cell surface (fig. 3). Wnt signalling in osteoblasts exerts its effects in bone primarily by regulating osteoclast formation through modulating the production of osteoprotegerin (OPG), the soluble inhibitor of the RANK signalling pathway [19]. Gain of function mutations in LRP5 result in high bone mass disorders (table 1) while loss of function mutations in LRP5 result in the low bone mass disorder osteoporosis-pseudoglioma syndrome (table 1).

Bone and Cartilage Matrix Collagens and Their Modifying Enzymes

The major function of osteoblasts is to create a mineralised bone matrix which, until mineralised, is called osteoid. Type 1 collagen accounts for about 90% of osteoid content, with the remainder composed largely of glycoproteins and proteoglycans. Other proteins that are important for mineralisation, including alkaline phosphatase, osteocalcin and osteopontin, are also secreted by osteoblasts into the newly forming matrix. The process of bone mineralisation is poorly understood. Collagens are a diverse family of structural proteins found in extracellular matrices. They are the most abundant proteins in the body and there are at least twenty-eight

40

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different collagens found in vertebrates. The characteristic feature of all collagens is a triple helical structure consisting of three interwoven α-chain polypeptides. The triple helical region of collagen α-chains consists of a repetitive series of amino-acid triplets – [Gly-X-Y]n – where Gly is glycine and X and Y are commonly proline or hydroxyproline. Collagens can be homo- or hetero-trimeric proteins, i.e. with three identical α-chains or with three α-chains encoded by either two or three unique genes respectively. During synthesis, carboxy pro-peptide regions of three pro-α-chains associate within the endoplasmic reticulum to initiate the formation of the triple helix which propagates towards the amino terminus. During assembly, many proline and lysine residues in fibrillar collagens become hydroxylated and some of the hydroxylysine residues are further modified by glycosylation. Intra- and inter-chain disulphide bonds are also formed during synthesis. Carboxy- and amino-propeptides are proteolytically cleaved extracellularly after secretion and the released monomers assemble into highly orientated, quarter-staggered fibrils which are held together through covalent cross-links promoted by the action of lysyl oxidase. The collagen triple helix is highly resistant to proteolytic cleavage by pepsin, trypsin and papain, and degradation of collagens is mediated by matrix metalloproteinases, cysteine proteinases (especially cathepsins B, K and L) and serine proteinases [20]. In bone, type I collagen is the most abundant fibrillar collagen. It is normally heterotrimeric, consisting of two α1(I) chains and one α2(I) chain. In the absence of pro-α2(I) chains, type I collagen α1(I) homotrimers can form. In bone, type I collagen forms heterotypic fibrils with type V collagen, a low-abundance fibrillar protein with three distinct α-chains – α1(V), α2(V) and α3(V). While the COL1A2 gene is not essential for survival, homozygous COL1A1 null mutations are not seen clinically. Skeletal phenotypes arising from mutations in type I collagen give rise to osteogenesis imperfecta and various forms of Ehlers-Danlos syndrome (EDS) (table 1), with mutations in type V collagen also giving rise to EDS (table 1). In cartilage, type II collagen is the most abundant fibrillar collagen, consisting of α1(II) homotrimers. Additionally, in cartilage, the pro-α1(II) chain is incorporated into heterotrimeric type XI collagen along with a pro-α1(XI) and a pro-α2(XI) chain. Type XI collagen is a low-abundance fibrillar collagen that forms heterotypic fibrils with type II collagen in cartilage. Yet another low abundance collagen – type IX, composed of three distinct α-chains – α1(IX), α2(IX) and α3(IX) – also forms heterotypic fibrils with type II collagen in cartilage. Type IX collagen has a triple helix that is interrupted by short non-helical regions which give the molecule some flexibility and it is classed as a fibril associated collagen with interrupted triple helices (FACIT). Mutations in type II collagen give rise to a variety of chondrodysplasias (table 1). Type IX collagen mutations give rise to various multiple epiphyseal dysplasias and Stickler syndrome, while mutations in type XI collagen give rise to Stickler and Marshall syndromes (table 1). Type X collagen is a homotrimeric protein found in the hypertrophic cartilage of the growth plate. Mutations in type X collagen result in Schmidt metaphyseal chondrodysplasia (table 1). Mutations that give rise to skeletal

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phenotypes have also been identified in some collagen modifying and processing enzyme genes including ADAMTS2, ADAMTS10, CRTAP, CTSK, LEPRE1, MMP2, MMP13, PLOD1, PLOD2 and PLOD3 (table 1). For a more comprehensive review of the clinical genetics of collagen disorders, see Byers [21].

Osteocytes and Bone Lining Cells

As bone matrix is being formed, some osteoblasts undergo a terminal differentiation event (fig. 2) and, instead of continuing to produce matrix, they become osteocytes, encapsulated in concentric layers within lacunae in the osteoid. The signals that initiate and control terminal osteocytic differentiation are not known. Numerous dendritic cellular processes connect osteocytes to each other through canaliculi, both laterally and between cell layers within bone. When osteoblasts stop creating bone, they turn into bone lining cells and remain on the bone surface. The transition from osteoblasts into bone lining cells is poorly understood. Osteocytes and bone lining cells account for the largest proportion of cells in mineralised bone but are probably the least characterised and understood cells of bone. An important development in osteocyte biology has been the identification of sclerostin, mutations in which cause sclerosteosis and Van Buchem disease (table 1), both characterised by progressive bone thickening [22, 23]. Sclerostin binds to a number of BMP growth factors and has been shown to inhibit BMP-mediated osteoblast differentiation [24]. It also binds directly to LRP5/6, preventing activation of the Wnt signalling pathway (fig. 3) [18].

Osteoclasts

Osteoclasts are large multinucleate cells found in close apposition to bone surfaces undergoing resorption. Osteoclast precursors share the same haematopoietic lineage as macrophages. One of the most significant breakthroughs in bone biology of the last fifteen years has been the identification and characterisation of the molecular pathway controlling osteoclastogenesis [25] (fig. 4). Osteoclast precursors express the receptor for macrophage colony stimulating factor (MCSF) which, when stimulated, promotes the expression of a TNF superfamily molecule, receptor activator of nuclear factor kappa-B (RANK). Osteoblasts control the differentiation of osteoclast precursors through production of RANK ligand (RANKL), a cell surface molecule that is the primary effector of the RANK receptor, and osteoprotegerin (OPG), a soluble decoy receptor for RANKL. The balance between RANKL and OPG concentrations regulates RANK activation. The RANK receptor can activate a network of intracellular pathways [25]. MCSF/RANKL stimulated osteoclast precursors form polykaryons through a process of cell membrane fusion that is poorly understood. RANK activation is also necessary for mature osteoclast activity. When settled onto bone, osteoclasts form a

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Fig. 4. Osteoclast differentiation. Osteoclasts arise from circulating haematopoietic cells of the monocyte/macrophage lineage (osteoclast precursors) that express c-fms, the receptor for macrophage colony-stimulating factor (MCSF). MCSF, produced by osteoblasts, stimulates expression of receptor activator of nuclear factor κ-B (RANK) on osteoclast precursors. The key regulator of osteoclastogenesis, RANK ligand (RANKL), is expressed by osteoblasts. RANKL binds to RANK and stimulates the fusion of osteoclast precursors to form multinucleated immature osteoclasts. Mature osteoclasts form a tight seal and generate a ruffled membrane border against the bone surface through which they secrete acid and proteolytic enzymes forming a resorption lacuna. RANKL acts on osteoclasts at all stages of differentiation. Osteoclastogenesis is regulated by osteoblasts through balancing the production of RANKL and Osteoprotegerin (OPG), a decoy receptor produced by osteoblasts to inhibit the interaction of RANKL and its receptor RANK.

region of tight contact between the cell and the bone surface known as the sealing zone, creating a tightly enclosed area underneath the osteoclast where bone resorption takes place. The cellular membranes within the sealing zone develop into a ruffled border, a structure of deeply folded cellular membranes adjacent to the bone surface, through which are secreted acid and proteolytic enzymes to mediate bone resorption. Defects in genes encoding the molecular pathway controlling acid production (CA2) and its secretion through ion channels (TCIRG1, CLCN7, OSTM1) have been identified as frequent causes of osteoclast rich infantile onset osteopetrosis (table 1; fig. 1, chapter 12) (reviewed in [26]). More recently, defects in RANKL [27] and in RANK [28] have been identified in cases of infantile onset osteopetrosis in which osteoclasts are completely absent (table 1; fig. 1, chapter 12). The ultimate fate of the osteoclast is apoptosis.

Hormones and Mineral Homeostasis

Parathyroid hormone (PTH), parathyroid hormone-related peptide (PTH-rP) and vitamin D are the key hormonal regulators of mineral homeostasis. PTH acts in bone to stimulate the release of calcium and phosphate, while at the same time acting on

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the kidney to enhance calcium reabsorption and to inhibit reabsorption of phosphate. PTH also enhances the renal production of 1,25(OH)2vitamin D, which acts on the intestine to enhance calcium absorption. The molecular mechanisms regulating intestinal absorption and renal reabsorption of calcium have recently been identified. Two calcium selective ion channels, TRPV5 and TRPV6, mediate epithelial transcellular transport of calcium from the intestinal or renal tubular lumen to the extracellular fluid, by way of intracellular calcium binding proteins and cell surface ion pumps (reviewed in Nijenhuis et al. [29]). FGF23 was identified as the gene responsible for autosomal dominant hypophosphataemic rickets (ADHR) (table 1) [30]. FGF23–/– mice have hyperphosphataemia, high renal reabsorption of phosphate and high circulating levels of 1,25(OH)2D [31, 32]. The receptor for FGF23 is a heterocomplex of the canonical FGF receptor and klotho [33]. Interestingly, klotho has β-gluguronidase enzymatic activity which is responsible for hydrolysing external sugar residues of TRPV5, resulting in trapping and activation of this calcium selective ion channel at the cell surface [34].

The Nervous System and Bone

Although nerves are found throughout the periosteum and near metabolically active parts of bone, until very recently little thought was given to the potential role of the nervous system in bone. Evidence for nerve fibres that signal through the neuropeptides calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and substance P began to emerge in the 1980s (reviewed in Jones et al. [35]). The discovery of a glutamate/aspartate transporter molecule GLAST in bone, previously associated with glutamatergic neuronal signalling [36], energised the search for glutamate signalling mechanisms acting on bone formation and resorption. Osteoblasts, osteocytes and osteoclasts have all been shown to express ionotropic and metabotropic glutamate and NMDA receptors, and ion channel controlled electrical currents consistent with these receptors have been measured in osteoclasts [37]. The inhibitory action on bone formation of leptin, a hormone involved in controlling body mass, has been shown to be mediated through adrenergic signalling resulting from leptin acting in the hypothalamus [38, 39].

Functional Adaptation of Bone to Load Bearing

Bones need to be stiff enough to bear the loads they are commonly subjected to without deforming or breaking under load [40]. Bone strength is a function of its stiffness and is dependent on several factors including size, shape and material composition/ spatial distribution. The concept of functional adaptation of the skeleton, consolidated in Wolff ’s law [41], was developed into the mechanostat hypothesis [2] based

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on observations that load bearing vertebrate bones undergo relatively few spontaneous as opposed to traumatic fractures, indicating that bones adapt their strength to be able to endure typical peak mechanical loads without fracturing while having a sufficiently large safety margin to endure occasional supra-normal loads. In the mechanostat hypothesis, Frost [2] describes a series of rules by which mechanical competence may be achieved and maintained in load bearing bones (which includes many non weight bearing bones e.g. mandibles), providing tissue-level mechanisms for functional adaptation of the skeleton to the loads placed upon it and presenting predictable hypotheses concerning the mechanical implications of bone disease. The key element is the biological machinery to sense the level of loading and to respond by increasing or decreasing the mechanical competence. To date, the nature of the mechanostat in bone is still unknown. Evidence dictates that such machinery is local to the bone under load, since bones respond to local or asymmetrical loads. This is typically seen in athletes such as tennis players in whom bones of the playing arm are strengthened preferentially [42]. A mechanostat model also implies upper and lower thresholds within which bone is sensed to be under normal loading, with loads above or below triggering a response to model or remodel the bone. Lanyon et al. [43] in the 1970s developed the concept that bone cells respond to the magnitude of the strain experienced by a bone under load (the ratio of change in bone length divided by original length) while, more recently, work in experimental animals and humans has shown that other factors relating to strain are also important, including the strain direction, rate of strain change, duration of loading, the number of loading cycles, frequency, repetition and rest within and between cycles [44]. Whether bone cells sense mechanical loads through direct cellular deformation or through shear strains induced by interstitial fluid flow resulting from bone deformation is unclear, but molecular mechanisms capable of strain detection have been described in osteoblasts, osteocytes, osteoclasts and vascular endothelial cells, with ion channels, integrins and associated proteins, connexins, cell surface structures, the cytoskeleton and nitric oxide as potential molecular mediators [45, 46]. The mechanostat hypothesis is consistent with observations that maternal environment and intrauterine muscular activity influence load bearing bones by the time of birth, and it makes provision for the influence of non-mechanical factors in bone growth, development and maintenance.

Healthy Bones for Life: Nutrition and Exercise in Bone Physiology

The impact of bone loss in the ageing population on quality of life and in health service provision has in the last 20–30 years introduced the consideration of public policy strategies to maximise bone mass accrual during childhood and adolescence by manipulation of diet or through exercise. Almost half of our adult bone mineral mass is accrued by the skeleton in the 3–4 years following the onset of puberty [47, 48],

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making adolescence one of the most critical periods for skeletal development. Peak bone mass, achieved during the third decade, is a powerful predictor of postmenopausal osteoporosis [49, 50] and it is widely assumed that optimising peak bone mass accrual during childhood and adolescence will produce bones that are better equipped to handle the inevitable loss of bone in later life. The two simplest ways to influence bone mass accrual are through nutritional intervention and exercise. A recent systematic review of 22 controlled trials concluded that weight-bearing exercise in children and adolescents leads to modest increases in bone parameters over 6 months [51]. However, it is difficult to assess which exercise activities are most appropriate, or the time frame within which they should be undertaken. Growth occurs heterogeneously in the skeleton throughout childhood where, for example, the longitudinal growth velocity of the legs in infancy is about twice that in the spine until puberty [52]. Benefits of exercise are likely to be site specific, depend on the type of exercise (weight bearing, high impact) and may be influenced by dietary and hormonal factors [53], and even by conditions experienced while in utero [52]. The mechanostat theory predicts that bone strength will decrease on disuse, but evidence is emerging from studies in former athletes and animals that skeletal benefits may persist despite lack of exercise [54]. Well-designed long-term studies are needed to see if such benefits may be achievable in children.

Conclusions

Advances in understanding the molecular mechanisms and pathways regulating bone cell function bring with them opportunities for the development of novel, rational approaches to treat disorders of bone cell dysfunction in children. Knowledge gained from systematic study of children’s bone disorders also provides insights into the physiology of healthy bones. Improving our understanding of the physiology of bone growth and development during childhood will lead to better prospects for finding early pharmacological, physiotherapeutic and nutritional strategies to optimise and maintain bone health from childhood into adulthood, which in turn may help to reduce the burden of bone loss and its related ill-health in old age.

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37 Laketic-Ljubojevic I, Suva LJ, Maathuis FJ, Sanders D, Skerry TM: Functional characterization of N-methyl-d-aspartic acid-gated channels in bone cells. Bone 1999;25:631–637. 38 Ducy P, Amling M, Takeda S, et al: Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell 2000;100:197– 207. 39 Takeda S, Elefteriou F, Levasseur R, et al: Leptin regulates bone formation via the sympathetic nervous system. Cell 2002;111:305–317. 40 Currey JD: Bones: Structure and Mechanics. Princeton, Princeton University Press, 2002. 41 Wolff J: Das Gesetz der Transformation der Knochen. Berlin, Hirschwald, 1892. 42 Haapasalo H, Kontulainen S, Sievanen H, Kannus P, Jarvinen M, Vuori I: Exercise-induced bone gain is due to enlargement in bone size without a change in volumetric bone density: a peripheral quantitative computed tomography study of the upper arms of male tennis players. Bone 2000;27:351–357. 43 Clark EA, Goodship AE, Lanyon LE: Locomotor bone strain as the stimulus for bone’s mechanical adaptability. J Physiol 1975;245:57P. 44 Skerry TM: One mechanostat or many? Modifications of the site-specific response of bone to mechanical loading by nature and nurture. J Musculoskelet Neuronal Interact 2006;6:122–127. 45 Rubin J, Rubin C, Jacobs CR: Molecular pathways mediating mechanical signaling in bone. Gene 2006; 367:1–16. 46 Malone AM, Anderson CT, Tummala P, Kwon RY, Johnston TR, Stearns T et al.: Primary cilia mediate mechanosensing in bone cells by a calcium-independent mechanism. Proc Natl Acad Sci USA 2007; 104:13325–13330.

47 Bailey DA: The Saskatchewan Pediatric Bone Mineral Accrual Study: bone mineral acquisition during the growing years. Int J Sports Med 1997; 18(suppl 3):S191–S194. 48 Bailey DA, Martin AD, McKay HA, Whiting S, Mirwald R: Calcium accretion in girls and boys during puberty: a longitudinal analysis. J Bone Miner Res 2000;15:2245–2250. 49 Hui SL, Slemenda CW, Johnston CC Jr: The contribution of bone loss to postmenopausal osteoporosis. Osteoporos Int 1990;1:30–34. 50 Seeman E: Reduced bone density in women with fractures: contribution of low peak bone density and rapid bone loss. Osteoporos Int 1994;4(suppl 1):15–25. 51 Hind K, Burrows M: Weight-bearing exercise and bone mineral accrual in children and adolescents: a review of controlled trials. Bone 2007;40:14–27. 52 Cooper C, Westlake S, Harvey N, Javaid K, Dennison E, Hanson M: Review: developmental origins of osteoporotic fracture. Osteoporos Int 2006;17:337– 347. 53 Loud KJ, Gordon CM: Adolescent bone health. Arch Pediatr Adolesc Med 2006;160:1026–1032. 54 Ducher G, Bass SL: Exercise during growth: compelling evidence for the primary prevention of osteoporosis? BoneKEy – Osteovision 2007;4:171– 180. 55 Superti-Furga A, Unger S: Nosology and classification of genetic skeletal disorders: 2006 revision. Am J Med Genet A 2007;143:1–18.

Peter Grabowski, PhD Faculty of Medicine, Dentistry and Health, Academic Unit of Child Health, University of Sheffield Sheffield S10 2RX (UK) Tel. +44 0114 2711798, Fax +44 0114 2755463, E-Mail [email protected]

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Chapter 4 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 49–57

Bone Biopsy: Indications and Methods Frank Rauch Genetics Unit, Shriners Hospital for Children and McGill University, Montréal, Qué., Canada

Abstract In the context of metabolic bone disorders, obtaining biopsies of iliac bone can be useful for establishing a diagnosis in an individual patient or for investigating pathomechanisms when a series of samples is examined. Although bone specimens are usually decalcified for routine pathology to facilitate sample processing, when investigating metabolic bone disorders it is usually much more informative to analyse undecalcified samples. Biopsy samples can be assessed qualitatively and quantitatively. Quantitative analysis by computerised histomorphometry of undecalcified bone biopsy samples is a key tool for studying bone metabolism and, to a lesser extent, bone mass and structure. Standard histomorphometric analyses focuses on trabecular bone and therefore mainly provides information on trabecular remodelling. Remodelling activity changes markedly with age during development. This has to be taken into account when histomorphometry is used in the paediatric setting. Children and adolescents with severe bone fragility should have a bone biopsy for diagnostic purposes unless the diagnosis is obvious from non-invasive examinations. Quantitative histomorphometric analysis of transiliac bone biopsy samples is especially valuable in clinical studies, as this method provides safety and efficacy data that can not be obtained in any other way. Copyright © 2009 S. Karger AG, Basel

In the context of metabolic bone disorders, obtaining biopsies of iliac bone can be useful for establishing a diagnosis in an individual patient or for investigating pathomechanisms when a series of samples is examined. Biopsy samples can be used for qualitative assessment – similar to the pathologist’s evaluation of other tissue specimens – and for quantitative analysis, called histomorphometry. Bone tissue is very hard and for that reason is more difficult to process than soft tissue. In routine pathology, bone tissue is therefore often decalcified and thus converted into a soft tissue. However, this leads to the loss of important information about bone mineralisation and bone cell activity. To assess metabolic bone disorders, it is therefore generally more informative to analyse samples undecalcified. Computerised quantitative histomorphometry of undecalcified bone biopsy samples is a method to obtain direct quantitative information on bone tissue. When tetracycline labelling is performed prior to biopsy, bone cell function can be studied in

vivo. Important for paediatric use, bone histomorphometric results are not directly influenced by the growth process. In contrast to some currently popular indirect methods of bone analysis, histomorphometry yields results with a known meaning. Knowledge of bone tissue is also crucial for interpreting the findings of molecular and cellular studies. Despite these advantages, bone histomorphometry is underused in paediatrics. This may be partly due to the fact that histomorphometry requires an invasive procedure to obtain a bone sample, is labour intensive, and needs special equipment and expertise. Other reasons may include overestimation of the utility of non-invasive bone diagnostics and lack of information about what bone histomorphometry does. The present contribution tries to address this latter point. More detailed information on paediatric histomorphometry is available elsewhere [1].

Bone Biopsy Procedure

Bone histomorphometry was first developed to study rib bone samples. This was soon abandoned because the ilium proved to be a much more convenient site for obtaining bone samples. In principle, histomorphometric analysis can be performed in any bone. In clinical paediatrics, however, the utility of samples from nonstandard sites is limited because reference data are only available for the ilium. Quantitative bone histomorphometry requires an intact biopsy specimen of good quality. This implies that the transiliac sample must be obtained under standardised conditions and with appropriate tools. It is essential that the sample is not fractured or crushed and contains two cortices separated by a trabecular compartment. These requirements are often quite difficult to meet in small or very osteopaenic children. Bone specimens for histomorphometric evaluation are horizontal, full-thickness biopsy samples of the ilium from a site 2 cm posterior from the anterior superior iliac spine. This bone is easily accessible, does not require extensive surgery, and is associated with few postoperative complications. Also, this is the only site for which paediatric histomorphometric reference data have been published [2]. A correctly performed biopsy procedure should yield a sample containing two cortices that are separated by a trabecular compartment (fig. 1). Vertical samples (from the iliac crest downwards, also called Jamshidi approach) are of questionable utility because of the presence of the growth plate at the top of the iliac crest. Turnover is very high and cortical thickness is very low in bone tissue below the growth plate and results are therefore not representative. Thus, the often-used term ‘iliac crest biopsy’ is a misnomer, as the iliac crest actually should be avoided during the biopsy procedure. The more accurate term is ‘transiliac biopsy’. The most widely used bone biopsy instrument is the Bordier needle. The inner diameter of the needle should be 5 mm (which we use in patients up to the age of 12 years) or 6 mm (for patients older than 12 years). The size of the needle diameter is

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Fig. 1. Section of an entire iliac biopsy specimen of a 9-year-old boy without metabolic bone disease. In this section core width is 7.2 mm, cortical width (i.e. the average length of the arrows indicated in the two cortical compartments) is 894 μm and bone volume per tissue volume in the trabecular compartment is 24.5%. Osteoid and cellular structures can not be identified at this magnification.

Cortical compartment

Trabecular compartment

Core width

important, because an appropriately large sample area must be available for histomorphometric analyses to obtain representative measures. A smaller needle diameter means that a smaller bone sample is obtained and that the margin of error of the histomorphometric analysis is wider. Most children younger than 14 years of age require general anaesthesia for the procedure. Local anaesthesia can be sufficient for older adolescents. Patients are allowed to get out of bed after 3 h and can usually be discharged on the same day. Another prerequisite for histomorphometric evaluation is bone labelling. Dynamic parameters of bone cell function can only be measured when the patient has received two courses of tetracycline label prior to biopsy. Tetracycline compounds form calcium chelate complexes that bind to bone surfaces. These complexes are buried within the bone at sites of active bone formation, whereas they redissociate from the other bone surfaces once serum tetracycline levels decrease. The tetracycline trapped at formation sites can then be visualised under fluorescent light (fig. 2). The most widely used tetracycline compound is demeclocycline hydrochloride (DeclomycinT, Ledermycin®). Two labelling cycles are given before the biopsy procedure, each one lasting for 2 days. Declomycin is given orally in two doses per day with a daily dose of 15–20 mg/kg body weight (maximum dosage: 900 mg/day). The first labelling course is given on days 17 and 16 before the biopsy procedure, the second course is given on days 5 and 4 before the procedure. The two courses are thus separated by an interlabel time of 10 days. Although children and adolescents generally tolerate tetracycline double labelling well, some side effects, such as allergic reactions, vomiting, and photosensitivity, might be observed. Administering the drug after meals can diminish gastrointestinal side effects. It is important that these meals do not include milk or other dairy products because tetracycline complexes with calcium contained in the food and is not absorbed adequately. Sun exposure must be

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Fig. 2. Trabecular remodelling. Multiple short tetracycline double-labels are present. Dynamic bone formation parameters are derived from the length of the tetracycline labels and the distance between the labels.

avoided while taking tetracyclines. Tetracycline use is generally not recommended for children younger than nine years of age because discolouration of teeth may occur. However, the previously mentioned schedule appears to be safe in this respect. At the Montreal Shriners Hospital, it has been used for more than 350 biopsies in children younger than 9 years of age and tooth discolouration has never been observed.

Sample Processing

The biopsy sample should be placed in a fixative solution as soon as possible after the procedure. The fixation process aims at the preservation of bone tissue constituents by inactivating lysosomal enzymes. The choice of fixative and temperature at which the sample should be kept depends on the planned staining techniques. For routine histomorphometry, 70% ethanol or 10% buffered formalin at room temperature can be used. The duration of fixation should be at least 48 h but should not exceed 10 days because the tetracycline labels are washed out when fixation is too long. Once in fixative, the sample can be sent to the laboratory where samples are cut and stained and where the histomorphometric analyses are performed.

Histomorphometric Parameters

Histomorphometrists use standardised terminology and clear definitions that were established by a working group of the American Society for Bone and Mineral Research [3]. According to these definitions, ‘bone’ is bone matrix, whether it is mineralised or not. Unmineralised bone matrix is called osteoid. The term ‘tissue’ refers to both bone and associated soft tissue, such as bone marrow. Histomorphometric measurements

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are performed in two-dimensional sections. Nevertheless, in order to stress the threedimensional nature of bone, the terminology committee favoured a three-dimensional nomenclature for reporting histomorphometric results [3]. Thus, what appears as a line in a microscopic bone section is called a surface, whereas what is visible as an area under the microscope is referred to as a volume. This is done simply by convention, and should not be mistaken as actual three-dimensional measurements. Histomorphometric parameters can be classified into four categories (table 1): Structural parameters, static bone formation parameters, dynamic formation parameters, and bone resorption parameters. Structural parameters describe the size and the amount of bone. The outer size of a transiliac biopsy specimen is called core width, a measure which reflects the thickness of the ilium. Cortical width is the average width of the two cortices. Bone volume per tissue volume of trabecular bone represents the proportion of the marrow cavity which is occupied by bone. In trabecular bone, bone volume per tissue volume can be schematically separated into two components, trabecular thickness and trabecular number. The group of static formation parameters comprises the surface extent, thickness and relative amount of osteoid, as well as the surface extent of osteoblasts (called osteoid surface per bone surface, osteoid thickness, osteoid volume per bone volume and osteoblasts surface per bone surface, respectively; table 1). Wall thickness reflects the amount of bone that is created by an osteoblast team during a remodelling event. Wall thickness should not be confused with cortical thickness, with which is does not have any relationship. Dynamic bone formation parameters yield information on in vivo bone cell function and can only be measured when patients have received two courses of tetracycline label prior to biopsy (table 1). The two basic parameters are the surface extent of mineralisation activity (mineralising surface per bone surface) and the speed of mineralisation in a direction perpendicular to the bone surface (mineral apposition rate). From these primary measures, mineralisation lag time and bone formation rate per bone surface are derived mathematically. It should be noted that a high bone formation rate does not necessarily lead to a net gain of bone. If the remodelling balance is zero, the amount of bone will remain unchanged even if bone formation rate is very high. The combination of a negative remodelling balance and high bone formation rate will even lead to rapid bone loss. Thus, bone formation rate per bone surface in trabecular bone indicates the activity of bone turnover rather than bone gain [4]. Bone resorption can only be quantified with static parameters, which makes evaluation of bone resorption the least informative aspect of histomorphometric analysis. It is possible to quantify the extent of bone surface that is covered by osteoclasts or which looks eroded (osteoclast surface per bone surface and eroded surface per bone surface, respectively), but it is not possible to tell from these measures how much bone resorption is actually going on. This may be an issue in the evaluation of renal bone disease. In chronic renal failure, osteoclasts resorb bone more slowly than normal, so that the extent of osteoclast and eroded surfaces overestimates the rate of bone resorption [5].

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Table 1. The most commonly used histomorphometric parameters Parameter Structural parameters Core width, mm Cortical width, μm Bone volume/tissue volume, %

Trabecular thickness, μm Trabecular number, /mm

Static formation parameters Osteoid thickness, μm Osteoid surface/bone surface, % Osteoid volume/bone volume, % Osteoblast surface/bone surface, % Wall thickness, μm Dynamic formation parameters Mineralising surface/bone surface, % Mineral apposition rate, μm/day Mineralisation lag time, days Bone formation rate/bone surface, μm3*μm-2*y-1 Static resorption parameters Eroded surface/bone surface, % Osteoclast surface/bone surface, %

Significance

overall size of the biopsy specimen distance between periosteal and endocortical surfaces space taken up by mineralised and unmineralised bone relative to the total size of a bone compartment self-explanatory number of trabeculae that a line through trabecular compartment would hit per millimetre of its length distance between the surface of the osteoid seam and mineralised bone percentage of bone surface covered by osteoid percentage of bone volume consisting of unmineralised osteoid percentage of bone surface covered by osteoblasts mean thickness of bone tissue that has been deposited at a remodelling site percentage of bone surface showing mineralising activity distance between two tetracycline labels divided by the length of the labelling interval time interval between the deposition and mineralisation of matrix amount of bone formed per year on a given bone surface percentage of bone surface presenting a scalloped appearance percentage of bone surface covered by osteoclasts

Bone Metabolism in Children and Adolescents

The volume of trabecular iliac bone increases markedly between 2 and 20 years of age [2]. This increase is entirely explained by trabecular thickening, whereas there is no change in trabecular number. Iliac trabeculae probably become thicker during development

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because bone remodels with a positive balance [6]. It has been estimated that, during a remodelling cycle, osteoblasts lay down about 5% more bone than osteoclasts resorb. In other words, 95% of the bone formation activity is required just to replace the bone that has been previously removed by the osteoclasts. Since the difference between resorption and formation is very small, a high remodelling activity is necessary to make trabeculae noticeably thicker. Remodelling activity is indeed elevated in young children, decreases until the age of 8 or 9 years, and increases again during puberty. After the age of puberty, remodelling activity declines into the much lower adult range [6].

Indications for Bone Biopsy in Paediatrics

The main use of iliac bone biopsies is to provide diagnostic clues in unclear bone fragility disorders. For example, some forms of osteogenesis imperfecta can be diagnosed on the basis of a characteristic histologic pattern [7]. Polyostotic fibrous dysplasia is sometimes difficult to distinguish from osteogenesis imperfecta on clinical grounds, but the diagnosis is usually quite obvious on bone histology. This has therapeutic implications, as children with osteogenesis imperfecta usually respond much better to bisphosphonate treatment than patients with fibrous dysplasia [7, 8]. Thus, children with multiple long-bone fractures or vertebral body compressions without adequate trauma should have a bone biopsy unless the diagnosis is obvious from non-invasive examinations. Another indication for bone biopsy is progressive bone deformity, which may sometimes arise without clear history of fractures. A bone biopsy sample allows evaluation of trabecular and cortical bone structure, the mineralisation process, bone lamellation (woven bone vs. lamellar bone, the appearance of lamellae), the presence of calcified cartilage, the activity of bone metabolism and the appearance of bone cells. All of this information is important in the assessment of skeletal disease processes, but none of it is reflected in ‘bone density’, whatever technique is used to measure it. These considerations are particularly relevant in the context of renal bone disease, which is a frequent but understudied problem after juvenile renal failure [9]. When the only aim is to assess an individual patient, it is not absolutely necessary to analyse the sample with quantitative histomorphometry. A qualitative evaluation of the histological appearance may be sufficient in such cases. However, a quantitative analysis is necessary in clinical research settings, when numbers are needed to describe the average effect of a disease or a treatment in a group of patients. Histomorphometric evaluation of bone biopsy samples should be a standard feature of studies that evaluate experimental drugs to treat bone disorders in children and adolescents. Current noninvasive methods for studying the amount, distribution, and metabolism of bone are fraught with technical limitations and uncertainties regarding the interpretation of results. The availability of histomorphometric data allows judging treatment effects in a rational way.

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The most important argument for performing bone biopsies in paediatric studies probably concerns patient safety. This is especially true when children and adolescents are treated with long-acting drugs, such as bisphosphonates. Analysis of bone samples provides safety measures that can not be obtained in any other way. For example, bone histologic studies have demonstrated that bisphosphonate treatment in children can lead to accumulation of calcified cartilage material in bone tissue, a disquieting finding that calls for caution in the use of these drugs in growing patients with minor skeletal symptoms [10, 11]. Thus, including bone biopsies in study protocols is crucial for documenting the efficacy of therapy as well as its safety.

Conclusions

Standard histomorphometric analysis of transiliac bone biopsies mainly provides information on trabecular remodelling. Assessment of cortical modelling processes is feasible but has rarely been used until now. When histomorphometric studies are performed in children and adolescents, it is important to take the age dependency of many histomorphometric parameters into account. When new treatments of bone disorders are studied, analysis of transiliac bone biopsy samples provides safety and efficacy data that can not be obtained in any other way. Children with multiple longbone fractures or vertebral body compressions that are not explained by adequate trauma should have a bone biopsy unless the diagnosis is obvious from non-invasive examinations.

Acknowledgements Thanks go to Mark Lepik for preparing the figures. The author is a Chercheur-Boursier Clinicien of the Fonds de la Recherche en Santé du Québec. This work was supported by the Shriners of North America.

References 1 Rauch F: Bone histomorphometry; in Glorieux FH, Pettifor J, Jueppner H (eds): Pediatric Bone. San Diego, Academic Press, 2003, pp 359–374. 2 Glorieux FH, Travers R, Taylor A, et al: Normative data for iliac bone histomorphometry in growing children. Bone 2000;26:103–109. 3 Parfitt AM, Drezner MK, Glorieux FH, et al: Bone histomorphometry: standardization of nomenclature, symbols, and units: report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 1987;2:595–610.

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4 Parfitt AM: Renal bone disease: a new conceptual framework for the interpretation of bone histomorphometry. Curr Opin Nephrol Hypertens 2003;12: 387–403. 5 Jaworski ZF, Lok E, Wellington JL: Impaired osteoclastic function and linear bone erosion rate in secondary hyperparathyroidism associated with chronic renal failure. Clin Orthop Relat Res 1975; 107:298– 310.

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6 Parfitt AM, Travers R, Rauch F, Glorieux FH: Structural and cellular changes during bone growth in healthy children. Bone 2000;27:487–494. 7 Rauch F, Glorieux FH: Osteogenesis imperfecta. Lancet 2004;363:1377–1385. 8 Plotkin H, Rauch F, Zeitlin L, Munns C, Travers R, Glorieux FH: Effect of pamidronate treatment in children with polyostotic fibrous dysplasia of bone. J Clin Endocrinol Metab 2003;88:4569–4575.

9 Groothoff JW, Offringa M, Eck-Smit BLF, et al: Severe bone disease and low bone mineral density after juvenile renal failure. Kidney Int 2003;63:266– 275. 10 Rauch F, Travers R, Plotkin H, Glorieux FH: The effects of intravenous pamidronate on the bone tissue of children and adolescents with osteogenesis imperfecta. J Clin Invest 2002;110:1293–1299. 11 Whyte MP, Wenkert D, Clements KL, McAlister WH, Mumm S: Bisphosphonate-induced osteopetrosis. N Engl J Med 2003;349:457–463.

Frank Rauch Genetics Unit, Shriners Hospital for Children 529 Cedar Avenue Montréal, Qué., H3G 1A6 (Canada) Tel. +1 514 842 5964, Fax +1 514 842 5581, E-Mail [email protected]

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Chapter 5 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 58–72

Bone Densitometry: Current Status and Future Perspectives Nicola Crabtreea ⭈ Kate Wardb a Department of Nuclear Medicine, Queen Elizabeth NHS Foundation Trust Hospital, Birmingham, and bMRCHuman Nutrition Research, Elsie Widdowson Laboratory, Cambridge, UK

Abstract In this chapter we discuss the concept of what determines bone strength and fracture risk and how this can be quantified using current technologies. We describe bone densitometry measurement techniques that are currently available and consider the strengths and limitations of each technique, with particular relevance to paediatric scanning. Magnetic resonance imaging is reviewed as one of the newer technologies applied to the assessment of the growing skeleton. The role of dual energy X-ray absorptiometry (DXA) and quantitative computed tomography (QCT) in the clinical assessment of bone health in children is considered and current diagnostic application reviewed. Copyright © 2009 S. Karger AG, Basel

What Is Bone Mineral Density?

When describing the densitometric properties of bone the terms ‘bone mineralisation’ and ‘bone density’ are often interchanged and incorrectly used. The term ‘mineralisation’ refers to the incorporation of additional bone mineral (calcium, phosphorus and other minerals) into an existing extracellular matrix and the term ‘density’, as defined by Archimedes as the ratio of bone mass to the volume (g/cm3). Whilst there is a definite meaning to the term mineralisation, the term ‘bone density’ is less well defined and dependent on the region being assessed and the technique used. Quantitative computed tomography (QCT) measures volumetric density (BMDv) whereas dual energy X-ray absorptiometry (DXA) measures areal density (BMDa), which is not a physical density but the ratio of the total amount of bone tissue within the projected area of bone (g/cm2). The basic model of the bone describes three types of density, ‘material’, ‘compartment’ and ‘total’ all of which will have a significant function in the determination of bone strength and fracture risk (fig. 1; table 1) [1]. The three densities of bone are:

Fig. 1. Schematic representations of the ‘different’ densities of bone. Material density, calculated as the mass of bone illustrated by the grey shading divided by the volume defined by the outer pale grey border excluding Haversian canals, blood vessels and osteons (a). Compartmental density of cortical (b1) or trabecular (b2) bone density, calculated as the grey shaded mass of bone divided by the volume defined by the outer pale grey border. Total bone density calculated as the mass of both cortical and trabecular bone divided by the total volume of the bone (c). Adapted from Rauch and Schoenau [1].

(a) Material density

(b1) Cortical (b2) Trabecular compartmental compartmental bone density bone density

(c) Total bone density

Table 1. Assessment of the three ‘densities’ of bone by DXA and pQCT BMDtotal

BMDcompartment

BMDmaterial

DXA

 (estimate)

only in area of the long bone which are comprised predominantly of cortical bone (e.g. radius)

QCT/pQCT



 cortical and trabecular

the resolution required to measure BMDmaterial is not possible with current noninvasive densitometric techniques; BMDmaterial can only be determined from specimens taken at bone biopsy, which is an invasive procedure

• BMDtotal – the mineral density of all the material contained within the periosteal envelope and/or articular surfaces. • BMDcompartment – the amount of mineral contained within the trabecular, or the cortical, compartments, i.e. the mass of mineral per unit volume of trabecular or cortical bone. • BMDmaterial – the degree of mineralisation of the organic bone matrix. It is important to understand which ‘density’ each measurement technique is assessing and how it contributes to skeletal development, bone strength and fracture risk.

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Assessment of Bone Density

The rationale for measuring bone strength in childhood is multi-factorial but can be broadly grouped into the following areas: • Assessment of the impact of chronic disease in childhood. • Monitoring the effect of modifiable factors in healthy growing children (e.g. physical activity, nutrient intake, etc.). • Predicting current and future risk of fragility fracture. Ideally, the measurement technique would be able to measure bone strength directly or, alternatively, important components of bone strength, i.e. bone mass/density, bone material and geometric properties. The technique would not be affected by growth, be safe and be readily performed in all children. Several techniques for measuring bone have been used over the past few decades, the most common of which are the technologies developed using X-rays (ionising radiation) which assess bone mass and bone density in vivo. These are DXA and QCT (both axial and peripheral).

Measurement Principles

The measurement of bone density and bone mass by ionising radiation is based upon the differential attenuation of the X-radiation beam through different tissues of the body. As X-rays pass through the body they will be attenuated (reduced in strength). The extent of attenuation varies with the energy of the photons and the nature and depth of the material they pass through. As such, the proportion of detected X-rays relates directly to the attenuation values and the area density of the body tissue through which it has passed i.e. bone or soft tissue. Using image processing techniques an attenuation/density map can be converted to a grey scale to produce an image which, with edge detection algorithms, can be used to calculate parameters such as bone size, bone area cortical width, etc. The most widely accessible and minimally invasive technique currently available to measure paediatric bone health is dual energy X-ray absorptiometry (DXA) which has the ability to measure bone mass and BMDatotal, both of which have been demonstrated to be associated with fracture risk [2].

Dual-Energy X-Ray Absorptiometry

Dual-energy X-ray absorptiometry (DXA) has been available since the late 1980s when it was principally introduced to measure and monitor the course of osteoporosis in post menopausal women [3]. Since the introduction and increased availability of DXA, there has been a dramatic rise in its use in paediatric research and clinical practice. The fundamental principle of DXA is the measurement of transmission of X-rays, produced from a stable X-ray source, at high and low energies. The two

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Fig. 2. Lumbar spine and whole body DXA scan of a 9-year-old child with a history of multiple low trauma fractures.

energies allow the discrimination of soft tissue and bone and, by calculating the mass attenuation of the two materials and using sophisticated edge detection techniques, it is possible to measure BMDatotal, bone mineral content (BMC) and the projectional area of the bone. Modern-day DXAs achieve fast, precise measurements with low levels of radiation (0.1–6 μSv) and reasonable image resolution, all of which are of utmost importance when measuring children [4] (fig. 2). DXA measurements can be made at the spine, hip, whole body and forearm. For clinical practice in children, the spine and whole body less head are recommended sites. This chapter focuses on the whole body and spine as these are currently the most common sites used clinically. Although DXA is the most commonly employed method for the assessment of bone health, it does have several limitations, which are particularly relevant to the measurement of children. These include: • size dependence of BMDatotal, • inability to separate trabecular and cortical compartments, • inaccuracies due to changes in body composition,

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2 × 2 × 2 cm

3 × 3 × 3 cm

Projected area

Bone mineral content Volume Projected area Volumetric density Areal density

Projected area

=8g = 8 cm3 = 4 cm2 = 1 g/cm3 = 2 g/cm2

Bone mineral content Volume Projected area Volumetric density Areal density

= 27 g = 27 cm3 = 9 cm2 = 1 g/cm3 = 3 g/cm2

Fig. 3. The two cubes represent vertebrae of different sizes and demonstrate the size dependence of areal BMD. The cubes have identical volumetric densities (1 g/cm3) but the smaller cube has considerably lower areal density than the larger cube on the right.

• software being designed for use in adults, • limited reference data. Additionally, there may be practical difficulties in acquiring DXA scans in young children, or children with marked learning difficulties as they may not be able to stay still for the duration of the scan [5]. The most significant of the limitations is that DXA is a projectional technique whereby a three-dimensional object is analysed using a two-dimensional projection. As mentioned, the attenuation of the X-rays is a function of the nature and depth of the material they pass through. Consequently, X-rays will be more highly attenuated in a large bone due to them travelling through more bone; therefore they will appear denser than a smaller bone with the same physical density (fig. 3). A direct consequence of the inability to measure bone depth is that the BMDatotal value calculated by DXA, is highly dependent on bone size [4, 6]. The consequence of the size dependence of BMDatotal means that DXA will inherently underestimate bone density in a short child, with smaller bones, and overestimate bone density in a tall child, with bigger bones, despite the fact that they may have identical volumetric densities. This technical problem can have serious consequences when measuring and monitoring children with chronic diseases, where the disease has also had an impact on their normal growth and development or their tempo of skeletal maturation, resulting in them being either tall or, more frequently, small for their chronological age. To avoid misdiagnosis of reduced BMDatotal due to variations in body stature rather than genuine deficiencies in bone mass, reference values for bone mineral density in children should either be independent of bone size,

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if presented according to age, or, alternatively, account for the variations in bone and body size, to reduce any measurement ambiguity. Several size adjustment techniques have been developed and are listed below.

Volumetric Bone Density for Age

The most frequently used method of size adjustment in paediatric DXA is the calculation of bone mineral apparent density (BMAD) or estimated volumetric bone density. At the lumbar spine, BMAD is calculated by using either vertebral width or projected area from the DXA scan to estimate the vertebral depth and thereby enabling calculation of volumetric BMD (g/cm3) [6, 7]. The relative size independence of this parameter makes this a useful marker for reduced absolute bone density and, along with BMD for age, it has also been shown to be related to fracture risk in children [8]. This density is sometimes inappropriately referred to in the literature as ‘true bone density’. Currently, this method of size adjustment is not feasible for the whole body DXA scan.

Bone Mineral Content for Height

Reporting BMC for height is the simplest of all of the size adjustment methods since it requires no assumptions about bone size. Currently, this size adjustment has not been related to fracture risk, although it has been shown to be useful in comparing populations with diminished stature. It also correlates with the estimated bone strength measured by pQCT in children [9].

Allometric Approach

The ‘Mølgaard’ model is a simple allometric approximation that provides a threestage assessment to explain reduced bone density. The three-stage model assesses height for age (short bones), bone area for height (narrow bones), and BMC for bone area (light bones) [10]. The important diagnostic value of this is that it provides both geometric and densitometric information to give a better understanding of the bones underlying fragility. For example, thin gracile bones in cerebral palsy would have reduced strength due to reduced mineralisation and size.

Regression Models

‘Size-adjusted BMC’, is calculated using a regression, or a multivariate, statistical model to adjust BMC for confounders, such as projected BA, overall body height,

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weight and pubertal stage [11–13]. This approach is often used in research studies. However, it should be borne in mind that body height and weight might not completely control for all relevant differences in size and shape of the skeletal region of interest. Furthermore, differences in bone size and shape may have important implications for bone strength, independently of adjusted or unadjusted BMC/ BMDatotal.

Mechanostat Functional Model

The mechanostat, or functional model, uses an alternative approach to size adjustment based on the relationship between muscle and bone [14]. The two-stage algorithm proposed by Schoenau et al. [15] was first applied to pQCT and then extended to DXA. Both techniques are based on the assumptions that BMC acts as a surrogate for bone strength and lean body mass as a surrogate for muscle load [16]. From this, four differential diagnoses can be made which potentially relate to risk of fracture. The two stages of assessment are: (1) whether the child has sufficient muscle for their height, and (2) whether they have sufficient bone for that muscle. This leads to four outcomes: (1) ‘Normal’ appropriate muscle mass for height and appropriate bone mass for muscle mass. (2) Primary bone defect, where the child has sufficient muscle for height but insufficient bone mass for muscle. (3) Primary muscle defect, where the child has reduced muscle for height but sufficient bone for muscle mass. (4) A mixed muscle and bone defect, where muscle and bone are both reduced. Currently, there is no consensus as to the best method of size adjustment, or which will best predict current or future fracture risk, or whether any of the different size adjustment techniques can improve the diagnostic capability of DXA. However, in a child with short stature, any of the size adjustment techniques will improve the diagnostic specificity of DXA and reduce the possibility of misdiagnosis due to body size [17].

Diagnostic Potential of Dual-Energy X-Ray Absorptiometry

The diagnostic potential of DXA was recently highlighted in a large prospective fracture study [18] and many other case control studies [2, 8, 19, 20]. The ISCD has recently produced recommendations for the clinical use of DXA as part of a comprehensive skeletal health assessment in patients with increased fracture. The guidelines recommend using DXA to measure the lumbar spine and whole body (less head) in patients

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with primary bone diseases or potential secondary bone diseases, and that the term ‘osteoporosis’ should not be used in context of densitometry measurements alone. They also recommend that results should be size adjusted in children of reduced stature and reported as ‘low BMC or bone mineral density for chronological age’ if the BMC or BMDatotal Z-score is less than or equal to –2.0’ [21]. However, bone assessment by DXA is only part of a comprehensive skeletal assessment and, as such, the DXA results should be combined with a full clinical history and any other tests carried out.

Quantitative Computed Tomography

The main limitation of DXA in children is using information gained from a 2-dimensional projection to assess a 3-dimensional structure. Consequently, a measurement technique that collects information in all dimensions may potentially overcome this limitation. The demand for such information in paediatric bone density assessment has led to a renewed interest in computed tomography, particularly peripheral computed tomography. The main principle of CT measurements are that the linear X-ray absorption coefficient are transformed into a CT number (Hounsfield Units) which are reconstructed to form the CT image. The CT number is then transformed to BMD and geometry is calculated using image processing. For standalone CT scanners a bone equivalent phantom is required for quantification of mean volumetric BMDcompartment (mg/cm3) from the image. QCT can be applied to axial or peripheral skeletal sites. Radiation exposure is much lower in pQCT than axial QCT [5]. The most important feature of QCT is that it provides size-independent measures of volumetric BMDtotal or BMDcompartment of the trabecular and cortical bone. The trabecular BMDcompartment measured by QCT is a composite of the amount of bone and marrow per voxel. The reason for this is the relatively small size of trabeculae compared to the voxel, resulting in marrow being included in the measurement (fig. 4). As trabecular bone is generally more metabolically active than cortical bone, trabecular BMDcompartment, as measured by QCT, is likely be more sensitive to change than cortical BMDcompartment measurements [22]. QCT quantifies other important features of bone strength and measures bone size and geometry. It also provides estimates of in vivo bone strength, which relate well to fracture load [23]. More recently, high-resolution techniques (HR-CT) allow in vivo finite element modelling to be performed and to study the loading conditions to which the bone is subjected, which may improve the fracture prediction of the technique. To date, this has only been applied in adults [24, 25]. Axial QCT of the spine was first described in the late 1970s [26], and became more widely used during the 1980s [22] until DXA was introduced in 1988. The original body CT scanners used rotate-translate technology and permitted only 2D slices to be obtained and the procedure took about 15 min. The evolution of spiral and multi-

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Fig. 4. Lumbar spine QCT and calibration block.

slice CT now allows the rapid, precise acquisition of volumetric scans of the spine, hip and peripheral sites. In a scan time as little as 10 s, a volume of data can be acquired. Quantitative skeletal assessment does not require the image quality required for conventional CT and therefore radiation dose can be minimised through use of a low dose technique [27]. Gathering a block of data makes the technique particularly useful in longitudinal studies as it make it easier to relocate the previous scan site. QCT has been applied in research studies to monitor response to intervention in central and peripheral sites [28, 29]. The limitations of axial QCT include: • approximately 10- to 12-fold greater dose of ionising radiation than DXA for spine scans, • the demand for CT equipment is much greater than for DXA, • specialist bone equivalent phantoms and software are required to perform bone measurements, • there is a lack of commercial analysis packages, • there is only one, small published reference dataset for paediatric use [30]. The high radiation dose but inherent advantages of QCT led to the development of stand alone pQCT scanners. Peripheral QCT first became commercially available in the early 1990s [31], the most commonly used technique is the single slice pQCT. The sites of measurement are the radius, tibia and femur and the radiation dose is much lower than axial QCT. For single slice techniques this is <1 μSv (fig. 5). Trabecular BMDcompartment measurements by QCT are less influenced by size than DXA BMDatotal and therefore use of agematched reference ranges should be appropriate. However, geometric and strength parameters should either be adjusted for height or limb length or a reference range for height be used [9, 32, 33].

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a

b Fig. 5. Tibial pQCT. Distal metaphysis (a), and (b) mid-diaphysis of a child with Duchenne muscular dystrophy.

Fig. 6. Sclerotic metaphyseal lines observed on a pQCT scan of the distal radius from intermittent bisphosphonate treatment.

There are important technical limitations to consider when using single-slice pQCT in children, particularly when considering reference line placement and measurement site and how to ensure precise relocation of measurement site in longitudinal measurements. It is essential that the growth plate, growth arrest lines or sclerotic metaphyseal lines from intermittent bisphosphonate use are avoided as they would produce falsely high measures (fig. 6) [34]. Anatomical abnormalities such as Madelung’s deformity may also cause difficulties in positioning the reference line. Movement artifact can often be a problem in young children or in those with physical disabilities as the scan time can take between 2 and 3 min per slice. HR-CT may overcome some of the limitations of the single slice technique as it gathers a volume of data (90 mm) and should therefore make repositioning easier. The particular advantage is the greater resolution, in-plane resolution of 82 μm, which provides the ability to study trabecular microstructure. In adults, this has led

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to a greater understanding of the age- and gender-related changes in bone morphometry and density [35]. The higher resolution also allows more detailed study of the cortical compartment of the metaphysis. The radiation exposure is approximately 3 μSv which is higher than single-slice but less than a day’s background radiation in the UK. The main limitation is movement artifact due to scan time.

Diagnostic Potential of Quantitative Computed Tomography

The diagnostic ability of axial or peripheral QCT has yet to be fully elucidated and it is used clinically in only a few specialist centres. The ISCD has recently produced guidelines for the clinical use of pQCT with recommendations to measure distal and diaphyseal sites. However, there can be no complete consensus for the application of the technique until fracture prediction data are available [33]. QCT is less widely used due to its greater radiation dose, but the acquisition of a volume of data should make reproducibility of longitudinal measurements sites better than single slice technique. Reference data are available for the spine [30] and the technique has been applied clinically to provide a more sensitive measure of bone response to treatment than BMAD by DXA [34]. It is imperative to try to minimise the limitations of the technique previously discussed, in particular the single slice methods, for pQCT or QCT to become accepted clinical tools for diagnosis or fracture prediction [33].

Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) is the most recently developed technique for skeletal assessment in children and can be applied to the peripheral or axial skeleton. The whole bone or specific regions can be measured [36, 37]. There is a stand alone high resolution MR scanner available for studies of the peripheral skeleton [38]. By measuring the signal from lipid and water protons, an image of the bone is obtained. Bone marrow provides the majority of the signal and so, in an image, marrow is white and bone black. Similar to QCT, MRI distinguishes trabecular from cortical bone compartments and provides measures of bone morphometry, so bone strength and trabecular architecture can be calculated. BMD has been measured and correlates well to ash weight and 3D-QCT [39]. MRI provides a volumetric measure without the use of ionising radiation, imaging in multiple anatomical planes is possible without having to reposition the subject and simultaneous scanning of several limbs is also feasible. There are a few limitations of MRI. The equipment is noisy and can be claustrophobic for the subject with scanning taking as long as 20–30 min and sedation may be necessary. To date, MRI has been used only in research protocols and its applicability in clinical practice has yet to be assessed.

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Quantitative Ultrasound

Quantitative ultrasound (QUS) was developed as a method of assessing bone status in 1984 [40]. The measurements obtained from QUS are based upon the attenuation of the ultrasound beam as it passes through the bone, either an entire bone as in transverse transmission, or along the cortical bone, for axial transmission (single probe). QUS measures broadband ultrasound attenuation (BUA dB/MHz), in the transverse transmission techniques only, and/or speed of sound (SOS, m/s). The main advantages of QUS in paediatrics are that it is non-ionising, non-invasive, portable and available at a relatively low-cost; reference data are available e.g. [41–43]. The main limitations are the lack of consensus as to what is actually being measured [44, 45], whether there is added benefit to DXA measurements [46], technical limitations and the commercial development of scanners being limited in recent years. The fracture prediction by QUS in children has yet to be fully assessed [46]. Recent data show calcaneal QUS discriminates between children with a history of upper limb fracture and those who do not [47]. QUS has been applied in clinical research to detect differences between bone status in children with disease compared to healthy controls [48–52] and the newer axial transmission techniques used in neonatal setting [44, 53–55].

Guidelines

To summarise, a bone scales allometrically during growth such that deviations from normal growth and mineral accrual may help to identify children with reduced bone strength and therefore increased risk of low trauma fracture. However, current in vivo measuring techniques, such as DXA and/or pQCT/QCT provide only one part in the diagnosis of osteoporosis in children. Current paediatric bone densitometry guidelines highlight that DXA is the only recommended method of clinical assessment of bone density measurement and that any decision to initiate therapeutic intervention should only be made in conjunction with a full skeletal health assessment, i.e. DXA measurements alone cannot be used to diagnose or treat a patient at risk of fracture (www.iscd.org/Visitors/pdfs/ISCD2007OfficialPositions-Pediatric. pdf) [21]. The guidelines also state that a diagnosis of osteoporosis requires the presence of both a clinically significant fracture history (long bone fracture of the lower extremities, or a vertebral compression fracture or two or more long bone fractures of the upper extremities) and low bone mineral content or bone mineral density, adjusted for age and/or body size. Furthermore, it is also important to consider less quantifiable factors, such as lifestyle and prolonged immobility, risk taking behaviour and/or potential abnormal loading conditions, when assessing fracture risk.

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Finally, although paediatric DXA it is now readily accessible to most clinical specialties, if there is any doubt about whom to refer, what to measure or how to interpret the result, it is important to consult a paediatric metabolic bone disease specialist, prior to requesting and performing the DXA scan [5].

References 1 Rauch F, Schoenau E: Changes in bone density during childhood and adolescence: an approach based on bone’s biological organization. J Bone Miner Res 2001;16:597–604. 2 Clark EM, Tobias JH, Ness AR: Association between bone density and fractures in children: a systematic review and meta-analysis. Pediatrics 2006;117:e291– e297. 3 Mazess RB, Barden HS: Measurement of bone by dual-photon absorptiometry (DPA) and dualenergy X-ray absorptiometry (DEXA). Ann Chir Gynaecol 1988;77:197–203. 4 Fewtrell MS: Bone densitometry in children assessed by dual X ray absorptiometry: uses and pitfalls. Arch Dis Child 2003;88:795–798. 5 National Osteoporosis Society: A Practical Guide to Bone Densitometry in Children. Bath, 2004. 6 Carter DR, Bouxsein ML, Marcus R: New approaches for interpreting projected bone densitometry data. J Bone Miner Res 1992;7:137–145. 7 Kroger H, Vainio P, Nieminen J, Kotaniemi A: Comparison of different models for interpreting bone mineral density measurements using DXA and MRI technology. Bone 1995;17:157–159. 8 Goulding A, Jones IE, Taylor RW, Manning PJ, Williams SM: More broken bones: a 4-year double cohort study of young girls with and without distal forearm fractures. J Bone Miner Res 2000;15:2011– 2018. 9 Leonard MB, Shults J, Elliott DM, Stallings VA, Zemel BS: Interpretation of whole body dual energy X-ray absorptiometry measures in children: comparison with peripheral quantitative computed tomography. Bone 2004;34:1044–1052. 10 Molgaard C, Thomsen BL, Prentice A, Cole TJ, Michaelsen KF: Whole body bone mineral content in healthy children and adolescents. Arch Dis Child 1997;76:9–15. 11 Nevill AM, Holder RL, Maffulli N, et al: Adjusting bone mass for differences in projected bone area and other confounding variables: an allometric perspective. J Bone Miner Res 2002;17:703–708.

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12 Prentice A, Parsons TJ, Cole TJ: Uncritical use of bone mineral density in absorptiometry may lead to size-related artifacts in the identification of bone mineral determinants. Am J Clin Nutr 1994;60:837– 842. 13 Warner JT, Cowan FJ, Dunstan FDJ, Evans WD, Webb DKH, Gregory JW: Measured and predicted bone mineral content in healthy boys and girls aged 6–18 years: adjustment for body size and puberty. Acta Paediatr 1998;87:244–249. 14 Frost HM: The mechanostat: a proposed pathogenic mechanism of osteoporoses and the bone mass effects of mechanical and nonmechanical agents. Bone Miner 1987;2:73–85. 15 Schoenau E, Neu CM, Beck B, Manz F, Rauch F: Bone mineral content per muscle cross-sectional area as an index of the functional muscle-bone unit. J Bone Miner Res 2002;17:1095–1101. 16 Crabtree NJ, Kibirige MS, Fordham JN, et al: The relationship between lean body mass and bone mineral content in paediatric health and disease. Bone 2004;35:965–972. 17 Gordon CM, Bachrach LK, Carpenter TO, et al: Dual energy X-ray absorptiometry interpretation and reporting in children and adolescents: the 2007 ISCD Pediatric Official Positions. J Clin Densitom 2008;11:43–58. 18 Clark EM, Ness AR, Bishop NJ, Tobias JH: Association between bone mass and fractures in children: a prospective cohort study. J Bone Miner Res 2006;21:1489–1495. 19 Goulding A, Cannan R, Williams SM, Gold EJ, Taylor RW, Lewis-Barned NJ: Bone mineral density in girls with forearm fractures. J Bone Miner Res 1998;13:143–148. 20 Manias K, McCabe D, Bishop N: Fractures and recurrent fractures in children: varying effects of environmental factors as well as bone size and mass. Bone 2006;39:652–657. 21 Lewiecki EM, Gordon CM, Baim S, et al: International Society for Clinical Densitometry 2007 Adult and Pediatric Official Positions. Bone 2008;43:1115–1121.

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22 Genant HK, Cann CE, Ettinger B, Gordan GS: Quantitative computed tomography of vertebral spongiosa: a sensitive method for detecting early bone loss after oophorectomy. Ann Intern Med 1982;97:699–705. 23 Augat P, Gordon CL, Lang TF, Iida H, Genant HK: Accuracy of cortical and trabecular bone measurements with peripheral quantitative computed tomography (pQCT). Phys Med Biol 1998;43:2873– 2883. 24 Diederichs G, Link T, Marie K, et al: Feasibility of measuring trabecular bone structure of the proximal femur using 64-slice multidetector computed tomography in a clinical setting. Calcif Tissue Int 2008;83:332–341. 25 Melton LJ, III, Riggs BL, van Lenthe GH, Achenbach SJ, Muller R, Bouxsein ML, et al: Contribution of in vivo structural measurements and load/strength ratios to the determination of forearm fracture risk in postmenopausal women. J Bone Miner Res 2007; 22:1442–1448. 26 Isherwood I, Rutherford RA, Pullan BR, Adams PH: Bone-mineral estimation by computer-assisted transverse axial tomography. Lancet 1976;ii:712– 715. 27 Cann CE: Low-dose CT scanning for quantitative spinal mineral analysis. Radiology 1981;140:813– 815. 28 Gilsanz V, Wren TA, Sanchez M, Dorey F, Judex S, Rubin C: Low-level, high-frequency mechanical signals enhance musculoskeletal development of young women with low BMD. J Bone Miner Res 2006;21:1464–1474. 29 Ward K, Alsop C, Caulton J, Rubin C, Adams J, Mughal Z: Low magnitude mechanical loading is osteogenic in children with disabling conditions. J Bone Miner Res 2004;19:360–369. 30 Gilsanz V, Gibbens DT, Roe TF, et al: Vertebral bone density in children: effect of puberty. Radiology 1988;166:847–850. 31 Schneider P, Borner W: Peripheral quantitative computed tomography for bone mineral measurement using a new special QCT-scanner. Methodology, normal values, comparison with manifest osteoporosis. Rofo 1991;154:292–299. 32 Ashby RA, Ward KA, Roberts SA, Edwards L, Mughal MZ, Adams JE: A reference database for the Stratec XCT-2000 peripheral quantitative computed tomography scanner in healthy children aged 6–19 years. Osteoporos Int 2008;10.1007/s00198–008– 0800–2. 33 Zemel B, Bass S, Binkley T, et al: Peripheral quantitative computed tomography in children and adolescents: the 2007 ISCD Pediatric Official Positions. J Clin Densitom 2008;11:59–74.

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34 Ward KA, Adams JE, Freemont TJ, Mughal MZ: Can bisphosphonate treatment be stopped in a growing child with skeletal fragility? Osteoporos Int 2007;18:1137–1140. 35 Riggs BL, Melton LJ, Robb RA, et al: A populationbased assessment of rates of bone loss at multiple skeletal sites: evidence for substantial trabecular bone loss in young adult women and men. J Bone Miner Res 2008;23:205–214. 36 Daly RM, Saxon L, Turner CH, Robling AG, Bass SL: The relationship between muscle size and bone geometry during growth and in response to exercise. Bone 2004;34:281–287. 37 Hogler W, Blimkie CJ, Cowell CT, et al: A comparison of bone geometry and cortical density at the mid-femur between prepuberty and young adulthood using magnetic resonance imaging. Bone 2003;33:771–778. 38 Wehrli FW: Structural and functional assessment of trabecular and cortical bone by micro magnetic resonance imaging. J Magn Reson Imaging 2007;25: 390–409. 39 Hong J, Hipp JA, Mulkern RV, Jaramillo D, Snyder BD: Magnetic resonance imaging measurements of bone density and cross-sectional geometry. Calcif Tissue Int 2000;66:74–78. 40 Langton CM, Palmer SB, Porter RW: The measurement of broadband ultrasonic attenuation in cancellous bone. Eng Med 1984;13:89–91. 41 Baroncelli GI, Federico G, Vignolo M, et al: Crosssectional reference data for phalangeal quantitative ultrasound from early childhood to young-adulthood according to gender, age, skeletal growth, and pubertal development. Bone 2006;39:159–173. 42 Mughal MZ, Ward K, Qayyum N, Langton CM: Assessment of bone status using the contact ultrasound bone analyser. Arch Dis Child 1997;76:535– 536. 43 Sawyer A, Moore S, Fielding KT, Nix DA, Kiratli J, Bachrach LK: Calcaneus ultrasound measurements in a convenience sample of healthy youth. J Clin Densitom 2001;4:111–120. 44 Fewtrell MS, Loh KL, Chomtho S, Kennedy K, Hawdon J, Khakoo A: Quantitative ultrasound (QUS): a useful tool for monitoring bone health in preterm infants? Acta Paediatr 2008;97:1625–1630. 45 Fricke O, Tutlewski B, Schwahn B, Schoenau E: Speed of sound: relation to geometric characteristics of bone in children, adolescents, and adults. J Pediatr 2005;146:764–768. 46 Wang Q, Nicholson PH, Timonen J, et al: Monitoring bone growth using quantitative ultrasound in comparison with DXA and pQCT. J Clin Densitom 2008;11:295–301.

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47 Jones G, Boon P: Which bone mass measures discriminate adolescents who have fractured from those who have not? Osteoporos Int 2008;19:251– 255. 48 Ahuja SP, Greenspan SL, Lin Y, Bowen A, Bartels D, Goyal RK: A pilot study of heel ultrasound to screen for low bone mass in children with leukemia. J Pediatr Hematol Oncol 2006;28:427–432. 49 Di Iorgi N, Muratori T, Secco A, et al: Quantitative ultrasound detects bone impairment after bone marrow transplantation in children and adolescents affected by hematological diseases. Bone 2008;43: 177–182. 50 Mainz JG, Sauner D, Malich A, et al: Cross-sectional study on bone density-related sonographic parameters in children with asthma: correlation to therapy with inhaled corticosteroids and disease severity. J Bone Miner Metab 2008;26:485–492. 51 Mughal MZ, Langton CM, Utretch G, Morrison J, Specker BL: Comparison between broad-band ultrasound attenuation of the calcaneum and total body bone mineral density in children. Acta Paediatr 1996;85:663–665.

52 Valerio G, Spadaro R, Iafusco D, et al: The influence of gluten free diet on quantitative ultrasound of proximal phalanxes in children and adolescents with type 1 diabetes mellitus and celiac disease. Bone 2008;43:322–326. 53 McDevitt H, Ahmed SF: Quantitative ultrasound assessment of bone health in the neonate. Neonatology 2007;91:2–11. 54 McDevitt H, Tomlinson C, White MP, Ahmed SF: Changes in quantitative ultrasound in infants born at less than 32 weeks’ gestation over the first 2 years of life: influence of clinical and biochemical changes. Calcif Tissue Int 2007;81:263–269. 55 Mercy J, Dillon B, Morris J, Emmerson AJ, Mughal MZ: Relationship of tibial speed of sound and lower limb length to nutrient intake in preterm infants. Arch Dis Child Fetal Neonatal Ed 2007;92:F381– F385.

Dr. Nicola Crabtree Department of Nuclear Medicine, Queen Elizabeth NHS Foundation Trust Hospital Edgbaston Birmingham B15 2TH (UK) Tel. +44 121 6272591, Fax+44 121 6272591, E-Mail [email protected]

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Chapter 6 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 73–92

A Practical Approach to Hypocalcaemia in Children Nick Shaw Department of Endocrinology, Birmingham Children’s Hospital, Birmingham, UK

Abstract Hypocalcaemia is one of the commonest disorders of mineral metabolism seen in children and can be a consequence of several different aetiologies. These include a failure of secretion or action of parathyroid hormone, disorders of vitamin D metabolism and abnormal function of the calciumsensing receptor. A practical approach to the investigation, diagnosis and subsequent management Copyright © 2009 S. Karger AG, Basel of hypocalcaemic disorders is presented.

Hypocalcaemia is one of the commonest disorders of calcium and phosphate metabolism seen in children. It may be asymptomatic or manifest with a variety of different symptoms which can vary with the age of the child. Although the differential diagnosis is quite wide the cause can usually be categorised into one of a small list of broad aetiologies. An understanding of the key determinants of the regulation of plasma calcium and the normal physiological response to hypocalcaemia will lead to an appropriate interpretation of investigations and subsequent diagnosis and management.

Physiological Response to Hypocalcaemia

A fall in plasma calcium will lead to several physiological changes which, acting in conjunction, lead to a rapid restoration of plasma calcium into the normal range. These changes take place in the three key organs that are involved in the maintenance of plasma calcium, i.e. the kidney, bone and the small intestine (fig. 1). Thus, a fall in the level of ionised calcium is detected by the calcium-sensing receptor located in the parathyroid glands and the renal tubules. In the parathyroid glands this leads to the secretion of parathyroid hormone (PTH) and the synthesis of additional parathyroid hormone. The increased circulating levels of parathyroid hormone then acts in three different ways.

Low serum Ca2+

FCa reabsorption

FPTH

FCa2+ and PO4 release

F Phosphate excretion F1⬀hydroxylase activity

F 1,25 (OH)2D3

FCa and PO4 absorption

Serum Ca2+F · Serum PO4 r or f

Fig. 1. Physiological response to hypocalcaemia.

1 It alters the renal tubular reabsorption of calcium in the kidney leading to more of the filtered plasma calcium being reabsorbed and less excretion in the urine. In doing this there is a reciprocal effect on the tubular reabsorption of phosphate with a fall in this leading to increased urinary phosphate excretion. Thus, one of the markers of a raised serum parathyroid hormone is a low plasma phosphate and tubular reabsorption of phosphate (TRP). 2 Parathyroid hormone acts on the skeleton stimulating osteoclasts to increase bone resorption leading to the release of calcium from bone into the circulation. 3 The increased circulating parathyroid hormone also stimulates the activity of the 1α-hydroxylase enzyme in the proximal renal tubule leading to increased secretion of 1,25(OH)2D which then increases intestinal calcium absorption. Thus alterations in renal calcium reabsorption, bone resorption and intestinal calcium absorption result in a restoration of the ionised calcium level. Although the synthesis and secretion of parathyroid hormone is the key factor in this response, it can be appreciated that this also requires a functional calcium-sensing receptor, appropriate synthesis of 1,25(OH)2D and a normal response of peripheral tissues to the secreted PTH.

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Signs and Symptoms of Hypocalcaemia

The symptoms of hypocalcaemia often reflect the key role of calcium in the processes of nerve conduction and muscle function with a low plasma calcium resulting in increased neuromuscular excitability. Paraesthesia, a tingling sensation, usually present around the mouth, fingers and toes is a common symptom of hypocalcaemia. Muscle cramps, which in some children may progress to tetany, which is due to sustained muscle contractions, often in the hands, can be experienced. Convulsions, which may be either focal or generalised, can be a manifestation of hypocalcaemia at any age but are particularly recognised during infancy and adolescence. Other less common symptoms include laryngospasm, stridor and apnoea in neonates. Hypocalcaemia can also lead to disturbances of cardiac rhythm and prolongation of the QT interval on an electrocardiograph. Chronic hypocalcaemia can lead to calcification of the basal ganglia, subcapsular cataracts, papilloedema and dental enamel hypoplasia, particularly of the primary dentition. Two manifestations of latent hypocalcaemia that can be evoked on clinical examination are the signs of Chvostek and Trousseau. The former consists of gentle repeated tapping with a forefinger on the lateral cheek over the course of the facial nerve 0.5– 1.0 cm below the zygomatic process and 2 cm anterior to the ear lobe. A positive sign is twitching of the corner of the mouth on the ipsilateral side due to contractions of the circumoral muscles. However, it is not a reliable marker of hypocalcaemia as one study has shown it to be negative in 29% of individuals with laboratory confirmed hypocalcaemia [1]. Trousseau’s sign is evoked when a sphygmomanometer cuff is inflated above systolic pressure and maintained for three minutes. A positive sign is the adoption of the ‘main d’accoucheur’ position with flexion of the wrist and metacarpophalangeal joints and extension of the interphalangeal joints and adduction of the fingers due to carpopedal spasm. Note that this sign can be painful for the individual if sustained for too long. It is believed to be a more specific marker of hypocalcaemia than Chvostek’s sign with one study showing that 94% of individuals with hypocalcaemia had a positive sign [2].

Investigations

There are several important investigations required in the management of a child with hypocalcaemia, the majority of which are biochemical in nature (table 1). It is usually the total plasma calcium that is measured in the blood although some laboratories and near patient testing facilities will measure the ionised calcium which is usually 50% of the total plasma calcium. Calcium is predominantly bound to albumin in the plasma and therefore deviation of the plasma albumin from the normal range will affect the measured total plasma calcium. Some laboratories will automatically adjust for this and quote a corrected plasma calcium. However, in the absence of this facility

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Table 1. Investigations in hypocalcaemia Plasma calcium, phosphate and magnesium Plasma albumin Plasma or serum alkaline phosphatase Plasma creatinine Plasma 25-hydroxyvitamin D Serum parathyroid hormone Store serum, e.g. for 1,25-dihydroxyvitamin D3 Send blood for DNA studies as appropriate Urine for calcium/creatinine ratio X-ray of wrist or knee

there are several simple correction factors that can be applied. One of the most well known is to correct the plasma calcium by 0.02 mmol/l for every 1 g/l that the plasma albumin deviates from the normal value of 40 g/l, e.g. total plasma calcium: 2.2 mmol/l, plasma albumin: 30 g/l, corrected calcium: 2.2–0.02 (30–40) = 2.4 mmol/l. A measurement of the plasma phosphate is useful as an indirect index of parathyroid hormone activity with a low phosphate reflecting a raised serum PTH and a high phosphate a reduced serum PTH. Hypomagnesaemia is a rare but important cause for hypocalcaemia and therefore plasma magnesium should be included in the list of investigations. Plasma or serum alkaline phosphatase is usually included as part of the standard bone profile provided by many laboratories as a marker of bone turnover. This is often raised when hypocalcaemia is secondary to a disorder of vitamin D and within the normal range when secondary to hypoparathyroidism. Plasma creatinine is an essential investigation to exclude the possibility of renal failure as the cause for hypocalcaemia. Measurement of 25OHD is an important routine investigation given the frequency of disorders of vitamin D in the aetiology of hypocalcaemia in children. It is the main circulating form of vitamin D and the metabolite that best reflects an individual’s vitamin D status. Although a level less than 50 nmol/l (20 ng/ml) would be regarded as consistent with vitamin D deficiency, hypocalcaemia does not usually occur until levels are less than 25 nmol/l (10 ng/ml). Once vitamin D deficiency has been excluded as a cause of the hypocalcaemia, most of the remaining causes have a genetic basis and blood should be taken for appropriate genetic analysis. Serum PTH is the most critical investigation in determining the aetiology of hypocalcaemia and, as indicated subsequently in this chapter, this is used here as the main means of classification. There are three additional investigations that may prove to be helpful. Additional serum should be routinely obtained when the initial investigations are performed and stored in the laboratory. This can then be used for

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Table 2. Aetiology of hypocalcaemia PTH disorder Reduced PTH secretion Impaired PTH action Vitamin D disorder Vitamin D deficiency Impaired vitamin D metabolism Impaired renal function Abnormality of the calcium-sensing receptor Gain of function mutations in the gene for the calcium-sensing receptor Antibodies to the calcium-sensing receptor

subsequent analysis, such as measurement of 1,25(OH)2D, when the initial investigations have not clarified the aetiology of the hypocalcaemia. Attempting to measure such a metabolite at a later stage when a child has been commenced on initial treatment is often compromised by the effect of the treatment. A measurement of the urine calcium/creatinine ratio, ideally on a second morning urine sample obtained in the fasting state, is a useful marker of renal calcium excretion and conversely of renal tubular calcium reabsorption [3]. Finally an X-ray of the metaphysis of a long bone such as at the wrist or knee may identify previously unsuspected pathology, such as the presence of rickets or of a dense skeleton in an infant with osteopetrosis.

Aetiology of Hypocalcaemia – Classification

It is possible to divide the causes of hypocalcaemia into three broad categories that reflect the three main important components in the regulation of plasma calcium, i.e. serum parathyroid hormone, vitamin D and the calcium-sensing receptor (table 2). Within each of these categories, it is possible to subdivide further. Thus, the category relating to PTH would encompass a reduction of PTH secretion, as seen in hypoparathyroidism, and an impairment of PTH action as seen in pseudohypoparathyroidism. Within the category relating to vitamin D this can again be divided into a lack of the essential substrate, 25OHD, as seen in vitamin D deficiency, or an impairment of the metabolism, as in 1α-hydroxylase deficiency, or end organ action of vitamin D. The category relating to disorders of the calcium-sensing receptor encompasses congenital disorders due to mutations in the relevant gene, as seen in autosomal dominant hypocalcaemia (ADH), and acquired disorders such as antibodies to the calcium-sensing receptor. However, a more practical approach in determining the aetiology of hypocalcaemia is to adopt a classification dependent on the level of circulating serum PTH in the presence of hypocalcaemia (table 3). Thus, a serum PTH that is either undetectable or low can be seen either in hypoparathyroidism or hypomagnesaemia. An

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Table 3. Categorisation of cause of hypocalcaemia Undetectable or low PTH Hypoparathyroidism Hypomagnesaemia Normal PTH Abnormality of the calcium-sensing receptor Raised PTH Pseudohypoparathyroidism Vitamin D deficiency Impaired vitamin D metabolism Chronic renal failure Osteopetrosis

inappropriately normal serum PTH is often seen when there is an abnormality affecting the function of the calcium-sensing receptor. Finally, an elevated serum PTH, which would be the normal physiological response, is seen when there is a disorder of vitamin D or a failure of end-organ action of PTH as seen in pseudohypoparathyroidism or osteopetrosis. The next section of this chapter will examine this classification in more detail with descriptions of the individual conditions.

Hypocalcaemia with Low or Undetectable PTH

Congenital Hypoparathyroidism This may occur as an isolated defect or in association with other developmental defects. A number of genetic abnormalities have now been identified that can be broadly divided into those that encode abnormal forms of PTH, defects in intracellular transcription factors or defects that prevent normal development of the parathyroid glands.

Isolated Congenital Hypoparathyroidism Isolated congenital hypoparathyroidism (#146200) may be sporadic or familial with autosomal-dominant (168450.0001), recessive (168450.0002) and X-linked recessive (%307700) forms being recognised. The gene for preproPTH is located on chromosome 11p15 (*168450) with homozygous mutations responsible for an autosomal-recessive form [4], whilst the autosomal-dominant inherited form is due to point mutations in the signal sequence of preproPTH preventing processing and translocation of PTH across the endoplasmic reticulum and cell membrane for exocytosis [5]. Another form of isolated hypoparathyroidism, which is inherited in an autosomal-recessive manner

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has been shown to be due to mutations of the transcription factors GCMB (glial cell missing B), also known as GCM2 (glial cell missing 2), (*603716), located on chromosome 6p24.2, which is responsible for parathyroid gland development [6]. Occasionally, some heterozygous individuals also have a mild degree of hypoparathyroidism which is inherited in an autosomal-dominant manner (see chapter 15, cases 1 and 2). The X-linked recessive form has now been identified due to an interstitial deletioninsertion involving chromosomes 2p25.3 and Xq27.1 [7]. This is thought to affect the function of SOX3, a transcription factor expressed in the developing parathyroid glands. All of these conditions present during the neonatal period or childhood without any signs of other organs being affected and treatment of the hypocalcaemia with vitamin D analogues is usually all that is required.

The 22q Deletion (Di George) Syndrome

The most well-known syndrome associated with congenital hypoparathyroidism is the Di George syndrome (DGS) (#188400) which overlaps with the velocardiofacial syndrome (#192430). It is the commonest chromosome deletion syndrome and affects 1 in 4,000–5,000 livebirths. Maldevelopment of the 3rd and 4th branchial pouches causes hypoplasia of the parathyroid glands and thymus in association with congenital conotruncal cardiac defects and a distinctive facial phenotype. Most cases are sporadic but familial cases with apparent autosomal-dominant inheritance are described. The majority are due to deletions at chromosome 22q11 although deletions at a second locus at 10p13 have been found in some patients. The deletion in chromosome 22q is hemizygous and encompasses a variable length of the chromosome. However, it seems that, when hypoparathyroidism is present, there is a common deletion of the TBX1 gene (*602054) which is a transcription factor involved in development of the pharyngeal arches, pouches and otic vesicles. Clinical features of the DGS are variable. Hypocalcaemia may be present in the neonatal period but is often transient although it may recur at puberty or in adulthood [8] – times when growth is most rapid and the demand for calcium is greatest. Occasionally, when hypocalcaemia presents clinically at adolescence, retrospective assessment of the case history strongly suggests that symptoms have been present for several years. However, hypocalcaemia may be overlooked in the neonatal period if the condition is accompanied by serious cardiac anomalies. It is thought that up to 70% of patients who survive the neonatal period have some degree of parathyroid hypoplasia (see chapter 15, case 3).

Other Forms of Familial Hypoparathyroidism

The hypoparathyroid, deafness, renal anomalies (HDR) (#146255) syndrome is inherited in an autosomal-dominant manner and is due to mutations in the gene coding

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for the transcription factor GATA3 on chromosome 10p14–10pter (*131320) which is critical for parathyroid, kidney and otic vesicle development [9]. Affected individuals usually have hypoparathyroidism in association with bilateral sensorineural deafness and renal anomalies. Another syndrome that includes hypoparathyroidism, mental and growth retardation and dysmorphic features (HRD syndrome) (#241410) is inherited in an autosomal recessive manner. It includes the Kenny-Caffey syndrome (KCS) (#244460) (short stature, osteosclerosis and ocular abnormalities) and the Sanjad-Sakati syndrome (growth failure, ocular malformations, microcephaly and mental retardation). The genetic defect for these was localised to chromosome 1q42-q43 and was subsequently identified as due to mutations in TBCE (*604934) (tubulin-specific chaperone E), which cause loss of function and probable altered microtubule assembly in affected tissues [10]. Finally, a number of mitochondrial disorders are associated with congenital hypoparathyroidism. These include Kearns-Sayre syndrome (KSS) (#530000) (progressive external ophthalmoplegia, heartblock or cardiomyopathy), MELAS (#540000) (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) and MTPDS (#609015) (mitochondrial trifunctional protein deficiency syndrome). As they are mitochondrial gene defects they are maternally inherited. In all of these conditions the hypoparathyroidism is often a relatively minor feature and may be overlooked in the light of the other problems present (see chapter 15, case 4). A rare form of congenital, though not genetically mediated, hypoparathyroidism has been reported secondary to maternal hyperparathyroidism [11]. Affected infants have presented in the neonatal period with hypocalcaemia due to transient hypoparathyroidism which, on investigation of the mother, has identified previously unrecognised hyperparathyroidism which presumably suppresses the foetal parathyroid glands in utero.

Acquired Hypoparathyroidism

This can be a consequence of surgery on the thyroid gland (e.g. total thyroidectomy for thyrotoxicosis) or parathyroid glands (e.g. primary hyperparathyroidism) due to their inadvertent removal or damage to the blood supply. Rarely, in children it may be related to radical neck dissection for malignancy. It should therefore be a routine that plasma calcium is estimated following thyroidectomy in children. Hypoparathyroidism may also be a complication of iron deposition in the parathyroid glands in children with thalassaemia major who receive repeated blood transfusions. This usually presents in the second decade often in conjunction with other end organ complications such as hypogonadism and diabetes mellitus. It has also been reported as a rare complication of Wilson’s disease due to copper deposition or iodine-131 therapy for thyroid disease.

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Autoimmune hypoparathyroidism can be either isolated or as part of the autoimmune polyendocrinopathy type 1 syndrome (APS1) (#240300) [12]. This latter condition may be sporadic or familial with an autosomal-recessive mode of inheritance. There is a triad of principal features, mucocutaneous candidiasis, hypoparathyroidism and adrenal insufficiency. Ectodermal dystrophy of the nails is often present leading to the alternative term APECED (autoimmune polyendocrinopathy-candidiasisectodermal dystrophy) for this syndrome. It usually presents in early childhood with mucocutaneous candidiasis (mean age 5 years) with hypoparathyroidism (mean age 9 years) being the earliest endocrine manifestation with subsequent development of adrenal insufficiency (mean age 14 years). Additional autoimmune features that may occur include malabsorption, chronic active hepatitis, thyroid disease, hypogonadism and diabetes mellitus. Antibodies against the parathyroid, thyroid and adrenal glands are present in many patients. The underlying genetic defect is due to mutations in the autoimmune regulator (AIRE) gene on Chromosome 21q22.3 (*607358) [13]. There are reports of affected individuals who have not been recognised to have this condition dying of adrenal insufficiency. It is therefore important that all children with apparent idiopathic acquired hypoparathyroidism have regular assessments of adrenal function. In affected children with APECED who are receiving treatment for hypoparathyroidism the onset of hypercalcaemia may be the first manifestation of adrenal insufficiency due to volume depletion as a consequence of mineralocorticoid deficiency. It is also important to be aware that many affected individuals have functional hyposplenism and are more vulnerable to pneumococcal infections and therefore should be immunised with Pneumovax [14].

Hypomagnesaemia

A rare but important cause of hypocalcaemia in a child is a low plasma magnesium. Magnesium is essential for PTH secretion and activation of the PTH receptor by ligand. There is an inadequate PTH response to hypocalcaemia in the presence of hypomagnesaemia which is corrected when the plasma magnesium is brought back into the normal range. This may present as a congenital defect with neonatal hypocalcaemia or may be an acquired defect in an older child. The causes for hypomagnesaemia can be broadly divided into those affecting intestinal magnesium absorption and those producing an excessive leak of magnesium from the renal tubules. In the context of a child with hypocalcaemia secondary to a low plasma magnesium, the easiest way to distinguish between these two possible causes is to assess urinary magnesium excretion with a urine magnesium/creatinine ratio on a spot urine sample for which normative paediatric data are available [3]. The normal response in the face of hypomagnesaemia is for the kidneys to reabsorb as much magnesium as possible and therefore a high magnesium/creatinine ratio would point to a renal tubular leak. Hypercalciuria is also present in some of these conditions and this is not always associated with hypermagnesuria.

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Hypomagnesaemia with Hypomagnesuria

Hypomagnesaemia with secondary hypocalciuria (HOMG1) (#602014) is caused by a selective defect of magnesium absorption in the small intestine with no evidence of any additional malabsorption. Affected infants present with hypocalcaemic fits in the first few weeks of life and are found to have remarkably low plasma magnesium values of <0.5 mmol/l [15]. They may initially require parenteral magnesium treatment and can then be maintained on an oral magnesium preparation which will keep the plasma calcium in the normal range despite the fact that the plasma magnesium remains subnormal at around 0.5 mmol/l (see chapter 15, case 5). The inheritance of this condition appears to be autosomal recessive and often occurs in consanguineous families. The genetic defect has been identified as due to mutations in TRPM6 on Chromosome 9q22 (*607009) which is expressed in intestinal epithelia and renal tubules [16] (see chapter 2 for further details). Renal calcium excretion is low in this condition.

Hypomagnesaemia with Hypermagnesuria

Associated with Hypocalciuria Hypomagnesaemia with associated hypocalciuria (HOMG2) (#154020) is the other main congenital form of hypomagnesaemia. The hypomagnesaemia is associated with isolated renal magnesium loss with high urinary magnesium excretion. This is an autosomal-dominant disorder due to mutations in the FXYD2 gene on Chromosome 11q23 [17] (*601814). This gene codes for the γ-subunit of the Na+/K+-ATPase on the inner membrane of the renal tubule and causes hypomagnesaemia with reduced urinary calcium excretion. The clinical manifestations are generally milder than those of HOMG1 and may not become apparent until adulthood. Isolated recessive renal hypomagnesaemia (IRH, HOMG4) (#611718) is a rare autosomal-recessive condition caused by a mutation in the epidermal growth factor (EGF) gene (*131530) which controls magnesium reabsorption via TRPM6 (see chapter 2 for details). Isolated hypomagnesaemia is associated with normal plasma and urine calcium, but the patients have psychomotor retardation and seizures with brisk reflexes, presumably as a result of other effects of EGF. Gitelman syndrome (#263800) has some overlap with the Bartter syndrome although it is a separate entity. Mutations in the thiazide-sensitive sodium chloride transporter (SLC12A3) (*600968) result in hypokalaemic alkalosis with salt wasting, hypomagnesaemia and hypocalciuria. Patients usually present after the age of five years with episodes of muscle weakness, lethargy, tetany and muscle cramps. Dermatitis may be present and, although Gitelman’s is described as being benign, a prolonged cardiac Q-T interval may give rise to arrhythmias and syncopal attacks. Chondrocalcinosis is a feature which these patients share with others with chronic

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hypomagnesaemia. Urinary calcium excretion is low. Treatment consists of correcting the biochemical abnormalities, particularly the potassium and magnesium deficiencies, with oral supplementation.

Associated with Hypercalciuria Hypermagnesuria with hypercalciuria and nephrocalcinosis (HOMG3) (#248250) is caused by mutations in the gene for claudin 16 (Paracellin 1) (*603959), which is located in the tight junctions of the epithelium of ascending loop of Henle. As a consequence, there is excessive excretion of both magnesium and calcium. Several different homozygous or compound heterozygous mutations have been described and the severity of the condition varies according to genotype. In some cases the problem is self-limiting whilst in others renal failure may ensue. Hypocalcaemia is occasionally present. Renal hypomagnesaemia with ocular involvement (#248190) is also autosomal recessive and is similar to HOMG3 but also includes ocular abnormalities such as coloboma, myopia and horizontal nystagmus. No mutations are found in the Claudin 16 gene but they are found in the similar claudin 19 gene (*610036). This is located mainly in the collecting ducts of the renal tubule.

Acquired Hypomagnesaemia

Hypomagnesaemia may also occur as a consequence of either malabsorption, as in Crohn’s disease or Whipple’s disease, or as an acquired renal defect secondary to certain drugs inducing excess renal magnesium wasting. These include the drugs cisplatinum, amphotericin B, cyclosporin and tacrolimus. Severe burn injury has also been reported to cause hypocalcaemia secondary to hypomagnesaemia [18].

Hypocalcaemia with Normal Parathyroid Hormone

Familial Hypocalcaemia with Hypercalciuria Individuals with this condition, autosomal-dominant hypocalcaemia (ADH) (included in #146200) were often previously felt to have idiopathic hypoparathyroidism with a serum PTH that was inappropriately normal in the face of hypocalcaemia. Then linkage analysis in large families with autosomal-dominant hypoparathyroidism mapped a candidate gene to a locus on chromosome 3q13 which corresponded to the region known to include the gene coding for the calcium-sensing receptor. Subsequently, it was identified that this condition was due to gain of function mutations in the gene for the calcium-sensing receptor [19] (*603959). Most of these mutations alter the set point

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of the calcium-sensing receptor in the parathyroid glands and the kidneys leading to a lower plasma calcium prior to PTH release and a lowered tubular calcium reabsorption causing relative hypercalciuria (see chapter 2 for details). One mutation causes constitutive activation of the CaSR that prevents PTH secretion even at low calcium concentrations [20]. A low plasma magnesium is often also present in this condition. Many subjects with this condition are asymptomatic but some individuals, particularly children during febrile episodes or neonates, exhibit hypocalcaemic symptoms particularly seizures and neuromuscular irritability. Treatment should be reserved for symptomatic subjects only as there is a significant risk of hypercalciuria and nephrocalcinosis. Although the condition is rare it may account for a significant proportion of cases of idiopathic hypoparathyroidism. In one study of 19 unrelated cases of isolated hypoparathyroidism in France, 8 subjects (42%) had activating mutations of the calcium-sensing receptor [21]. Forty-four mutations have now been described with the majority present in the extracellular or transmembrane domains. A web site is maintained to keep track of these mutations (http://www.casrdb.mcgill.ca/). Thiazide diuretics have been used with effect to reduce the hypercalciuria in treated individuals. An alternative option to consider if treatment is required is the use of synthetic PTH which has been used with apparent benefit in 1 child [22] (see chapter 15, cases 6 and 7).

Antibodies to the Calcium-Sensing Receptor

A biochemical picture similar to ADH has also been reported in individuals who have been found to have antibodies to the calcium-sensing receptor often in the context of other autoimmune disease [23]. In at least one individual the hypocalcaemia was transient.

Hypocalcaemia with a High Parathyroid Hormone

Pseudohypoparathyroidism This condition was first reported in 1942 and was the first example of hormone resistance identified in man [24]. It has similar biochemical features to hypoparathyroidism with hypocalcaemia and hyperphosphataemia with the exception that serum levels of PTH are elevated. Pseudohypoparathyroidism (PHP) refers to several distinct but related disorders in which resistance to PTH is the predominant feature. Unlike most hormone-resistant conditions, the defect is not in the PTH receptor but in the signalling protein Gsα which is downstream of many different G protein coupled hormone receptors and acts by stimulating the production of adenyl cyclase which activates the second-messenger cyclic AMP. Resistance to the action of PTH in this condition appears to be mainly in the proximal renal tubule and therefore other

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actions of PTH, e.g. on bone, are unaffected. Thus affected individuals can have normal levels of plasma calcium for many years despite elevated PTH levels. It will often present in mid childhood with hypocalcaemic fits or muscle spasms. PHP is subdivided into two types dependent on the renal tubular response to infused exogenous PTH. Type I is where there is blunting of both cyclic AMP generation and urinary phosphate excretion whereas type II demonstrates impaired urinary phosphate excretion only. The category of type I PHP is subdivided into PHP types Ia, Ib and Ic. PHP Type Ia (#103580) In this subtype affected individuals, in addition to PTH resistance, have features of Albright’s hereditary osteodystrophy (AHO), which is a constellation of physical features including round face, truncal obesity, short stature, shortening of the 4th and 5th metacarpals and metatarsals, heterotopic ossification and/or mental retardation. Evidence of additional hormone resistance, particularly hypothyroidism and hypogonadism, is usually present and there may also be evidence of growth hormone deficiency due to resistance at the GHRH receptor. The underlying genetic defect is due to heterozygous mutations in the gene GNAS on chromosome 20q13.2 (+139320) which encodes for the Gsα subunit [25]. Skin fibroblasts and erythrocytes from affected individuals have a 50% reduction in Gsα mRNA. A related disorder is pseudopseudohypoparathyroidism (PPHP) (#612463) in which individuals have AHO features but no evidence of hormone resistance. Heterozygous mutations in the GNAS gene are also present and affected individuals can be found in the same kindreds as those with PHP type Ia. The phenotype expressed is dependent on the gender of the parent transmitting the gene defect with paternal transmission causing PPHP and maternal transmission PHP type Ia [26]. This is due to the imprinted nature of the Gsα protein in different tissues with the maternal allele being predominantly expressed in the proximal renal tubules, thyroid gland, gonads and pituitary. Therefore, an individual carrying a maternally transmitted Gsα mutation will exhibit hormone resistance but an individual carrying a paternally expressed mutation, which is not expressed in the proximal renal tubule, will have no hormone resistance as only the maternally expressed allele, which is normal, is expressed (fig. 2). PHP Type Ib (#603233) This second subtype of type I pseudohypoparathyroidism is characterised by PTH resistance in the absence of AHO features. Additional hormone actions such as those of TSH can also be impaired. Although most cases are sporadic, familial cases with autosomal-dominant inheritance are reported. The severity of the condition can vary significantly. As in PHP type Ia PTH resistance only occurs when there is maternal transmission. However, in contrast, mutations in the GNAS gene are not present in the majority of subjects with PHP type Ib although the disease gene maps to a region on chromosome 20q containing the GNAS locus. Current evidence indicates that the

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p

Hormone resistance

m

Fig. 2. Schematic representation of imprinting of Gsα mutation expression in renal tubules (adapted from Wilson et al. [26]). The black square represents a Gsα mutation.

p m

Normal hormone response

most likely cause is mutations in regulatory regions of the GNAS gene inherited from the mother that interfere with parent specific methylation of the gene [27]. PHP Type Ic (#612462) These patients have the characteristic features of AHO with multiple hormone resistance but have not been shown to have mutations in the GNAS gene. However, it is not clear whether or not this is a separate entity. PHP Type II (%203330) This is a rarely reported condition with as yet no genetic basis. These patients do not have features of AHO and the resistance to PTH is confined to the phosphaturic response with a normal cyclic AMP response. It has been pointed out that a biochemical picture similar to this condition can be seen in severe vitamin D deficiency with hypocalcaemia and a high plasma phosphate despite an elevated serum PTH suggesting renal resistance to PTH [28]. As these abnormalities rapidly respond to administration of vitamin D, it has to be speculated as to whether PHP type II actually exists or may just be another manifestation of vitamin D deficiency. The genetic conditions relating to hypoparathyroidism and pseudohypoparathyroidism are summarised in table 4.

Disorder of Vitamin D Supply or Metabolism

Vitamin D Deficiency Although vitamin D deficiency in children will usually present with rickets, symptomatic hypocalcaemia may also be the first manifestation. This is particularly the case during infancy and puberty where the rapid growth that characterises these periods may be responsible for increased requirements for calcium for bone mineralisation

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Table 4. Summary of genetic causes of hypocalcaemia Disorder

Abbreviation OMIM

Conditions associated with low PTH levels Familial Isolated FIH hypoparathyroidism

Other forms of familial hypoparathyroidism 22q deletion (Di George) DGS1 syndrome Di George-like syndrome DGS2 Hypoparathyroid sensorineural HDR deafness renal anomalies Hypoparathyroid mental and HRD growth retardation Kenneydysmorphic features Caffey SanjadSakati Mitochondrial disorders Progressive external ophthalmoplegia, heartblock or cardiomyopathy (Kearns-Sayre) Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes Mitochondrial trifunctional protein deficiency syndrome Autoimmune polyendocrine syndrome type 1

Gene

OMIM

Gene location

168450.0001

AD

*168450

11p15.3-p15.1

168450.0002 168450.0003 %307700

AR AR XLR deletion/ insertion AR (usually)

prepro PTH PTH PTH ?SOX3

*168450 *168450 *313430

11p15.3-p15.1 11p15.3-p15.1 Xq27.1/2p25.3

GCMB (GCM2)

*603716

6p24.2

TBX1

*602054

22q11.2

#188400

AD but mostly sporadic

#146255

AD

? GATA3

*131320

10p13 10p15

#241410 #244460

AR

TBCE

*604934

1q42-q43

KSS

#530000

mitochondrial

MELAS

#540000

mitochondrial

MTPDS

#609015

mitochondrial

APS1 APECED

#240300

AR

AIRE

*607358

21q22.3

#602014

AR

TRPM6

*607009

9q22

#154020

AD

#611718

AR

FXYD2 (Na/ *601814 K-ATPase) EGF *131530

Hypomagnesaemia with hypomagnesuria Hypomagnesaemia with HOMG1 secondary hypocalciuria Hypomagnesaemia with hypermagnesuria Hypomagnesaemia with HOMG2 associated hypocalciuria Isolated recessive renal IRH hypomagnesaemia HOMG4

Hypocalcaemia

Inheritance

11q23 4q25

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Table 4. Continued Disorder

Abbreviation OMIM

Inheritance

Gene

OMIM

Gene location

#263800

AR

*600968

16q13

#248250

AR

thiazidesensitive NaCl transporter (SLC12A3) claudin 16

*603959

3q27

#248190

AR

claudin 19

*610036

1p34.2

Hypocalcaemia with normal PTH Autosomal-dominant ADH hypocalcaemia

#146200

AD

CaSR

+601199

3q13.3-q21

Hypocalcaemia with raised PTH Pseudohypoparathyroidism type Ia

PHP1A

#103580

GNAS complex

+139320

20q13.2

Pseudohypoparathyroidism type Ib

PHP1B

#603233

AD (maternally inherited) or sporadic AD (maternally inherited)

Pseudohypoparathyroidism type Ic Pseudopseudohypoparathyroidism Pseudohypoparathyroidism type II

PHP1C

#612462

PPHP

#612463

PHP2

%203330

Gitelman syndrome

Hypermagnesuria with hypercalciuria and nephrocalcinosis Renal hypomagnesaemia with ocular involvement

HOMG3

?AD maternally inherited AD (paternally inherited) ?

GNAS +139320 complex methylation defects ?GNAS complex GNAS +139320 complex ? ?

20q13.32, 20q13.2, 20q13.2

20q13.2 ?

The OMIM numbers of the various conditions and those of their related genes, where known, are shown in columns 3 and 6, respectively. The chromosomal locations of the various genes are shown in column 7.

and longitudinal growth [29]. It is often the case in these groups that radiological evidence of rickets is absent at the time of presentation. Another phenomenon often seen in these age groups is the presence of a high plasma phosphate despite a high circulating level of PTH. This would imply that there is resistance to the action of PTH in the renal tubules. There is evidence to suggest that this occurs as a consequence of dietary calcium deficiency which corrects when adequate calcium intake is supplied [30]. Any of the additional forms of ‘calciopaenic’ rickets caused by defects in Vitamin D metabolism or action can have hypocalcaemia as a feature. These are described in more detail in chapter 8.

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Chronic renal failure and chronic liver disease can also present with a biochemical picture of hypocalcaemia with a high circulating level of serum PTH. In the former hypocalcaemia is a consequence of the failure of adequate synthesis of 1,25(OH)2D and the rise in plasma phosphate due to failure to excrete a phosphate load. This is usually managed by the use of a phosphate binder such as calcium carbonate in conjunction with a vitamin D analogue such as alfacalcidol. In chronic liver disease, hypocalcaemia occurs in combination with rickets. It is primarily because of malabsorption of vitamin D and calcium rather than a failure of activity of the 25-hydroxylase enzyme in the liver which only occurs with end stage liver failure. This is often treated with a vitamin D analogue such as alfacalcidol or calcitriol.

Osteopetrosis

A rare but important cause of hypocalcaemia presenting in the neonatal period is infantile osteopetrosis [31]. In this condition there is a failure of osteoclast action and therefore a lack of bone resorption. There have been several reports of infants with this condition who initially present with neonatal hypocalcaemic convulsions indicating the importance of bone resorption to maintain adequate levels of plasma calcium in the neonatal period. Affected infants usually have raised levels of serum PTH which is unable to produce bone resorption due to the osteoclast defect as another form of PTH resistance. An X-ray of a wrist or knee will demonstrate the characteristic dense bones. It is an important condition to diagnose early as preservation of eyesight is dependent on the timing of diagnosis and the availability of bone marrow transplantation (see chapter 12 for a more detailed description of the various forms of osteopetrosis).

Treatment

The treatment of hypocalcaemia is dependent on two factors: 1 whether the hypocalcaemia is causing severe symptoms such as convulsions, and 2 the underlying cause for the hypocalcaemia. If urgent correction of the plasma calcium is required, an intravenous bolus of 10% calcium gluconate 0.5 ml/kg (0.11 mmol/kg) to a maximum of 20 ml/kg over 5–10 min should be administered, followed by a continuous intravenous infusion over 24 h of 1.0 mmol/kg (maximum 8.8 mmol). It is important to note that extravasation of intravenous calcium can cause a considerable reaction and subsequent scarring in the skin and subcutaneous tissues and therefore the infusion site should be regularly checked. In addition, once the severe symptoms have resolved, the intravenous route should be discontinued in favour of oral calcium supplements. For a child where urgent correction of the plasma calcium is not required oral calcium

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supplements 0.2 mmol/kg to a maximum of 10 mmol should be administered four times daily. In the management of hypoparathyroidism or pseudohypoparathyroidism the use of a vitamin D analogue such as 1α-hydroxyvitamin D (alphacalcidol) or 1,25(OH)2D3 (calcitriol) in a dose of 25–50 ng/kg/day is the most appropriate way of increasing intestinal calcium absorption. The aim is to maintain the plasma calcium at the lower end of the normal range between 2.0 and 2.2 mmol/l as the renal calcium reabsorption will be low in these conditions due to the lack of action of PTH with the risk of hypercalciuria. Monitoring should include periodic assessment of a urine calcium/creatinine ratio and renal ultrasonography to detect nephrocalcinosis. In theory recombinant PTH could be used instead of a vitamin D analogue but would require daily parenteral injection and therefore the oral route is preferred. Hypomagnesaemia will usually respond to an oral magnesium supplement such as magnesium glycerophosphate in a dose of 0.2 mmol/kg three times daily. The use of oral magnesium salts is sometimes limited by diarrhoea. If hypomagnesaemic symptoms are severe and do not respond to oral magnesium supplements, intramuscular injection of a 50% solution of magnesium sulphate (MgSO4.7H2O) can be given. This contains 2 mmol/ml. Vitamin D deficiency should be treated with ergocalciferol (D2) or colecalciferol (D3) as the fastest way to replenish deficient stores of 25OHD. There is a liquid preparation suitable for infants and young children containing 3,000 units per ml. A dose of 3,000 units daily for infants less than 6 months and 6,000 units daily for age 6 months to 12 years given for an initial period of 6 weeks is often adequate. In the adolescent with hypocalcaemia secondary to vitamin D deficiency there is a capsule of ergocalciferol containing 10,000 units. Calcium supplements may also be required in the initial management of these disorders until the plasma calcium has returned to the normal range.

Conclusions

As can be seen from this chapter, there are many different disorders that can cause hypocalcaemia in a child. It is important that all the appropriate investigations are undertaken prior to the initiation of any treatment. A stepwise logical approach in the interpretation of the relevant investigations will often lead to the correct diagnosis. Some of these conditions are congenital in origin, e.g. congenital hypoparathyroidism, infantile osteopetrosis and are likely to present early in life, whereas some of the conditions are acquired, e.g. vitamin D deficiency, autoimmune polyendocrinopathy and can therefore present at any time during childhood or adolescence. Some of the conditions have important associated features, e.g. adrenal insufficiency and hyposplenism in autoimmune polyendocrinopathy which need to be watched for in the long-term management of the condition. Finally, many of the disorders have a genetic

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basis which has now been identified and therefore it is important, once a provisional diagnosis is made, to undertake genetic studies so as to confirm the suspected diagnosis and to inform appropriate genetic counselling.

References 1 Fonseca OA, Calverley JR: Neurological manifestations of hypoparathyroidism. Arch Intern Med 1967;120:202–206. 2 Schaaf M, Payne CA: Effect of diphenylhydantoin and phenobarbital on overt and latent tetany. N Engl J Med 1966;274:1228–1233. 3 Shaw NJ, Wheeldon J, Brocklebank JT: Indices of intact serum parathyroid hormone and renal excretion of calcium, phosphate, and magnesium. Arch Dis Child 1990;65:1208–1211. 4 Parkinson DB, Thakker RV: A donor splice site mutation in the parathyroid hormone gene is associated with autosomal recessive hypoparathyroidism. Nat Genet 1992;1:149–152 5 Datta R, Waheed A, Shah GN, Sly WS: Signal sequence mutation in autosomal dominant form of hypoparathyroidism induces apoptosis that is corrected by a chemical chaperone. Proc Natl Acad Sci USA 2007;104:19989–19994. 6 Ding C, Buckingham B, Levine MA: Familial isolated hypoparathyroidism caused by a mutation in the gene for the transcription factor GCMB. J Clin Invest 2001;108:1215–1220. 7 Bowl MR, Nesbit MA, Harding B, et al: An interstitial deletion-insertion involving chromosomes 2p25.3 and Xq27.1, near SOX3, causes X-linked recessive hypoparathyroidism. J Clin Invest 2005; 115:2822–2831. 8 Taylor SC, Morris G, Wilson D, Davies SJ, Gregory JW: Hypoparathyroidism and 22q11 deletion syndrome. Arch Dis Child 2003;88:520–522. 9 Van Esch H, Groenen P, Nesbit MA, et al: GATA3 haplo-insufficiency causes human HDR syndrome. Nature 2000;406:419–422. 10 Parvari R, Hershkovitz E, Grossman N, et al: Mutation of TBCE causes hypoparathyroidismretardation-dysmorphism and autosomal recessive Kenny-Caffey syndrome. Nat Genet 2002;32:448– 452. 11 Poomthavorn P, Ongphiphadhanakul B, Mahachoklertwattana P: Transient neonatal hypoparathyroidism in two siblings unmasking maternal normocalcemic hyperparathyroidism. Eur J Pediatr 2008;167:431–434. 12 Perheentupa J: Autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy. J Clin Endocrinol Metab 2006;91:2843–2850.

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13 Pearce SH, Cheetham T, Imrie H, Vaidya B, Barnes ND, Bilous RW, et al: A common and recurrent 13-bp deletion in the autoimmune regulator gene in British kindreds with autoimmune polyendocrinopathy type 1. Am J Hum Genet 1998;63:1675–1684. 14 Pearce SH, Cheetham TD: Autoimmune polyendocrinopathy syndrome type 1: treat with kid gloves. Clin Endocrinol (Oxf) 2001;54:433–435. 15 Shalev H, Phillip M, Galil A, Carmi R, Landau D: Clinical presentation and outcome in primary familial hypomagnesaemia. Arch Dis Child 1998; 78:127–130. 16 Schlingmann KP, Weber S, Peters M, et al: Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat Genet 2002;31:166– 170. 17 Meij IC, Koenderink JB, van Bokhoven H, Assink KF, Groenestege WT, de Pont JJ, et al: Dominant isolated renal magnesium loss is caused by misrouting of the Na(+),K(+)-ATPase gamma-subunit. Nat Genet 2000;26:265–266. 18 Klein GL, Nicolai M, Langman CB, Cuneo BF, Sailer DE, Herndon DN: Dysregulation of calcium homeostasis after severe burn injury in children: possible role of magnesium depletion. J Pediatr 1997;131:246– 251. 19 Pearce SH, Williamson C, Kifor O, et al: A familial syndrome of hypocalcemia with hypercalciuria due to mutations in the calcium-sensing receptor. N Engl J Med 1996;335:1115–1122. 20 Zhao XM, Hauache O, Goldsmith PK, Collins R, Spiegel AM: A missense mutation in the seventh transmembrane domain constitutively activates the human Ca2+ receptor. FEBS Lett 1999;448:180–184. 21 Lienhardt A, Bai M, Lagarde JP, et al: Activating mutations of the calcium-sensing receptor: management of hypocalcemia. J Clin Endocrinol Metab 2001;86:5313–5323. 22 Mittelman SD, Hendy GN, Fefferman RA, et al: A hypocalcemic child with a novel activating mutation of the calcium-sensing receptor gene: successful treatment with recombinant human parathyroid hormone. J Clin Endocrinol Metab 2006;91:2474– 2479.

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23 Kifor O, McElduff A, LeBoff MS, et al: Activating antibodies to the calcium-sensing receptor in two patients with autoimmune hypoparathyroidism. J Clin Endocrinol Metab 2004;89:548–556. 24 Albright FBC, Smith PH, Parson W: Pseudohypoparathyroidism: an example of ‘Seabright-Bantam syndrome’. Endocrinology 1942;30:922–932. 25 Weinstein LS, Gejman PV, Friedman E, Kadowaki T, Collins RM, Gershon ES, et al: Mutations of the Gs alpha-subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA 1990;87: 8287–8290. 26 Wilson LC, Oude Luttikhuis ME, Clayton PT, Fraser WD, Trembath RC: Parental origin of Gs alpha gene mutations in Albright’s hereditary osteodystrophy. J Med Genet 1994;31:835–839. 27 Bastepe M, Juppner H: GNAS locus and pseudohypoparathyroidism. Horm Res 2005;63:65–74.

28 Rao DS, Parfitt AM, Kleerekoper M, Pumo BS, Frame B: Dissociation between the effects of endogenous parathyroid hormone on adenosine 3⬘,5⬘-monophosphate generation and phosphate reabsorption in hypocalcemia due to vitamin D depletion: an acquired disorder resembling pseudohypoparathyroidism type II. J Clin Endocrinol Metab 1985;61:285– 290. 29 Ladhani S, Srinivasan L, Buchanan C, Allgrove J: Presentation of vitamin D deficiency. Arch Dis Child 2004;89:781–784. 30 Khadikar AHH, Sayyad M, Sanwalka N, Khadikar V, Mughal MZ: Oral calcium supplementation reverses the biochemical picture of parathyroid hormone resistance in underprivileged Indian toddlers. Arch Dis Child 2008;93(suppl 1):A1. 31 Srinivasan M, Abinun M, Cant AJ, Tan K, Oakhill A, Steward CG: Malignant infantile osteopetrosis presenting with neonatal hypocalcaemia. Arch Dis Child Fetal Neonatal Ed 2000;83:F21–F23.

Dr. N.J. Shaw Department of Endocrinology, Birmingham Children’s Hospital Steelhouse Lane Birmingham B6 4NH (UK) Tel. +44 0121 333 8189, Fax +44 0121 333 8191, E-Mail [email protected]

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Chapter 7 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 93–114

A Practical Approach to Problems of Hypercalcaemia Justin H. Davies Department of Child Health, Southampton University Hospital Trust, Southampton, UK

Abstract Hypercalcaemia is a rarer problem in children than that of hypocalcaemia. However, when it does occur, it is a condition that requires proper diagnosis before correct treatment can be instituted. Problems may arise either because of excess PTH secretion, e.g. because of parathyroid tumour, or because of inactivating mutations of the calcium-sensing receptor or because some other factor, such as vitamin D or PTHrP, causes hypercalcaemia independently of PTH. In the latter instance, PTH secretion is suppressed. It is often useful to get a clue to the aetiology by examining the urine calcium concentration as this may guide one towards the correct diagnosis. Treatment is aimed at either removing the source of the excess PTH or whichever other factor is involved. In some cases treatment is not necessary as the hypercalcaemia remains asymptomatic and does not cause any problems. If the underlying problem cannot be treated directly, measures can often be taken to reduce the plasma calcium by medical means which can sometimes be used as an interim measure Copyright © 2009 S. Karger AG, Basel before definitive treatment is undertaken.

Children present with hypercalcaemia much less frequently than do adults. Hypercalcaemia is also, in contrast to adults, less common a problem than that of hypocalcaemia. Nevertheless, when it does occur, it can be a serious condition which requires correct diagnosis in order to provide appropriate treatment. The commonest causes of hypercalcaemia in adulthood are malignancy and primary hyperparathyroidism. In children, the causes are more diverse and, particularly in neonates and infancy, are often exclusive to that age range. When considering a practical approach to these problems the causes can most conveniently be divided according to the accompanying levels of parathyroid hormone (PTH).

Steady State Calcium Homeostasis

For a detailed discussion of calcium homeostasis, see chapter 2. The circulating concentration of calcium is initially sensed by a calcium-sensing receptor (CaSR). As a

result, a cascade of events is established that includes secretion of PTH, which acts via a second-messenger system on the target organs, the principal ones of which are bone and kidney. Abnormalities in any part of this system can cause hypercalcaemia. In addition, abnormalities of vitamin D supply or metabolism can also cause hypercalcaemia. PTHrP occasionally causes hypercalcaemia but the fourth hormone involved in calcium metabolism, calcitonin, which has a hypocalcaemic effect, is not thought to cause such problems. Extracellular calcium homeostasis is required for normal muscle and nerve function, bone quality and blood coagulation. Disturbances in the plasma concentration of calcium cause symptoms that are principally related to changes in these functions. In general, hypercalcaemia leads to a reduction in nerve and muscle activity. The extracellular CaSR is a G-protein coupled receptor consisting of a large extracellular domain and a seven transmembrane-spanning region with an intracellular C-terminal tail. Ligand-binding results in a conformational change of the intracellular loops and activation of phospholipase C, resulting in increased intracellular free calcium which activates protein kinase C activity. The CaSR is a critical regulator for the maintenance of constant serum calcium levels and acts by inhibiting PTH secretion and promoting renal calcium excretion in response to increased calcium levels. It is expressed in many cells that participate in calcium homeostasis including C cells of the parathyroid, kidney cells, osteoblasts and intestinal cells. The main systemic regulators of calcium homeostasis are PTH, 1,25(OH)2D and, to a lesser extent, calcitonin. PTHrP may contribute to local calcium levels at the cellular level. Normally, PTH stimulates release of calcium from bone, promotes reabsorption of calcium at the loop of Henle and distal tubule in the kidney and catalyses conversion of 25OHD to 1,25(OH)2D by stimulating renal 1α-hydroxylase activity. Primary hyperparathyroidism therefore causes hypercalcaemia via all these mechanisms. It also lowers serum phosphate levels by inhibiting renal phosphate reabsorption. 1,25(OH)2D promotes hypercalcaemia by a number of different mechanisms. It increases calcium and phosphate absorption from the intestine. It also increases mineralisation of bone, possibly by increasing intracellular transport of calcium ions and by increasing circulating concentrations of mineralisation substrates calcium and phosphate. 1,25(OH)2D increases calcium reabsorption in the distal tubule of the kidney. 1,25(OH)2D levels are reduced during hyperphosphataemia and by corticosteroids. Hypercalcaemia resulting from increases in 1,25(OH)2D occurs independently of PTH which is therefore suppressed. PTHrP is made by a variety of tissues and acts in a paracrine and autocrine manner to regulate local tissue calcium concentrations. Evidence is accumulating that it has a significant role in calcium homeostasis during foetal life and is involved in placental calcium transport and foetal chondrocyte maturation. PTHrP may also have a role in mobilising calcium from maternal bones during lactation. Its actions are mediated

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through the PTH/PTHrP type 1 receptor and its principal clinical significance is as a mediator of humoral hypercalcaemia of malignancy.

Pathophysiology of Hypercalcaemia

In the setting of a rise in plasma calcium in an individual with normal regulatory mechanisms, hypercalcaemia suppresses the secretion of PTH. PTH maintains calcium levels but only in the narrow range of serum calcium levels from 2.1–2.6 mmol/l. Levels above or below this range are relatively ineffective at further stimulating or suppressing PTH and rely on direct exchange of calcium between bone and extracellular fluid. When PTH is suppressed because of hypercalcaemia, levels of 1,25(OH)2D decline and intestinal calcium absorption is reduced [1]. Other factors leading to hypercalcaemia include metabolic alkalosis, which causes increased tubular calcium reabsorption. Phosphate-induced hypocalcaemia may increase PTH and cause hypercalcaemia. Stimulation of osteoclasts by cytokines, such as TNF-α, IL-1, and IL-6 may also lead to hypercalcaemia.

Clinical Features and Clinical Evaluation of Hypercalcaemia

These are shown in tables 1 and 2. There is often an insidious onset. When severe, the predominant symptoms are hypotonia from myopathy, features of dehydration and polyuria secondary to calcium-induced renal AVP resistance.

Investigation of Hypercalcaemia

Many children are not fully investigated for hypercalcaemia [2]. The underlying diagnosis must be determined to initiate the appropriate therapy. The investigations for hypercalcaemia are shown in table 3 and should be taken at the time of hypercalcaemia [3].

Specific Conditions Leading to Hypercalcaemia during Childhood

When considering the causes of hypercalcaemia, it is useful to divide them into those that are associated with either high, normal or suppressed levels of PTH. Thus, for instance, primary and tertiary hyperparathyroidism are associated with raised PTH, CaSR abnormalities generally have normal PTH values, whereas PTH levels are suppressed in those conditions resulting from vitamin D excess. The genetic conditions leading to hypercalcaemia are shown in table 4 and the secondary causes in table 5.

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Table 1. Symptoms most commonly associated with hypercalcaemia Nervous system Behavioural disturbance/personality changes Malaise Headache Hallucinations Unsteady gait Proximal muscle weakness/generalised myopathy Irritability/confusion Gastrointestinal system Paralytic ileus leading to: Abdominal cramping Constipation Anorexia Nausea Vomiting Pancreatitis secondary to ectopic calcification Renal system Dehydration Renal stones Polyuria and polydipsia Renal failure Musculoskeletal system Bone pain Ectopic calcification Miscellaneous Conjuctivitis Band keratopathy

Hypercalcaemia Associated with Raised Parathyroid Hormone

Primary Hyperparathyroidism Primary hyperparathyroidism during childhood is rare and the incidence is unknown. It is characterised by autonomous secretion of PTH, independent of circulating calcium levels, either due to parathyroid hyperplasia or adenoma. It occasionally leads to symptomatic hypercalcaemia in children whereas in adults it is a much more frequent cause of hypercalcaemia. In the largest paediatric series of over 20 years’ experience, 55 cases were identified in France [4]. Of these, 11 (20%) presented in the neonatal period, and the remaining 44 (80%) presented during adolescence. Clinical signs at presentation were usually related to nephrolithiasis. Bone pain, arthropathy

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Table 2. Clinical assessment of hypercalcaemia History Symptoms suggestive of hypercalcaemia Symptoms suggestive of malignancy Drug history, including vitamin D therapy, complementary alternative medicine, supplements Family history of renal stone, hypercalcaemia, parathyroidectomy, multiple endocrine neoplasia Examination Assess degree of dehydration Syndromic features Presence of ectopic/subcutaneous calcification/rash Short stature/disproportionate short stature Generalised lymphadenopathy, organomegaly Bone pain, fractures

Table 3. Investigations undertaken in hypercalcaemia Initial investigations at the time of hypercalcaemia Calcium Phosphate Alkaline phosphatase Electrolytes and creatinine PTH 25-Hydroxyvitamin D If appropriate, store serum for consideration of measurement at later date for: 1,25(OH)2D PTHrP DNA for genetic analysis Urine calcium:creatinine ratio Subsequent investigations to be considered Renal ultrasound scan Investigation of parents for abnormalities of calcium homeostasis Skeletal survey Parathyroid gland ultrasound scan Parathyroid gland SestaMIBI scan

and muscle aches induced gait abnormalities. Hypercalcaemia at presentation was a constant feature with a mean calcium of 3.2 mmol/l (range 2.8–4.3). The plasma PTH was often elevated but sometimes normal. Hypercalciuria was common (71%). Nephrocalcinosis occurred in 20% and renal insufficiency in 5%.

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Table 4 Genetic causes of hypercalcaemia Disorder

Abbreviation

Conditions associated with raised PTH levels Familial primary FIHP hyperparathyroidism (HRPT1) Multiple endocrine neoplasia MEN1 type I Multiple endocrine neoplasia MEN2A type IIA Multiple endocrine neoplasia MEN2B type IIB (no parathyroid (MEN3) involvement) Multiple endocrine neoplasia MEN2C type IIC (no parathyroid involvement) Multiple endocrine neoplasia MEN4 type IV Hyperparathyroid jaw-tumour HYP-JT syndrome (HRPT2) Familial isolated HRPT3 hyperparathyroidism Neonatal severe NSHPT hyperparathyroidism Parathyroid adenomas Parathyroid carcinoma Conditions associated with normal PTH levels Familial benign hypercalcaemia FBH1 (FHH) (familial hypocalciuric hypercalcaemia) type I Familial benign hypercalcaemia FBH2 (Familial Hypocalciuric Hypercalcaemia) Type II Familial benign hypercalcaemia FBH3 (familial hypocalciuric hypercalcaemia) type III Conditions associated with low PTH levels Williams-Beuren syndrome WBS Jansen’s metaphyseal chondrodysplasia Idiopathic hypercalcaemia of infancy

OMIM

Inheritance

Gene

OMIM

Gene location

#145000

AD

+131100

AD

MEN1, HRPT2, HRPT3 MEN1

131100.0020

11q13

#171400

AD

+164761

10q11.2

#162300

AD

Ret protooncogene Ret protooncogene

+164761

10q11.2

#155240

AD

Ret protooncogene

+164761

10q11.2

#610755

AD

CDKN1B

*600778

12p13

#145001

AD

HRPT2

*607393

1q25-q31

%610071

AD

?

#239200

AR

CaSR

+601199

3q13.3-q21

#608266

Somatic AD

PRAD1/PTH HRPT2

*168461 *607393

11q13 1q25-q31

#145980

AD

CaSR

+601199

3q13.3-q21

%145981

AD

?

19p13.3

%600740

AD

?

19q13

#194050

?AD

7q11.23

#156400

AD

elastin, LIM kinase, etc. PTH1R

143880

?

?

2p14-p13.3

*168468

3p22-p21.1

Each condition is shown with its gene (where known) and the chromosomal location of that gene. OMIM numbers are also shown for both condition and gene.

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Table 5. Causes of secondary hypercalcaemia Hypercalcaemia associated with acquired disease Malignancy Acute lymphoblastic leukaemia Acute myeloid leukaemia Ovarian dysgerminoma Hepatoblastoma Rhabdomyosarcoma Medulloblastoma Endocrine Thyrotoxicosis Addison’s disease Chronic renal failure Immobilisation Drug induced Vitamin D intoxication Vitamin A intoxication Thiazides Granulomatous disorders Subcutaneous fat necrosis of the newborn Sarcoidosis Tuberculosis Gestational maternal hypocalcaemia Renal tubular acidosis Phosphate depletion in severe prematurity Congenital hypothyroidism

Specific Causes of Hyperparathyroidism Familial Isolated Primary Hyperparathyroidism (#145000)

Any of the genetic causes of familial primary hyperparathyroidism (FIPH) may give rise to isolated disease. Thus, multiple endocrine neoplasia type 1 or the hyperparathyroid jaw-tumour syndrome may have no other manifestations, at least at the time of diagnosis (see below for a fuller description of these conditions). In addition, a locus, mapped to chromosome 2p14-p13.3, has also been described in association with FIPH, although the function of the gene is not known. The other forms of multiple endocrine neoplasia are also, though more rarely, associated with FIPH. Sporadic cases have been described in association with mutations in the PTH gene itself or in PRAD1 (see below). These genes are thought to be oncogenes or tumour suppressor genes. In sporadic cases, a ‘single hit’ mutation affects a proto-oncogene such as

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PRAD1 resulting in preferential growth of a single cell line. As a consequence, all of the familial forms of the condition are autosomal dominant. In the familial syndromes, a germline ‘first hit’ mutation affects a tumour suppressor gene and makes the parathyroid (and other) glands susceptible to a ‘second hit’ [5]. Tumours that arise in familial hyperparathyroidism are usually the result of hyperplasia whilst those occurring in sporadic cases are adenomas. However, these can be multiple and it is sometimes difficult to distinguish between the two.

Multiple Endocrine Neoplasia Type 1 (MENI) (+131100)

This is an autosomal-dominant condition in which parathyroid tumours are the most common, occurring in 90% of patients. Pancreatic tumours occur in 60% and anterior pituitary tumours in 30%. Less commonly, adrenocortical or carcinoid tumours are present as well as lipomas, angiofibromas and collagenomas [6]. Of these tumours, the parathyroids are usually the first to present, doing so in adolescence or early adulthood. The aetiology is probably an inactivating mutation of the MEN1 gene (131100.0020), located on chromosome 11q13, which normally codes for a tumour suppressor protein MENIN. Nonsense mutations, deletions, insertions, donor-splice site mutations, and missense mutations have all been described. If an MEN1 gene mutation is detected within a family, genetic counselling and early diagnosis can be offered. Penetrance is 7% by age 10 years rising to 52% by age 20 and eventually to 100% [6].

Multiple Endocrine Neoplasia Type II (MEN2)

Within this syndrome there are three different variants all of which are linked by mutations in the same gene, the c-ret proto-oncogene (*164761), that is located on chromosome 10cen-10q11.2. Several different mutations have been found in all three variants but some of these give rise to more aggressive tumour formation than others and this can be useful in family studies and in advising on such matters as prophylactic thyroidectomy for C cell carcinoma. Mutations in a second gene, NTRK1 (*191315), have also been shown to cause isolated medullary cell carcinoma of the thyroid. Only the commonest variant, MEN2A (#171400) is usually associated, in 20% of cases, with parathyroid tumours. MCT and phaeochromocytomas also occur. MEN2B (#162300) is not usually associated with parathyroid tumours but has an association with phaeochromocytomas, mucosal neurofibromas, and intestinal autonomic ganglion dysfunction. In the third variant, MCT-only (#155240), no tumours other than MCT occur.

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Multiple Endocrine Neoplasia Type IV (MEN4)

MEN4 (#610755) is caused by mutations in the CDKN1B gene (*600778). Pituitary, parathyroid and other tumours feature in this condition, although usually only in adulthood. The gene product is thought to be a suppressor of cell proliferation which acts via cyclin-dependent kinases. Allelic loss of chromosome 1p32-pter has been found in a number of cases of isolated sporadic parathyroid adenomas [7]. This region contains a putative tumour suppressor gene, but the gene product is unidentified.

Hyperparathyroid Jaw-Tumour Syndrome (HYP-JT) (#145001)

Hyperparathyroid jaw-tumour syndrome is an autosomal-dominant condition caused by mutations in the HRPT2 gene (*607393). This is found on chromosome 1q21q31. Inactivating mutations of this gene cause tumours not only of the parathyroid but also of the mandible and maxilla. The latter are fibro-osseous in nature. Renal tumours also occur within the same families. The HRPT2 gene codes for parafibromin which, together with the products of three other genes, LEO1 (*610507), PAF1 (*610506) and CTR9 (*609366), forms a parafibromin complex that normally associates with RNA polymerase II large subunit to act as a tumour suppressor [8]. When HRPT2 is mutated, tumour formation is more likely. Parathyroid adenomas usually do not occur until adulthood but may present during adolescence. Affected individuals should be screened regularly for tumour development.

Isolated Parathyroid Adenomas

Occasionally parathyroid adenomas have been found in association with abnormalities in the PRAD1 (cyclin D1) gene. Amongst other functions, this gene becomes associated with the PTH gene and acts as a tumour suppressor gene. Abnormalities result in over expression of PRAD1 resulting in deregulation of parathyroid gland development [9].

Parathyroid Carcinoma (#608266)

Parathyroid carcinoma forms only about 1% of cases of primary hyperparathyroidism. Most cases are associated with mutations in the HRPT2 gene and can occur in the same families as those with parathyroid adenomas in HYP-JT syndrome. The average age at which they occur is during the fifth decade and consequently are rarely seen in children [10] (see chapter 15, case 12).

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It can be difficult to distinguish carcinoma from adenoma both clinically and histologically but carcinoma tends to present with more aggressive hypercalcaemia and has an equal sex distribution.

Tertiary Hyperparathyroidism

Tertiary hyperparathyroidism is a condition that arises when a hypocalcaemic stimulus to PTH secretion has persisted for a sufficient period of time that the parathyroids become autonomous. The commonest situation in which this arises is chronic renal failure (see below). If the hypercalcaemia and hyperparathyroidism persist, it may be necessary to remove the parathyroids. However, it is important to identify and, if necessary, treat vitamin D deficiency before doing so as this is commonly present and may result in suppression of PTH values if treated appropriately [11]. Occasionally, tertiary hyperparathyroidism can occur when there has been chronic secondary hyperparathyroidism associated with vitamin D deficiency. Under these circumstances, the plasma calcium may remain normal in the presence of severe rickets until treatment with vitamin D is commenced, at which stage the hypercalcaemia becomes apparent (see chapter 15, case 13).

Neonatal Severe Hyperparathyroidism (NSHPT) (#239200)

Although the hypercalcaemia of NSHPT is associated with raised PTH, it will be discussed in the next section.

Hypercalcaemia Associated with Normal PTH Levels

Inactivating Calcium-Sensing Receptor Mutations Two disorders result from inactivating CaSR mutations – familial benign hypercalcaemia and neonatal severe hyperparathyroidism.

Familial Benign Hypercalcaemia (FBH) or Familial Hypocalciuric Hypercalcaemia (FHH) FBH comprises three distinct genetic disorders. Type 1 (#145980) is the commonest and is caused by inactivating CaSR (+601199) mutations. The causes of type 2 (%145981) (19p13.3) and type 3 (%600740) (19q13) have yet to be identified.

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Individuals with FBH are typically asymptomatic and have a PTH-dependent, hypercalcaemic disorder [12, 13]. Type 1 FBH results from heterozygous, autosomal dominant inactivating mutations of the CaSR. This results in a ‘right shift’ of the sigmoidal curve that describes the relationship between plasma calcium and PTH secretion (see chapter 2) and results in parathyroid and renal resistance to suppression of PTH release. Urinary calcium excretion is usually low. Some families have been reported with type 1 FBH and hypercalciuria and renal stone disease, which remitted following subtotal parathyroidectomy. Renal calcium handling responded normally to the calciuric action of hypercalcaemia despite abnormal calcium-regulated PTH release [14]. In these families, it seems that the mutations are more likely to be within the intracellular ‘tail’ of the CaSR molecule. Furthermore, some families with isolated familial hyperparathyroidism have CaSR mutations and thus the biochemical picture may be indistinguishable from primary hyperparathyroidism. Affected individuals exhibit mild-to-moderate hypercalcaemia (~3.00 mmol/l), typically normal PTH levels (i.e. not suppressed), high-to-normal range or mildly elevated serum magnesium concentrations (in contrast to primary hyperparathyroidism) and a reduced urine calcium:creatinine ratio. Approximately 80% have a urine calcium:creatinine ratio of less than 0.01 mmol/mmol creatinine [15]. Some patients have lipohyperplasia of their parathyroid gland. Another phenotypic variant of FHH may be presentation as neonatal hyperparathyroidism, which is distinct from neonatal severe hyperparathyroidism. Some infants present with moderate hypercalcaemia, elevated PTH, parathyroid bone disease and variable symptoms of hypercalcaemia [12]. Although the condition results from a heterozygous mutation of the CaSR, severe hypercalcaemia secondary to hyperparathyroidism results. This situation is likely to arise in neonates who have inherited their mutation from their father whilst their mother is normal [16]. Under these circumstances, the foetus considers the mother to be hypocalcaemic and responds by overproduction of PTH. If initial intensive medical management is successful in preventing complications the condition may revert to the clinical phenotype of FBH without having to resort to surgery. Cinacalcet may be helpful under these circumstances until the hyperparathyroidism settles [17]. Evidence is accumulating that FBH is associated with an increased risk of pancreatitis. Parathyroidectomy may be offered to affected individuals when there is recurrent pancreatitis. In vitro studies suggest that calcimimetics may sensitise the CaSR and improve the signal transduction in known mutant CaSRs [4] (see chapter 13). Calcimimetics are highly specific allosteric activators of CaSR without intrinsic activity in the absence of calcium. Their use in FBH is the subject of ongoing studies. Polymorphisms of the C-terminal domain of the CaSR may be associated with a marginally elevated calcium and increased risk of coronary artery disease. In the absence of CaSR mutations, autoantibodies directed at the calcium sensing receptor may also cause hypocalciuric hypercalcaemia [18].

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Neonatal Severe Hyperparathyroidism (#239200) This disorder results from homozygous inactivating CaSR mutations and usually presents in the neonatal period. The biochemical parameters show serum calcium levels >3.5 mmol/l, and may be as high as 5–6 mmol/l, very high PTH levels, and severe bone disease, often with multiple fractures. There is a variable phenotype with some affected individuals being identified in adulthood who are asymptomatic with normal renal function. In one case series, the presenting features were hypotonia (54%), thoracic deformities (36%), feeding difficulties (18%) and respiratory distress (27%) [4]. The serum calcium at presentation was a mean of 3.64 mmol/l (range 2.75–6.75). The mean serum phosphate concentration was 1.3 mmol/l (range 0.8–1.9). The plasma PTH concentration was always high with a mean of 536 pg/ml (range 56–2,214). 25OHD was often normal. Neck ultrasonography localised parathyroid abnormalities in only 27% of cases. Complications observed at diagnosis were skeletal demineralisation including metaphyseal irregularities, cortical dualisation, sub-periosteal erosion and bell-shaped thoracic deformation. Preoperative medical management may require hyperhydration with diuretics and bisphosphonate administration. Medical treatment with a calcium-poor diet may postpone the need for surgery. Surgical treatment of neonatal cases consists of total parathyroidectomy [4, 19, 20]. Postoperatively, these infants may have a ‘hungry bone’ syndrome and require large quantities of calcium, given by intravenous infusion if necessary, to prevent hypocalcaemia until the bones heal sufficiently for this no longer to be needed.

Hypercalcaemia Associated with Low Parathyroid Hormone Levels

Williams-Beuren Syndrome (#194050) Williams-Beuren syndrome is a multisystem disorder which is a contiguous gene deletion syndrome on chromosome 7q11.23 and occurs in 1 in 20,000 births. Hemizygosity of the elastin gene (#194050) defect is observed in over 90% of the cases. The deletion of the elastin gene accounts for the vascular and connective tissue abnormalities. Several other genes, including LIM kinase (*601329), may also be deleted. The genes that contribute to the features such as hypercalcaemia, dysmorphic facies, and mental retardation are unknown but presumably the variability in the phenotype depends on precisely which genes are deleted (c.f. 22q deletion syndrome – see chapter 6). Approximately two-thirds of infants are born small for gestational age and may have facial features such as depressed nasal bridge, epicanthic folds, and/or prominent lips. Approximately 70% have a cardiac malformation, most commonly supravalvular aortic stenosis. Peripheral pulmonary stenosis also occurs. When older,

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affected children have ‘elfin facies’ and a loquacious manner, the so-called ‘cocktail party’ affect. Tooth decay and enamel hypoplasia are more frequent. The hypercalcaemia, if present initially, rarely persists beyond the end of the first year of life but may progress to nephrocalcinosis. Elevated levels of 1,25(OH)2D have been observed in some infants. In others, some authors have hypothesised increased sensitivity to normal circulating levels [20] whilst others have demonstrated hypercalcaemia during puberty. Treatment of hypercalcaemia is with a calcium-restricted diet.

Jansen Metaphyseal Chondrodysplasia (#156400) This condition results from a heterozygous mutation in the PTHR1 gene which results in a constitutively activated PTH/PTHrP receptor [21]. The receptor is found in the kidney, bone and growth plate. Constitutive activity in bone causes hypercalcaemia and hypophosphataemia, despite normal or undetectable serum PTH or PTHrP, although this may be intermittent and particularly associated with episodes of intercurrent illness. Increased bone resorption of calcium from the kidney is life-long. The increased bone turnover leads to elevated serum alkaline phosphatase and osteocalcin. Following completion of linear growth, the calcium levels fall but remain elevated [20, 21] (see chapter 15, case 34). The biochemical abnormalities may not be apparent in the first week of life but are usually manifest by 3 months of age. The abnormal receptor in the growth plate causes delayed chondrocyte differentiation resulting in postnatal-onset short-limbed short stature. Choanal atresia and/or rib fractures may result in respiratory distress shortly after birth. There is often normal growth in infancy but short stature becomes more apparent during mid-childhood. The phenotype includes hypertelorism, mandibular hypoplasia and progressive disproportionate short stature. A bell-shaped thorax with widened costo-chondral junctions is seen. The legs are bowed, particularly the tibiae. Tooth and enamel development is normal. Feeding difficulties, recurrent vomiting and dehydration occur. Bone lesions are apparent on X-ray. These include rachitic changes, radiolucencies and irregular metaphyses of the long bones and also the metacarpals and metatarsals (distinct from rickets), and sclerotic changes at the basal skull. Several treatment modalities have been proposed but bisphosphonates may be of some benefit in reducing the rapid rate of bone turnover (see chapter 15, case 34).

Idiopathic Hypercalcaemia of Infancy (Lightwood Syndrome) These children present with hypercalcaemia in the first year of life, typically after 6 months of age. There are no dysmorphic features and the hypercalcaemia is more

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prolonged than that seen in Williams-Beuren syndrome. The PTH is suppressed and, in one study, affected children had an increase in N-terminal PTHrP at the time of hypercalcaemia, which became undetectable during normocalcaemia. Elevated 25OHD and high normal 1,25(OH)2D levels have been found in some patients at the time of hypercalcaemia and these abnormalities may persist even during normocalcaemia [22]. Non-progressive subclinical nephrocalcinosis may occur. Long-term follow-up of affected individuals has shown that nephrocalcinosis and hypercalciuria may persist [23]. There may be an increased incidence of behavioural problems and deficits in performance IQ [24].

Hypophosphatasia The various forms of hypophosphatasia are discussed in chapter 12. Hypercalcaemia can be a feature of these conditions at times but is not usually a major problem.

Secondary Hypercalcaemia

Malignancy Hypercalcaemia occurs in up to 30% of adult patients with cancer [25], but is seen much less frequently in childhood malignancy. Detection of hypercalcaemia often heralds a poor prognosis in adult patients but this is not the case with paediatric patients where it is most commonly observed at diagnosis. Hypercalcaemia associated with malignancy can be classified into four types (table 6) [25]. In children with local osteolytic hypercalcaemia, for example at presentation of acute lymphoblastic leukaemia, the hypercalcaemia results from an increase in osteoclastic bone resorption in areas surrounding the malignant cells in the marrow space [26]. The condition known as humoral hypercalcaemia of malignancy is caused by systemic secretion of PTHrP by malignant tumours and has been reported in childhood acute lymphoblastic leukaemia [27] and other childhood malignancies including medulloblastoma and hepatic sarcoma [28, 29]. PTHrP has similar actions to PTH and results in increased bone resorption and enhances renal retention of calcium. Any tumour that secretes 1,25(OH)2D causes hypercalcaemia as a result of a combination of enhanced osteoclastic bone resorption and enhanced intestinal absorption of calcium. The latter has been observed in children with ovarian dysgerminoma [30]. Ectopic secretion of PTH is a very rare cause of hypercalcaemia in adults but has not been observed in children (table 6). Children with hypercalcaemia of malignancy are often dehydrated as a result of the water-concentrating defect (nephrogenic diabetes insipidus) induced by

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Table 6. Types of hypercalcaemia associated with malignancy Type

Bone metastases

Causal agent

Typical tumours

Local osteolytic hypercalcaemia Humoral hypercalcaemia of malignancy

common extensive minimal or absent

cytokines, chemotherapy, PTHrP PTHrP

1,25-Dihydroxyvitamin D3-secreting lymphomas Ectopic hyperparathyroidism

variable

1,25-dihydroxyvitamin D3

ALL, AML, lymphoma ovarian dysgerminoma, medulloblastoma, hepatic sarcoma lymphoma

variable

PTH

variable

hypercalcaemia and by decreased oral hydration resulting from anorexia, nausea or vomiting. A rapid elevation in serum calcium results in neurological dysfunction whereas chronic hypercalcaemia may cause only minor symptoms. Intravenous bisphosphonate (pamidronate) is the most effective agent for children with hypercalcaemia of malignancy. In the US pamidronate and zoledronate and in the UK ibandronate and clodronate are licensed for use as treatment for malignant hypercalcaemia in adults. The majority of paediatric practice in the UK is with pamidronate. Bisphosphonate therapy should be started early. A reduction in serum calcium is usually observed in the first 24 h and the nadir of calcium generally occurs within 7 days and sometimes results in hypocalcaemia that requires oral calcium supplementation. For a more detailed description of the use of bisphosphonates and their potential problems, see chapter 13.

Endocrine Causes of Hypercalcaemia Thyrotoxicosis may result in mild hypercalcaemia by direct stimulation of osteoclast bone resorption by T3. Increased serum calcium level suppresses PTH and hypercalciuria results. Treatment should be directed at thyrotoxicosis with anti-thyroid drugs and beta-blockers where appropriate. Hypercalcaemia may rarely occur with acute adrenal insufficiency. The mechanism is uncertain but may relate to volume depletion as a result of the mineralocorticoid deficiency and changes in vitamin D metabolism secondary to glucocorticoid deficiency. Some patients with APECED syndrome who are hypoparathyroid may become hypercalcaemic, despite not changing their treatment, if adrenal insufficiency supervenes (see chapter 6).

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Chronic Renal Failure Acute and chronic renal failure may cause hypercalcaemia but by different mechanisms. In acute renal failure, hypercalcaemia occurs during the diuretic phase and in association with rhabdomyolysis. Hypocalcaemia may occur during acute renal failure and hypercalcaemia during recovery of renal function. It is thought that release of calcium from calcium deposits that have accumulated in muscle during the hypocalcaemic phase may occur. Release of 25OHD from damaged muscle may lead to an increase in available substrate for conversion to 1,25(OH)2D as the kidneys recover. The increase in serum calcium and 1,25(OH)2D returns to normal during the diuretic phase. Hypercalcaemia occurs in up to 20% of patients with chronic renal failure. Several mechanisms lead to hypercalcaemia. Secondary hyperparathyroidism with parathyroid gland hyperplasia occurs in response to hyperphosphataemia, hypocalcaemia and reduced 1,25(OH)2D levels. In some patients, despite treatment of secondary hyperparathyroidism with phosphate restriction, calcium binders and calcitriol, the PTH levels remain elevated. This results in tertiary hyperparathyroidism. These patients have increased parathyroid gland mass from hyperplasia. Proliferation of parathyroid cells may lead to somatic mutations and monoclonal expansion of the type seen in parathyroid adenomas. Following renal transplantation, hypercalcaemia may occur from elevated PTH as a result of slow involution of the hyperplastic parathyroid glands. The hypercalcaemia may resolve spontaneously over months.

Immobilisation Immobilisation from prolonged bed rest or secondary to other causes, for example severe disability, is associated with disordered calcium homeostasis. Hypercalciuria occurs in all individuals that are completely immobilised, but hypercalcaemia occurs only in those with a high bone turnover state. This is most commonly observed during adolescence especially in those with spastic quadriplegia or spinal cord injuries. Immobilisation-associated hypercalcaemia occurs in adults with high bone turnover states such as thyrotoxicosis, hyperparathyroidism or malignancy. Hypercalcaemia results from increased osteoclastic bone resorption and decreased bone formation. Prolonged immobilisation may lead to renal stone formation and osteopaenia. Serum PTH and 1,25(OH)2D levels are suppressed and hypercalcaemia and hypercalciuria may resolve with the onset of weight bearing. Bisphosphonates and calcitonin have been used to treat immobilisation-associated hypercalcaemia [31].

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Drug-Induced Hypercalcaemia Vitamin D intoxication results in hypercalcaemia by increasing intestinal calcium absorption an increasing osteoclastic bone resorption. Hyperphosphataemia may also occur. If prolonged, nephrocalcinosis and renal impairment occurs and may require treatment with bisphosphonate [32]. Vitamin A toxicity may also cause hypercalcaemia [33, 34]. Vitamin A analogues such as cis-retinoic acid are used as a treatment for acne and other retinoic acids are used in the treatment of certain malignancies. The mechanism of hypercalcaemia is unclear but vitamin A has direct effects to stimulate bone resorption. Thiazide diuretics may cause hypercalcaemia, particularly in those with primary hyperparathyroidism, by increasing renal tubular calcium reabsorption.

Granulomatous Disorders

Subcutaneous Fat Necrosis Newborns who develop subcutaneous fat necrosis (SCFN) usually are healthy at birth but may have predisposing factors including obstetric trauma, meconium aspiration, asphyxia, hypothermia, or hypoxia. Within the first few weeks of life, hard, indurated violaceous nodules and plaques with ill-defined overlying erythema develop on the trunk, arms, buttocks, thighs, or cheeks, which may be painful. Histological findings show a panniculitis. The pathophysiology is likely secondary to excessive endogenous synthesis of 1,25(OH)2D. This mechanism is supported by the finding that histological examination of skin biopsies taken from affected areas showed copious expression of 1α-hydroxylase in the inflammatory infiltrate [35]. Presentation is often with symptoms of hypercalcaemia especially hypotonia from myopathy. The calcium levels are often very high. Initial treatment is with fluids and pamidronate if hypercalcaemia is refractory [36, 37]. The hypercalcaemia may persist for as long as 1–2 years. Glucocorticoids have also been used (see chapter 15, case 15).

Other Granulomatous Disorders Sarcoidosis and other granulomatous diseases such as tuberculosis can occasionally give rise to hypercalcaemia. The mechanism is thought to be similar to that of subcutaneous fat necrosis.

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Gestational Maternal Hypocalcaemia If infants present with persistent hypercalcaemia, one of the differential diagnoses to be considered is maternal hypocalcaemia. Investigation of neonatal hypercalcaemia should include a measurement of plasma calcium in both parents. If the mother is hypocalcaemic (e.g. as a result of undiagnosed hypoparathyroidism) the infant may become temporarily hyperparathyroid in order to maintain adequate plasma calcium levels in utero. This can persist postnatally and may take several weeks to settle.

Treatment of Hypercalcaemia Treatment of hypercalcaemia is aimed at both lowering serum calcium concentration and correcting the underlying disease. Apart from general measures aimed at reducing calcium intake and increasing mobility, there are currently six approaches to correcting hypercalcaemia (table 7).

(1) Increase Urinary Calcium Excretion

Filtered calcium is principally reabsorbed in the proximal tubule and the ascending loop of Henle. This process is mainly passive whereas active resorption of calcium occurs in the distal loop under the influence of PTH and to a lesser degree 1,25(OH)2D. In hypercalcaemia, urinary calcium excretion can be increased by inhibiting proximal and loop sodium reabsorption, thus reducing passive reabsorption of calcium. Proximal reabsorption is inhibited by volume expansion from intravenous saline infusion, which increases delivery of sodium, calcium and water to the loop of Henle, and administration of a loop diuretic such as furosemide then blocks transport at this site. The majority of children with severe hypercalcaemia have volume contraction due to both decreased fluid intake and to the natriuretic effects of hypercalcaemia. Loop diuretics must be used with caution in the long term as they will increase the chances of nephrocalcinosis developing.

(2) Reduce PTH Secretion

In cases where excess PTH secretion is a cause of the hypercalcaemia, it may be possible to reduce secretion of the hormone. In NSHPT the calcimimetic agent, cinacalcet, reduces PTH secretion sufficiently to allow the hyperparathyroidism to settle without recourse to parathyroidectomy, particularly if the CaSR mutation is heterozygous. Similarly, in chronic renal failure, cinacalcet may help to reduce PTH secretion

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Table 7. Various treatments used in hypercalcaemia General measures Reduce calcium from parenteral feeding Discontinuation of oral calcium supplements Discontinue medications that independently lead to hypercalcaemia, e.g. thiazides, vitamin D, calcitriol Increase the weight bearing mobility of the patient Discontinuation of the use of the use of sedative drugs to promote mobility Specific measures Increase urinary calcium excretion Isotonic saline (0.9% saline) plus loop diuretic (furosemide) Reduce PTH secretion Cinacalcet Decrease intestinal calcium absorption Glucocorticoids in hypervitaminosis D secondary to granulomatous disease, increased intake, haematological malignancy Diminish bone resorption Bisphosphonates Calcitonin Dialysis Surgery

and reverse the hyperparathyroidism. At the same time, it is important to ensure that vitamin D deficiency is not also present as treatment of this may also help to reverse the high PTH levels [12].

(3) Decrease Intestinal Calcium Absorption

If hypercalcaemia is caused by raised levels of 1,25(OH)2D some effect can be achieved by administration of glucocorticoids which reduce the conversion of 25OHD to its active metabolite.

(4) Inhibit Bone Resorption

Bisphosphonates Bisphosphonates are extremely effective in children with moderate to severe hypercalcaemia [38]. Pamidronate is the drug of choice in children even in those with renal failure. It is given at a dose of 0.5–1.0 mg/kg as an infusion over 4–6 h. A reduction in calcium is observed 12–24 h after administration and may last for 2–4

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weeks. A sustained period of hypocalcaemia may follow the initial administration of pamidronate and additional calcium supplementation may be required. Calcitonin In the acute situation calcitonin injections can be effective in reducing plasma calcium. The effect is rapid but wears off after a while (see chapter 13 for further details).

(5) Dialysis

In resistant, life-threatening hypercalcaemia, haemodialysis against a low-calcium dialysate is more effective than peritoneal dialysis at lowering calcium levels.

(6) Surgery

Surgery is required for primary hyperparathyroidism. This should be undertaken by an experienced paediatric endocrine surgeon. Neonatal severe hyperparathyroidism requires total parathyroidectomy, which renders the child hypoparathyroid. The decision to operate for milder forms depends on the degree of hypercalcaemia, symptoms and potential for renal damage. Preoperative assessment of parathyroid adenomas can sometimes be successful either by use of Doppler ultrasound or with SestaMIBI scintigraphy. The latter is more successful where a single adenoma rather than more generalised hyperplasia is present. Rapid peroperative PTH measurements can be undertaken to determine whether or not parathyroidectomy has been effective. Pamidronate may be required preoperatively in those with severe or symptomatic hypercalcaemia.

Conclusions

Hypercalcaemia is a relatively rare problem in childhood when compared with that of hypocalcaemia. Nevertheless, it remains an important one that requires proper assessment and diagnosis before correct treatment can be given. The spectrum of disease is rather wider than that found in adults with the emphasis less specifically on hyperparathyroidism and malignancy and encompassing a wide range of conditions, many of them genetic in origin. Once a correct diagnosis has been made, it is usually possible to offer effective treatment.

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References 1 Bilezikian JP: Clinical review 51:management of hypercalcemia. J Clin Endocrinol Metab 1993;77: 1445–1449. 2 Hind E, Wood P, Walker V, Davies JH: The investigation of hypercalcaemia in children. Horm Res 2007;68(suppl 1):74. 3 Jacobs TP, Bilezikian JP: Clinical review: rare causes of hypercalcemia. J Clin Endocrinol Metab 2005; 90:6316–6322. 4 Mallet E: Primary hyperparathyroidism in neonates and childhood: the French experience (1984–2004). Horm Res 2008;69:180–188. 5 Bastepe M, Jüppner H, Thakker RV: Parathyroid disorders; in Glorieux FH, Pettifor JM, Jüppner H (eds): Pediatric Bone: Biology and Diseases. San Diego, Academic Press, 2003. 6 Bassett JH, Forbes SA, Pannett AA, et al: Characterization of mutations in patients with multiple endocrine neoplasia type 1. Am J Hum Genet 1998;62:232–244. 7 Williamson C, Pannett AA, Pang JT, et al: Localisation of a gene causing endocrine neoplasia to a 4 cM region on chromosome 1p35-p36. J Med Genet 1997;34:617–619. 8 Rozenblatt-Rosen O, Hughes CM, Nannepaga SJ, et al: The parafibromin tumor suppressor protein is part of a human Paf1 complex. Mol Cell Biol 2005; 25:612–620. 9 Fu M, Wang C, Li Z, Sakamaki T, Pestell RG: Cyclin D1: normal and abnormal functions. Endocrinology 2004;145:5439–5447. 10 Shane E: Clinical review 122: parathyroid carcinoma. J Clin Endocrinol Metab 2001;86:485–493. 11 K/DOQI clinical practice guidelines for bone metabolism and disease in children with chronic kidney disease. Am J Kidney Dis 2005;46:S1–S121. 12 Brown EM: Editorial: mutant extracellular calciumsensing receptors and severity of disease. J Clin Endocrinol Metab 2005;90:1246–1248. 13 Wystrychowski A, Pidasheva S, Canaff L, et al: Functional characterization of calcium-sensing receptor codon 227 mutations presenting as either familial (benign) hypocalciuric hypercalcemia or neonatal hyperparathyroidism. J Clin Endocrinol Metab 2005;90:864–870. 14 Carling T, Szabo E, Bai M, Ridefelt P, Westin G, Gustavsson P, et al: Familial hypercalcemia and hypercalciuria caused by a novel mutation in the cytoplasmic tail of the calcium receptor. J Clin Endocrinol Metab 2000;85:2042–2047. 15 Marx SJ, Spiegel AM, Brown EM, et al: Divalent cation metabolism: familial hypocalciuric hypercalcemia versus typical primary hyperparathyroidism. Am J Med 1978;65:235–242.

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16 Pearce S, Steinmann B: Casting new light on the clinical spectrum of neonatal severe hyperparathyroidism. Clin Endocrinol (Oxf) 1999;50:691–693. 17 Festen-Spanjer B, Haring CM, Koster JB, Mudde AH: Correction of hypercalcaemia by cinacalcet in familial hypocalciuric hypercalcaemia. Clin Endocrinol (Oxf) 2008;68:324–325. 18 Kifor O, Moore FD Jr, Delaney M, et al: A syndrome of hypocalciuric hypercalcemia caused by autoantibodies directed at the calcium-sensing receptor. J Clin Endocrinol Metab 2003;88:60–72. 19 Ghirri P, Bottone U, Coccoli L, et al: Symptomatic hypercalcemia in the first months of life: calciumregulating hormones and treatment. J Endocrinol Invest 1999;22:349–353. 20 Rodd C, Goodyer P: Hypercalcemia of the newborn: etiology, evaluation, and management. Pediatr Nephrol 1999;13:542–547. 21 Schipani E, Langman C, Hunzelman J, et al: A novel parathyroid hormone (PTH)/PTH-related peptide receptor mutation in Jansen’s metaphyseal chondrodysplasia. J Clin Endocrinol Metab 1999;84: 3052–3057. 22 Pronicka E, Rowinska E, Kulczycka H, Lukaszkiewicz J, Lorenc R, Janas R: Persistent hypercalciuria and elevated 25-hydroxyvitamin D3 in children with infantile hypercalcaemia. Pediatr Nephrol 1997;11: 2–6. 23 Huang J, Coman D, McTaggart SJ, Burke JR: Longterm follow-up of patients with idiopathic infantile hypercalcaemia. Pediatr Nephrol 2006;21:1676– 1680. 24 Udwin O, Yule W, Martin ND: Age at diagnosis and abilities in idiopathic hypercalcaemia. Arch Dis Child 1986;61:1164–1167. 25 Stewart AF: Clinical practice: hypercalcemia associated with cancer. N Engl J Med 2005;352:373–379. 26 Niizuma H, Fujii K, Sato A, Fujiwara I, Takeyama J, Imaizumi M: PTHrP-independent hypercalcemia with increased proinflammatory cytokines and bone resorption in two children with CD19-negative precursor B acute lymphoblastic leukemia. Pediatr Blood Cancer 2007;49:990–993. 27 Shimonodan H, Nagayama J, Nagatoshi Y, et al: Acute lymphocytic leukemia in adolescence with multiple osteolytic lesions and hypercalcemia mediated by lymphoblast-producing parathyroid hormone-related peptide: a case report and review of the literature. Pediatr Blood Cancer 2005;45:333– 339.

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28 Dharmaraj P, Ball S, Johnstone H, et al: Hypercalcaemia in relapsed medulloblastoma 8 years post-diagnosis; evidence to support PTHrP production by medulloblastoma cells. Horm Res 2006;66: 268–272. 29 Lakhdir F, Lawson D, Schatz DA: Fatal parathyroid hormone-related protein-induced humoral hypercalcemia of malignancy in a 3-month-old infant. Eur J Pediatr 1994;153:718–720. 30 Hibi M, Hara F, Tomishige H, et al: 1,25-Dihydroxyvitamin D-mediated hypercalcemia in ovarian dysgerminoma. Pediatr Hematol Oncol 2008;25:73–78. 31 Go T: Low-dose oral etidronate therapy for immobilization hypercalcaemia associated with GuillainBarre syndrome. Acta Paediatr 2001;90:1202–1204. 32 Chatterjee M, Speiser PW: Pamidronate treatment of hypercalcemia caused by vitamin D toxicity. J Pediatr Endocrinol Metab 2007;20:1241–1248. 33 Kimmoun A, Leheup B, Feillet F, Dubois F, Morali A: Hypercalcemia revealing iatrogenic hypervitaminosis A in a child with autistic troubles. Arch Pediatr 2008;15:29–32.

34 Nagasawa M, Okawa H: All-trans retinoic acid induced hypercalcemia in a patient with acute promyelocytic leukemia: its relation to increased PTH-rP. Int J Hematol 1994;59:143–144. 35 Farooque A, Moss C, Zehnder D, Hewison M, Shaw NJ: Expression of 25-hydroxyvitamin D(3)-1alphahydroxylase in subcutaneous fat necrosis. Br J Dermatol 2008. 36 Alos N, Eugene D, Fillion M, Powell J, Kokta V, Chabot G: Pamidronate: treatment for severe hypercalcemia in neonatal subcutaneous fat necrosis. Horm Res 2006;65:289–294. 37 Lombardi G, Cabano R, Bollani L, Del Forno C, Stronati M: Effectiveness of pamidronate in severe neonatal hypercalcemia caused by subcutaneous fat necrosis: a case report. Eur J Pediatr 2008. 38 Lteif AN, Zimmerman D: Bisphosphonates for treatment of childhood hypercalcemia. Pediatrics 1998;102:990–993.

Justin H. Davies, MD Department of Child Health, Southampton University Hospital Trust Tremona Road Southampton, SO16 6YD (UK) Tel. +44 2389 796985, Fax +44 2380 795230, E-Mail [email protected]

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Chapter 8 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 115–132

A Practical Approach to Rickets Jeremy Allgrove Royal London Hospital, Great Ormond Street Hospital, London, UK

Abstract Rickets is a condition in which there is failure of normal mineralisation (osteomalacia) of growing bone. Whilst osteomalacia may be present in adults, rickets cannot occur. It is generally caused by a lack of mineral supply which can either be as a result of deficiency of calcium (calciopaenic rickets) or of phosphate (phosphopaenic rickets) although, in addition, renal tubular acidosis may also interfere with the process of mineralisation and cause rickets. Only calciopaenic and distal renal tubular disorders will be discussed in this chapter. The commonest cause of rickets is still vitamin D deficiency which is also responsible for problems other than rickets. Disorders of vitamin D metabolism or responsiveness may also cause similar problems. Distal renal tubular acidosis may be caused by a variety of metabolic errors similar to those of osteoclasts. One form of DRTA also causes a form of osteopetrosis. This chapter describes these conditions in detail and sets out a logical approach to Copyright © 2009 S. Karger AG, Basel treatment.

Osteomalacia is a condition in which mineralisation of osteoid tissue fails to occur normally. This is usually because of a deficiency in the supply of mineral, calcium or phosphate, for one reason or another. Although osteomalacia can occur in any individual, rickets can only occur in the presence of unfused epiphyses as it manifests itself in the growth plate. Therefore, adults cannot suffer from rickets but may have osteomalacia. There are two principal causes of rickets, which are usually referred to as calciopaenic and phosphopaenic. In addition, a third group of conditions, related to distal renal tubular acidosis, also cause rickets although these are, in many ways related to the former group. Phosphopaenic rickets will not be considered in this chapter, as it is dealt with elsewhere, and will deal only with the various forms of calciopaenic rickets.

Vitamin D

For a detailed discussion of the physiology of vitamin D, see chapter 2. Vitamin D is a secosteroid which is derived principally in the form of cholecalciferol from

Table 1. Levels (in nmol/l) of vitamin D in plasma that denote various degrees of vitamin D status Toxic

>200

Fully replete Replete Insufficient Deficient Seriously deficient

70–200 50–70 30–50 15–30 <15

the action of ultraviolet light on dehydrocholesterol. It then undergoes two metabolic steps, firstly involving hydroxylation at the 25-position to form 25OHD, and secondly at the 1α position of the steroid molecule to form the active metabolite, 1,25(OH)2D. Following this activation, 1,25(OH)2D acts on receptors, which are classical steroid receptors, and which involve ligand binding followed by transfer to the nucleus with subsequent DNA binding. Defects in any of these processes can give rise to rickets and a thorough understanding of the various processes is required in order to be able to make a correct diagnosis so that appropriate treatment can be instigated.

Vitamin D Deficiency It is generally agreed that the circulating level of 25OHD is a reflection of vitamin D status. Recently, a consensus has emerged about what constitutes vitamin D deficiency [1]. The various stages of vitamin D status are summarised in table 1. 25OHD has a wide therapeutic index and, unless a situation is present in which the control mechanisms for the metabolism of 1,25(OH)2D are faulty (e.g. sarcoidosis or neonatal subcutaneous fat necrosis), wide fluctuations in 25OHD can occur without adverse effects. Levels below 200 nmol/l are generally non-toxic and levels between this and 70 nmol/l are regarded as being normal. Levels between 50 and 70 nmol/l are not usually associated with problems although it is believed by some that the higher levels are required to maintain the non-classical actions of vitamin D. These are not discussed in this chapter. Once levels fall below 50 nmol/l there is a progressive rise in PTH in order to maintain adequate calcium absorption and a condition of insufficiency develops. Levels below 30–35 nmol/l are described as being deficient and those below 15–20 nmol/l as severe deficiency. However, not everyone who has such low levels of 25OHD will necessarily develop symptoms and there are several other factors, particularly calcium intake, that influence their development.

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Stages of Vitamin D Deficiency In 1975, Arnaud et al. [1] described three stages in the development of vitamin D deficiency (fig. 1). In the first stage, when vitamin D deficiency is in its early stages, plasma calcium is maintained by a rise in PTH. However, PTH, like many polypeptide hormones has the property of modifying the response to itself and, in due course, a state of relative PTH resistance develops which results in hypocalcaemia and hyperphosphataemia. This can be confused clinically with pseudohypoparathyroidism (see chapter 6). At this stage, patients may present with hypocalcaemic convulsions. If this does not happen, stage 2 begins in which vitamin D levels continue to fall and PTH rises further. This overcomes the resistance to PTH and plasma calcium rises at the expense of hypophosphataemia. Rickets may begin to appear and alkaline phosphatase rises. 1,25(OH)2D levels may be maintained during this period and may even be elevated. If vitamin D levels fall further, stage 3 is entered. Insufficient vitamin D substrate is now present and 1,25(OH)2D levels begin to fall. Hypocalcaemia and hypophosphataemia occur in association with a further rise in PTH and alkaline phosphatase. At this stage rickets is usually severe. All of these clinical and biochemical changes are reversible with adequate vitamin D treatment.

Investigation of Child with Rickets

When a child presents with clinical rickets or is found, as a result of radiological investigation, to have rickets, it is important to undertake investigation in a logical order so that a correct diagnosis can be made. This should begin with basic biochemistry (calcium, phosphate, albumin, alkaline phosphatase, creatinine). 25OHD must always be measured and blood saved for later measurement of 1,25(OH)2D if this is appropriate. Note that the concentration of 25OHD may be low even if the remainder of the biochemistry is normal. It is not possible to make a diagnosis relating to rickets (or hypocalcaemia) if vitamin D deficiency is present and this must always be corrected before a definitive diagnosis can be made. PTH should also be measured. Urine should be taken for estimation of calcium, phosphate and creatinine. From these, the ‘corrected’ calcium (see chapter 6) and the fractional excretion, tubular reabsorption and tubular threshold for phosphate (TmPO4/GFR) can be calculated (see chapter 9). Further investigations will depend on the results of the preliminary tests and might include urinary amino acids, blood gases and DNA for appropriate genetic analysis. If X-rays have not already been taken, these will also be required.

Syndromes Associated with Vitamin D Deficiency

Five principal conditions are described in connection with vitamin D deficiency.

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Stage 1

Stage 2

Stage 3

PTH

Ca

25O HD

Fig. 1. Stages in the development of vitamin D deficiency. Adapted from Arnaud et al. [1].

Pi

Congenital Rickets Occasionally rickets may be present at birth [2]. Bone mineralisation is poor and the ribs are very soft. This gives rise to chest deformity which presents radiologically as a bell-shaped chest. Respiratory function is impaired and respiratory support may be required. Hypocalcaemia and hypophosphataemia are present, 25OHD is low, PTH elevated and maternal vitamin D levels are low or undetectable. It is important to distinguish this condition from others that cause a bell shaped chest and respiratory difficulties such as I cell disease or thanatophoric dwarfism, since the latter are usually fatal whilst congenital rickets, with appropriate treatment, eventually recovers (see chapter 15, case 16 for a clinical description). Treatment consists of providing adequate vitamin D and calcium supplements together with appropriate respiratory support. Recovery may take several months.

Dilated Cardiomyopathy A rare but potentially fatal consequence of vitamin D deficiency is dilated cardiomyopathy [3]. This presents within the first 6 months after birth. All of the patients described have been from ethnic minority ‘at-risk’ groups. Cardiac failure, which

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may result in cardio-respiratory arrest, is associated with hypocalcaemia and a raised PTH. The failure may be sufficiently severe as to warrant cardiac support in the form of extracorporeal membrane oxygenation (ECMO). Continued support with ventilation and continuous positive airway pressure (CPAP) may be needed for several weeks. As with congenital rickets, it is important to make the correct diagnosis, since other forms of dilated cardiomyopathy during the first few months usually carry a poor prognosis whilst that secondary to vitamin D deficiency usually eventually recovers although it may take several months to do so completely. During this time, cardiotropic drugs may continue to be needed (see chapter 15, case 17 for a clinical description).

Classical Rickets This usually presents in the toddler age group although it can present later. The most common observation is bowing of the legs, which tends to become apparent as the child starts to walk. However, motor development is frequently delayed. This is caused by a combination of muscle weakness, bone pain and bowing of the legs. In young children the bowing typically occurs at the lower ends of the tibiae but older children may demonstrate either genu valgum, genu varum or a combination of the two which gives a ‘windswept’ appearance to the legs. Other clinical manifestations include swelling of the wrists, poor growth, aches and pains, which may result in a miserable child, Harrison’s sulci (indrawing of the lower ribs as a result of softening) and a ‘rickety rosary’. This is caused by swelling of the costo-chondral junctions and is seen as an inverted ‘V’ parallel and lateral to the costal margins. It is not seen parallel to the sternum – a common misconception (see chapter 15, fig. 9, case 18). Very young infants may have a ‘ping pong’ skull, although demonstration of this should be avoided. Pelvic deformity, if persistent into adult life, may result in difficulties with delivery of infants as a result of narrowing of the birth canal in the mother. Radiological evidence is seen principally at the growth plates where the normal slightly convex clearly defined appearance of the metaphyses is replaced by a concave ‘moth-eaten’ appearance which is best seen at the radius and ulna, lower end of the femur and upper end of the tibia. A chest X-ray also shows the costochondral swelling. There may also be a generalised reduction in bone density. Whilst many of these appearances are the result of failure of normal mineralisation, there are additional changes caused by the concomitant hyperparathyroidism and the appearances of severe hyperparathyroidism may be confused with rickets. As the bones heal, the classical appearances are replaced by a dense line of calcification at the metaphyses and, if the child has been exposed to vitamin D prior to diagnosis, partial healing may be seen. Case 18 in chapter 15 describes a classic example.

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Hypocalcaemic Convulsions Vitamin D deficiency may present only with hypocalcaemic convulsions. This is particularly true of those who are either very young or who are adolescent [4]. Particularly in the latter group, there may be no radiological evidence of rickets. Symptoms may be present acutely or, in retrospect, have been present for several months. The clinical manifestations, such as tetany, convulsions and muscle aches and pains, can be very distressing and may have been interfering with normal activities for some while. These are accompanied by the biochemical changes of hypocalcaemia, hyperphosphataemia and raised PTH. These findings may be confused with those of pseudohypoparathyroidism (PHP) and patients are sometimes referred with this diagnosis. However, none of the other features of PHP is present (although this is also the case in PHP Type 1b) and 25OHD levels are low. It is therefore essential that, when a patients presents with hypocalcaemic convulsions and a raised PTH, 25OHD levels are measured and, if necessary, corrected before a diagnosis of PHP is contemplated. This situation represents the first stage in the development of vitamin D deficiency (see above) and it is thought that these age groups are particularly vulnerable to hypocalcaemia because of their more rapid growth rate than during mid childhood [4]. All the symptoms are correctable by treatment with vitamin D. However, in the acute situation, the neurological abnormalities can be corrected by an intravenous infusion of calcium which gives almost instant relief of the pain, for which patients can be extremely grateful (see chapter 15, case 19).

Generalised Aches and Pains Personal experience suggests that probably the commonest symptoms of vitamin D deficiency are those of vague aches and pains of a somewhat non-specific nature. These are not usually accompanied by any specific biochemical abnormalities apart from vitamin D deficiency, although PTH may be slightly elevated and plasma phosphate at the lower end of the normal range. Adequate treatment with vitamin D relieves the symptoms (see chapter 15, case 20).

Calcium Deficiency Rickets Calcium deficiency rickets has also been described, particularly from parts of South and West Africa. The patients tend to present later than those with vitamin D deficiency rickets and respond better to oral calcium than to vitamin D [5]. Calcium deficiency may also compound the problems of vitamin D deficiency and make it more likely that rickets will develop. Studies in the north of England and in India have

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1␣, 25 (OH)2D 200 180 160 140 120 100 80 60 40 20 0 –50

0

50

100

150

200

250

300

Days on treatment

Fig. 2. Response of 1,25(OH)2D to treatment with 3,000 IU vitamin D daily in a patient with vitamin D deficiency. There is a rapid rise in 1,25(OH)2D which peaks within 72 h and falls gradually over the next few months whilst remaining above the normal physiological range. Despite this, the patient did not become hypercalcaemic. Adapted from Papapoulos et al. [7].

demonstrated that, for the same degree of vitamin D deficiency, rickets is more likely to be present in Indian adolescent girls who have a lower calcium intake [6].

Treatment of Vitamin D Deficiency Rickets The treatment of choice for vitamin D deficiency is vitamin D either in the form of colecalciferol or ergocalciferol. Either preparation is effective if given in adequate dose although it is possible that ergocalciferol is slightly less effective. For a more detailed discussion of this and recommended doses, see chapter 13. Once treatment has been instituted, the biochemical abnormalities usually revert to normal within a few weeks, although 1,25(OH)2D levels may remain high for several months. PTH levels usually fall to within the normal range as normocalcaemia is restored. However, occasionally PTH may remain elevated and hypercalcaemia ensues as a state of tertiary hyperparathyroidism evolves. In this case parathyroidectomy is required (see chapter 15, case 13). The use of 1α-hydroxylated metabolites of vitamin D, either alfacalcidol or calcitriol, is not to be recommended in nutritional vitamin D deficiency. There are two reasons for this. Firstly, the use of these compounds does nothing to restore vitamin D levels. Secondly, levels of 1,25(OH)2D rise to supraphysiological amounts following the onset of treatment in severe rickets [7] (fig. 2). If only physiological amounts of 1α-hydroxylated vitamin D metabolites are given, these high levels in plasma are not

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achieved. If supraphysiological doses are used, they must be monitored to ensure that hypercalcaemia does not become a problem. Orthopaedic intervention is sometimes required especially if genu valgum or genu varum is sufficiently severe as to interfere with locomotion. However, even quite severe bone deformities may correct themselves adequately with appropriate therapy, especially in young children, and sufficient time must be given to allow for this to happen before referral to an orthopaedic surgeon.

Rickets Caused by Abnormalities of Vitamin D Metabolism The genetic causes of calciopaenic rickets are summarised in the first part of table 2.

Defects in 25-Hydroxylase Activity

Liver Disease The first stage of vitamin D metabolism, 25-hydroxylation, occurs principally in the liver. For a detailed description of this, see chapter 2. There are now thought to be at least four enzymes that are involved in 25-hydroxylation, two of which do so as a by-product of drug metabolism. Of the others, one is a high-capacity, low-affinity enzyme and the other a lowcapacity, high-affinity enzyme. Consequently, liver disease rarely causes significant problems with vitamin D metabolism if adequate supplies of vitamin D are available. Patients with chronic liver disease are usually given vitamin D supplementation. Occasionally, premature infants who develop a neonatal hepatitis may not respond adequately to supplementation with vitamin D or alfacalcidol and calcitriol therapy is required.

Selective 25-Hydroxy Vitamin D3 Deficiency (#600081) There is one description of two brothers who were thought to have a selective mutation in one of the 25-hydroxylase enzymes (CYP2R1) [8]. Mild hypocalcaemia and hypophosphataemia, raised alkaline phosphatase and low 25OHD levels, despite adequate vitamin D intake and no evidence of liver disease, were present. They required high vitamin D intake to maintain adequate 25OHD levels.

Vitamin D-Dependent Rickets Type 1 (VDDR-I, 1α-Hydroxylase Deficiency) (#264700) The second step in vitamin D metabolism is controlled by a highly regulated single enzyme. This condition was first reported in 1958 [9]. Successful treatment with

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calcitriol was reported in 1961 [10]. Mutations in this enzyme are responsible for the syndrome variously referred to as vitamin D-dependent rickets type 1, Prader rickets, pseudovitamin D deficiency rickets or 1α-hydroxylase deficiency. It is caused by mutations in the CYP27B1 gene (*609506). Patients with this condition usually present during the toddler age as they begin to walk [11]. The clinical and radiological features are similar to those of vitamin D deficiency rickets. The biochemical features of mild hypocalcaemia, hypophosphataemia and raised alkaline phosphatase and PTH are also present. However, 25OHD levels are normal whilst 1,25(OH)2D levels are low or (inappropriately) at the lower end of the normal range. They do not respond to treatment with vitamin D and require a 1α-hydroxylated compound, either alfacalcidol or calcitriol. The former, where available, is generally preferred as it only needs to be administered once a day. Initially, in order to mimic the raised levels of 1,25(OH)2D that occur following the start of treatment of vitamin D deficiency rickets, supraphysiological doses may be required until bone healing has occurred. Subsequently, these patients can be maintained on physiological doses (30– 50 ng/kg/day). Complete healing of bones occurs and remodelling to reverse bowing or genu valgum, etc. usually takes place satisfactorily if it not too severe at the time of diagnosis. However, it should be noted that, since many of these patients are from an ethnic minority, consanguineous families who are at an increased risk of vitamin D deficiency, vitamin D supplementation may also be required (see chapter 15, case 21).

Rickets Caused by Abnormalities of Vitamin D Responsiveness Hereditary 1α,25(OH)2D-resistant rickets (HVDRR) (#277440), sometimes referred to as vitamin D-dependent rickets type 2, is an autosomal-recessive condition caused by mutations in the vitamin D receptor (VDR) (*601769) and was first described in 1978. These mutations may cause abnormalities of either ligand binding (ligand binding negative) or DNA binding (ligand binding positive). In general, those patients who are ligand binding negative tend to be less severely affected and have normal hair. Those who are ligand binding positive often have an associated alopecia and are more severely affected although alopecia does not necessarily predict the severity of the condition [12]. Patients usually present during infancy or as toddlers with typical features of rickets and failure to thrive although milder cases may not present until adolescence. Radiological features of rickets are present and the biochemical features of hypocalcaemia, which may be quite severe, hypophosphataemia, raised alkaline phosphatase, and PTH are present. In contrast to patients with 1α-hydroxylase deficiency, 1,25(OH)2D levels are raised even if treatment has not yet been instituted. This is driven by a combination of a raised PTH, hypophosphataemia and deficiency of 24-hydroxylation. If treatment with 1α-hydroxylated compounds is attempted, 1,25(OH)2D levels rise even further and can achieve nanomolar concentrations.

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Table 2. Summary of the causes and principal features of the various inborn errors of metabolism of vitamin D, distal renal tubular acidosis and sundry other genetic causes of renal tubular disorders that are associated with either hypercalciuria, hyperphosphaturia, nephrocalcinosis or renal failure Clinical condition

OMIM

Location

Gene

Gene product

OMIM

Inheritance

Features

2q33-qter

CYP27A1

vitamin D 25-hydroxylase

*606530

AR

cerebellar ataxia, pseudobulbar palsy, premature atherosclerosis, cataracts; rickets not a feature.

25-Hydroxyvitamin #600081 D deficiency

11p15.2

CYP2R1

*608713

AR

rickets appearing in early childhood; responds to highdose vitamin D or calcitriol.

Nil

7q22.1

CYP3A4

*124010

drug metabolism

Nil

1p31.3p31.2

CYP2J2

*601258

drug metabolism

12q13.1q13.3

CYP27B1

25OHD-1α hydroxylase

*609506

20q13.2q13.3

CYP24A1

25OHD-24 hydroxylase

*126065

Inborn errors of vitamin D metabolism Cerebrotendinous xanthomatosis

#213700

#264700

Vitamin D-dependent rickets type 1

Nil

AR

classical rickets appearing in toddler age range; responds to 1α-hydroxylated vitamin D metabolites may be upregulated in some ethnic groups

1α,25(OH)2Dresistant rickets, ligand binding positive

#277440

12q12q14

VDR

vitamin D receptor

*601769

AR

severe rickets usually unresponsive to vitamin D metabolites; alopecia usual

1α,25(OH)2Dresistant rickets, ligand binding negative

#277440

12q12q14

VDR

vitamin D receptor

*601769

AR

severe rickets usually unresponsive to vitamin D metabolites; alopecia usually absent

Vitamin D-dependent rickets type II with normal vitamin D receptor

%600785 ?

VDR

vitamin D receptor

?

rickets with lower limb deformities; good physical condition; muscle weakness, alopecia, etc. not present

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Table 2. Continued Clinical condition

OMIM

Location

Gene

Gene product

OMIM

band 3 glycoprotein +109270

Inheritance

Features

AD

nephrocalcinosis, nephrolithiasis, rickets

Distal renal tubular acidosis Autosomaldominant distal renal tubular acidosis

#179800

17q21q22

SLC4A1

Distal renal tubular acidosis with progressive nerve deafness

#267300

2cen-q13

ATP6V1B1 B subunit of vacuolar proton pump

*192132

AR

nephrocalcinosis, rickets, sensorineural deafness

Autosomalrecessive distal renal tubular acidosis

#602722

7q33-q34

ATP6N1B

A subunit of vacuolar proton pump

*605239

AR

nephrocalcinosis, nephrolithiasis, rickets

Autosomalrecessive distal renal tubular acidosis

#602722

17q21q22

SLC4A1

band 3 glycoprotein +109270

AR

nephrocalcinosis, nephrolithiasis, rickets; elliptocytosis in some patients

Renal tubular acidosis III

267200

?

?

?

?AR, ?XL

rickets, nephrolithiasis, nephrocalcinosis

AD

hypercalciuria, recurrent calcium oxalate stones

?

hypercalciuria, nephrocalcinosis, dysmorphic features

?

Other renal tubular disorders causing proximal renal tubular acidosis, hypercalciuria, etc. Absorptive hypercalciuria 2

#143870

Absorptive hypercalciuria 1

SAC

soluble adenylyl cyclase

%607258 4q33-qter

?

?

Dent disease 1

#300009

Xp11.22

CLCN5

chloride channel 5

*300008

XLR

rickets, hypercalciuria, hyperphosphaturia, aminoaciduria, nephrolithiasis, renal failure

X-linked recessive nephrolithiasis

#310468

Xp11.22

CLCN5

chloride channel 5

*300008

XLR

nephrolithiasis, renal failure

Low-molecularweight proteinuria with hypercalciuria and nephrocalcinosis

#308990

Xp11.22

CLCN5

chloride channel 5

*300008

XLR

low-molecularweight proteinuria, hypercalciuria, nephrocalcinosis

Rickets

1q24

*605205

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Table 2. Continued Clinical condition

Location

Gene

Gene product

OMIM

Inheritance

Features

X-linked recessive #300554 hypophosphataemic rickets

Xp11.22

CLCN5

chloride channel 5

*300008

XLR

hypophosphataemic rickets ± nephrocalcinosis

Dent disease 2

#300555

Xq26.1

OCRL1

phosphatidylinositol *300535 4,5-bisphosphate-5phosphatase

XLR

similar to Dent 1

Lowe oculocerebrorenal syndrome

#309000

Xq26.1

OCRL1

phosphatidylinositol *300535 4,5-bisphosphate-5phosphatase

XLR

vitamin D-resistant rickets, ocular abnormalities, mental retardation

Wilson’s disease

#277900

13q14.3q21.1

ATP7B

copper transporting *606882 ATPase Beta polypeptide

AR

liver cirrhosis, neurological manifestations, low caeruloplasmin, hypercalciuria, nephrocalcinosis

IMAGE

300290

Chr.X

?

?

XLR

hypercalciuria, hypercalcaemia, IUGR, adrenal insufficiency, mild dysmorphism, hypogonadotrophichypogonadism

Fanconi-Bickel syndrome

#227810

3q26.1q26.3

GLUT2

Glucose transporter 2

AR

Hypophosphataemic rickets, hepatorenal glycogenosis, proximal renal tubulopathy

Fanconi renotubular syndrome

%134600 15q15.3

?

?

AD

Fanconi syndrome, mild rickets

Cystinosis

#219800

CTNS

cystinosin

AR

hypophosphataemic rickets, metabolic acidosis, photophobia, short stature, hypothyroidism, renal failure

126

OMIM

17p13

*138160

*606272

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Table 2. Continued Clinical condition

OMIM

Location

Gene

Gene product

OMIM

Inheritance

Features

Antenatal Bartter syndrome, type 1

#601678

15q15q21.1

SLC12A1

Sodium-potassiumchloride cotransporter-2

*600839

AR

Hypokalaemic hypochloraemic alkalosis, salt wasting, hypercalciuria, nephrocalcinosis, osteopaenia

Antenatal Bartter syndrome, type 2

#241200

11q24

KCNJ1

inward-rectifying apical potassium channel

*600359

AR

hypokalaemic hypochloraemic alkalosis, salt wasting, hypercalciuria, nephrocalcinosis, osteopaenia

Bartter syndrome, type 3

#607364

1p36

CLCNKB

kidney chloride channel B

*602023

AR

hypokalaemic hypochloraemic alkalosis, salt wasting, hypercalciuria, nephrocalcinosis, osteopaenia, occasional hypomagnesaemia

Infantile Bartter syndrome with sensorineural deafness, type 4

#602522

1p31

BSND

barttin

*606412

AR

hypokalaemic hypochloraemic alkalosis, salt wasting, hypercalciuria, nephrocalcinosis, osteopaenia

16q13

SLC12A3

thiazide-sensitive Na-Cl cotransporter

600968

AR

hypochloraemic, hypokalaemic alkalosis, hypocalciuria, renal magnesium wasting

Gitelman syndrome #263800

The conditions are shown with their genes and OMIM numbers as well as mode of inheritance and main clinical features. Note that not all of these conditions are associated with rickets.

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Several different treatment modalities have been used. Milder forms of the condition will sometimes respond to supraphysiological doses of vitamin D, alfacalcidol or calcitriol. In one report oral phosphate effected some improvement where calcitriol had failed to do so [13] and in another 24,25(OH)2D was apparently used with success [14]. However, in the absence of a significant response, intravenous calcium is the treatment of choice. This will usually need to be given via a central venous line by continuous infusion initially. Once bone healing has been achieved, it may be possible to reduce the time over which the calcium is infused and high-dose oral calcium supplementation may be sufficient to maintain normal biochemistry, at least temporarily, particularly at times of less rapid growth during childhood. At the onset of treatment, there may be reductions in both plasma phosphate and plasma magnesium, presumably in response to the bone mineralisation, and attention should be paid to ensuring that these are adequately corrected (see chapter 15, case 22).

Vitamin D-Dependent Rickets with Normal Vitamin D Receptor (%600785) One other form of vitamin D receptor defect has been described from South America [15]. These patients present with mainly lower limb deformities but are otherwise well with normal muscle power and none of the other features normally associated with rickets. Alopecia is not a feature. Plasma calcium levels are low or low-normal and 1,25(OH)2D and alkaline phosphatase elevated. No abnormalities have been demonstrated in the VDR [16] and post-translation defects leading to failure of normal protein binding are thought to be the cause [17].

Renal Tubular Acidosis

Two kinds of renal tubular acidosis (RTA) are described, both of which may be associated with rickets. Proximal RTA is caused by one of a variety of defects in bicarbonate reabsorption in the proximal renal tubule. It is usually a feature of the Fanconi syndrome, of which there is a variety of causes, and may occur as a secondary phenomenon in, for instance, vitamin D deficiency rickets as a result of the raised PTH. The presence of a mild metabolic acidosis in association with rickets should not automatically lead to a diagnosis of RTA. The acidosis corrects on treatment with vitamin D. Another feature of the Fanconi syndrome is hypophosphataemia and hyperphosphaturia. There are also several other renal tubular conditions that are variously associated with mostly proximal renal tubular abnormalities including acidosis, hypercalciuria, hyperphosphaturia, nephrocalcinosis and renal failure. These are not discussed here but the causes and principal features of these renal tubular disorders are shown in the third section of table 2.

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Distal RTA (DRTA) The mechanisms that are involved in acidifying the urine in order to excrete excess acid produced as a result of cellular metabolism are very similar to those that enable osteoclasts to produce acid as a means of dissolving bone mineral during the process of bone resorption. Indeed, some of the same enzymes play a part in both processes. These are summarized in figure 3. Hydrogen ions, together with bicarbonate ions, are generated by carbonic anhydrase (CA2) from water and carbon dioxide. The bicarbonate ions are reabsorbed from the renal tubular cells across the basement membrane in exchange for chloride ions via an anion exchanger (SLC4A1). The hydrogen ions are then excreted together with the chloride into the renal tubular lumen via a proton pump, which has two subunits, A1 and B1. Defects in any of these enzymes can cause DRTA. These patients can present at any age from the neonatal period onwards. Rickets and failure to thrive are the principal features. Radiological features are similar to those of vitamin D deficiency rickets. Biochemical features include a metabolic acidosis which, unlike proximal RTA, has no threshold for urine acidification. A notable feature is the presence of hypercalciuria which may lead to nephrocalcinosis and, ultimately, renal failure. It is essential to make a correct diagnosis as treatment with vitamin D analogues worsens the hypercalciuria whilst treatment with bicarbonate corrects it. Hypokalaemia may also sometimes be present. The causes and principal features of DRTA are shown in the second part of table 2.

Distal RTA without Progressive Deafness (RTADR) (#602722) This is an autosomal-recessive condition caused by mutations in the ATP6V0A4 gene (*605239) that codes for the A subunit of the proton pump. Acidosis may be present from as early as 3 weeks of age. Mild hearing loss may become a feature in later life although this is not a major feature of the condition [18]. Distal RTA with progressive nerve deafness (#267300) is also an autosomal-recessive condition caused by mutations in the ATP6B1 gene (*192132) which codes for the B subunit of the apical proton pump [19]. This enzyme is also present in the cochlea and progressive nerve deafness results from damage to this organ. The clinical course of this condition may vary even within the same families [20].

Distal RTA and Elliptocytosis Mutations in the anion exchanger SLC4A1 gene (+109270) have been shown to cause both autosomal-dominant and autosomal-recessive DRTA. This enzyme is also present in red cell membranes where it is known as band 3 protein. These mutations may

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Fig. 3. Schematic representation of the generation of acid urine by renal tubular cells showing the similarities between this and the generation of an acid environment by osteoclasts (see chapter 12). Defects in any of the enzyme systems can give rise to distal renal tubular acidosis. At least one of the enzymes, carbonic anhydrase 2, is shared with osteoclasts. Mutations in the gene for this enzyme cause osteopetrosis with DRTA.

ECF

Lumen

Carbonic anhydrase II* H2O + CO2 H– + HCO–3 H+

A B##

Anion exchanger

Proton pump

HCO–3

Cl–

#Also in cochlea

*Also in osteoclasts

also cause elliptocytosis. A wide variety of mutations has been described and the nature of the mutation determines whether DTRA or elliptocytosis is the predominant clinical feature. Most of these mutations have been described in South Asian patients and some of them may confer a degree of resistance to malaria [21].

Osteopetrosis Type 3 (OPTB3) (#259730) A mild form of DTRA is present in OPTB3, an osteoclast-rich form of osteopetrosis. It is caused by mutations in the carbonic anhydrase II (CA2) gene (*611492). This is present both in renal tubules, where it causes DTRA, and in osteoclasts where it generates the acid environment that allows bone resorption to be initiated [22]. It is autosomal recessive. The osteopetrosis usually presents in infancy with a history of fractures and the RTA develops later in adolescence or early adulthood.

Treatment of DRTA Acid excretion, in contrast to bicarbonate reabsorption, to maintain normal acid-base balance is relatively modest. Treatment of DRTA consists of oral supplementation with a suitable bicarbonate preparation in sufficient quantity to maintain a normal pH in plasma. Monitoring of renal calcium excretion is important to prevent progressive nephrocalcinosis and bicarbonate therapy needs to be maintained indefinitely. Vitamin D analogues should not be used.

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Conclusions

Rickets may result from a wide variety of causes. Vitamin D deficiency remains the single most common cause of rickets, even in developed countries and in those with a sunny climate. Whilst most of the other causes are rare, it is important to make a correct diagnosis since appropriate treatment can then be instituted without causing harm. It is also important to recognise that most forms of rickets can be adequately treated if a correct diagnosis is made. Making that diagnosis depends on a thorough understanding of the physiology of vitamin D and a logical approach to diagnosis.

References 1 Arnaud SB, Arnaud CD, Bordier PJ: The interrelationships between vitamin D and parathyroid hormone in disorders of mineral metabolism in man; in Norman AW (ed): Vitamin D and Problems of Uremic Bone Disease. Amsterdam, de Gruyter, 1975, pp 397–416. 2 Teotia M, Teotia SP: Nutritional and metabolic rickets. Indian J Pediatr 1997;64:153–157. 3 Maiya S, Sullivan I, Allgrove J, et al: Hypocalcaemia and vitamin D deficiency: an important, but preventable cause of life threatening infant heart failure. Heart 2008;94:581–584. 4 Ladhani S, Srinivasan L, Buchanan C, Allgrove J: Presentation of vitamin D deficiency. Arch Dis Child 2004;89:781–784. 5 Thacher TD, Fischer PR, Strand MA, Pettifor JM: Nutritional rickets around the world: causes and future directions. Ann Trop Paediatr 2006;26:1–16. 6 Khadilkar A, Das G, Sayyad M, et al: Low calcium intake and hypovitaminosis D in adolescent girls. Arch Dis Child 2007;92:1045. 7 Papapoulos SE, Clemens TL, Fraher LJ, Gleed J, O’Riordan JL: Metabolites of vitamin D in human vitamin-D deficiency: effect of vitamin D3 or 1,25-dihydroxycholecalciferol. Lancet 1980;ii:612– 615. 8 Casella SJ, Reiner BJ, Chen TC, Holick MF, Harrison HE: A possible genetic defect in 25-hydroxylation as a cause of rickets; in Norman AW (ed): Vitamin D and Problems of Uremic Bone Disease. Amsterdam, de Gruyter, 1994, pp 929–932. 9 Fraser D, Salter RB: The diagnosis and management of the various types of rickets. Pediatr Clin North Am 1958;417–441. 10 Prader A, Illig R, Heierli E: An unusual form of primary vitamin D-resistant rickets with hypocalcemia and autosomal-dominant hereditary transmission: hereditary pseudo-deficiency rickets. Helv Paediatr Acta 1961;16:452–468.

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11 Kitanaka S, Takeyama K, Murayama A, et al: Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene in patients with pseudovitamin D-deficiency rickets. N Engl J Med 1998; 338:653–661. 12 Fraher LJ, Karmali R, Hinde FR, et al: Vitamin D-dependent rickets type II: extreme end organ resistance to 1,25-dihydroxy vitamin D3 in a patient without alopecia. Eur J Pediatr 1986;145:389–395. 13 Rosen JF, Fleischman AR, Finberg L, Hamstra A, DeLuca HF: Rickets with alopecia: an inborn error of vitamin D metabolism. J Pediatr 1979;94:729– 735. 14 Liberman UA, Samuel R, Halabe A, et al: End-organ resistance to 1,25-dihydroxycholecalciferol. Lancet 1980;i:504–506. 15 Giraldo A, Pino W, Garcia-Ramirez LF, Pineda M, Iglesias A: Vitamin D dependent rickets type II and normal vitamin D receptor cDNA sequence: a cluster in a rural area of Cauca, Colombia, with more than 200 affected children. Clin Genet 1995;48:57– 65. 16 Hewison M, Rut AR, Kristjansson K, et al: Tissue resistance to 1,25-dihydroxyvitamin D without a mutation of the vitamin D receptor gene. Clin Endocrinol (Oxf) 1993;39:663–670. 17 Chen H, Hewison M, Hu B, Adams JS: Heterogeneous nuclear ribonucleoprotein (hnRNP) binding to hormone response elements: a cause of vitamin D resistance. Proc Natl Acad Sci USA 2003; 100:6109–6114. 18 Stover EH, Borthwick KJ, Bavalia C, et al: Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss. J Med Genet 2002;39:796– 803.

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19 Karet FE, Finberg KE, Nelson RD, et al: Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness. Nat Genet 1999;21:84–90. 20 Feldman M, Prikis M, Athanasiou Y, Elia A, Pierides A, Deltas CC: Molecular investigation and longterm clinical progress in Greek Cypriot families with recessive distal renal tubular acidosis and sensorineural deafness due to mutations in the ATP6V1B1 gene. Clin Genet 2006;69:135–144.

21 Bruce LJ, Wrong O, Toye AM, et al: Band 3 mutations, renal tubular acidosis and South-East Asian ovalocytosis in Malaysia and Papua New Guinea: loss of up to 95% band 3 transport in red cells. Biochem J 2000;350 Pt 1:41–51. 22 Sly WS, Whyte MP, Sundaram V, et al: Carbonic anhydrase II deficiency in 12 families with the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. N Engl J Med 1985;313:139–145.

Jeremy Allgrove, MD Department of Paediatric Endocrinology, First Floor, David Hughes Building Royal London Hospital, Whitechapel London E1 1BB (UK) Tel. +44 20 7377 7468, Fax +44 20 7943 1353, E-Mail [email protected]

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Chapter 9 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 133–156

Disorders of Phosphate Homeostasis and Tissue Mineralisation Clemens Bergwitza ⭈ Harald Jüppnerb a

Endocrine and bPediatric Units, Massachusetts General Hospital, Boston, Mass., USA

Abstract Phosphate is absorbed from the diet in the gut, stored as hydroxyapatite in the skeleton, and excreted with the urine. The balance between these compartments determines the circulating phosphate concentration. Fibroblast growth factor 23 (FGF23) has recently been discovered and is part of a previously unrecognised hormonal bone-kidney axis. Phosphate-regulating gene with homologies to endopeptidases on the X chromosome, and dentin matrix protein 1 regulate the expression of FGF23 in osteocytes, which then is O-glycosylated by UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyl-transferase 3 and secreted into the circulation. FGF23 binds with high affinity to fibroblast growth factor receptor 1c in the presence of its co-receptor Klotho. It inhibits, either directly or indirectly, reabsorption of phosphate and the synthesis of 1,25-dihydroxy-vitamin-D by the renal proximal tubule and the secretion of parathyroid hormone by the parathyroid glands. Acquired or inborn errors affecting this newly discovered hormonal system can lead to abnormal phosphate homeostasis and/or tissue mineralisation. This chapter will provide an update on the current knowledge of the pathophysiology, the clinical presentation, diagnostic evaluation and therapy of the disorders of phosphate homeostasis and tissue mineralisation. Copyright © 2009 S. Karger AG, Basel

In contrast to the regulation of calcium homeostasis, which has been extensively studied over the past several decades [1], relatively little is known about the regulation of phosphate homeostasis. Most important, yet often completely unexpected, insights into the regulation of phosphate homeostasis were obtained through the definition of genetic mutations underlying rare inherited disorders in humans. For example, mutations in phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) provided a molecular genetic explanation for X-linked hypophosphataemia (XLH), the most frequent form of renal phosphate-wasting [2]. Likewise, positional cloning strategies led to the identification of mutations in the chloride channel CLCN5 as the cause of X-linked recessive nephrolithiasis (Dent’s disease) [3], in fibroblast growth factor 23 (FGF23) as the cause

of autosomal-dominant hypophosphataemic rickets (ADHR) [4], in dentin matrix protein 1 (DMP1) as a cause of autosomal-recessive hypophosphataemia (ARHP) [5], and in UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) as the cause of a form of tumoral calcinosis [6]. These findings were extended by the generation of a number of animal models, including the Klotho-null mouse, which has led to particularly important insights [7]. This mouse, which has a phenotype resembling hyperphosphataemic familial tumoral calcinosis (HFTC) in humans, lacks the expression of alpha Klotho (KL), which has a single membrane-spanning domain, and was subsequently found to be a co-receptor of FGF23 [8, 9]. Based on the murine phenotype, KL mutations were recently identified by the candidate-gene approach as a cause of hyperphosphataemia in familial tumoral calcinosis type 3 [10] (see below). The circulating phosphate concentration is determined by the balance between intestinal absorption of phosphate from the diet, storage of phosphate in the skeleton, and reabsorption of phosphate from the urine. It is taken up from the circulation into cells via type II and type III sodium-phosphate co-transporters to facilitate cellular functions such as DNA and membrane lipid synthesis, generation of high-energy phosphate esters, and intracellular signalling. Only 30% of intestinal phosphate absorption occurs in a regulated, 1,25(OH)2D-dependent manner [11]. Consequently, reabsorption of phosphate from the urine in the renal proximal tubules via type II and type III sodium-phosphate co-transporters plays a key role in maintaining serum phosphate homeostasis, while excess phosphate is excreted (for recent reviews, see [12–20]). Renal phosphate reabsorption lies under tight hormonal control by PTH and FGF23 and, to a lesser extent, by insulin, by the hormones of the somatotropic pituitary axis [21], by FGF7 [22] and possibly by matrix extracellular phosphoglycoprotein (MEPE) and secreted frizzled-related protein 4 (sFRP-4) [20]. FGF23 is part of a newly discovered endocrine bone-kidney axis [23, 24]. PHEX and DMP1 regulate the expression of FGF23 in osteocytes, which is secreted into the circulation after undergoing O-glycosylation by GALNT3 [25]. FGF23 binds to FGFR1c and its co-receptor KL [9, 26] and activation of this receptor complex inhibits, either directly or indirectly, the reabsorption of phosphate by reducing the expression of the sodium-phosphate cotransporters NaPi-IIa and NaPi-IIc at the brush border membrane of the proximal renal tubules [27]. FGF23 also decreases the synthesis of 1,25(OH)2D in this portion of the renal tubules [28, 29] and it appears to reduce secretion of PTH by the parathyroid glands [30, 31]. For a more detailed description of phosphate metabolism, see chapter 3. This chapter will provide an update on the pathophysiology, clinical presentation, diagnostic evaluation and therapy of the disorders associated with these factors. Disorders affecting both calcium and phosphate homeostasis such as parathyroid dysfunction, disorders of vitamin D metabolism and action, and generalised proximal renal tubular dysfunction such as Fanconi syndrome or renal tubular acidosis are important considerations in the differential diagnosis, but will not be discussed here.

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Epidemiology

XLH is the most common inherited disorder of phosphate homeostasis affecting 1:20,000 births [32], while the other inherited forms are considered to be rare. Among the acquired disorders of phosphate homeostasis are post-renal transplant hypophosphataemia [33, 34], burn injury-related [35] and post-hepatectomy hypophosphataemia [36], which are increasingly recognised abnormalities. Patients affected by these disorders show an inappropriately high renal phosphate excretion, which appears to go along with elevated FGF23 levels in the case of post-renal transplant hypophosphataemia, while the role of FGF23 in the other two processes remains to be clarified. FGF23 levels are also increased in early chronic kidney disease (stages 2–3) before the development of anaemia, which may help to maintain normophosphataemia, but is likely to suppress 1α-hydroxylase and thus may also support the development of secondary hyperparathyroidism [37, 38]. Tumour-induced osteomalacia, on the other hand, is comparatively rare, with only a few hundred cases described in the literature to date [39, 40].

Clinical Assessment of Phosphate Homeostasis

The clinical assessment of phosphate homeostasis can be challenging: serum phosphate concentrations are influenced by the time of day, relationship to meals, and age of the subject, and none of the methods for determination of tubular reabsorption is entirely satisfactory. To determine the cause of abnormal serum phosphate levels in a patient who has normal parathyroid and renal function, we generally first assess his or her tubular reabsorption for phosphate (%TRP). For this purpose the patient is asked to collect a 3-hour timed urine for phosphate and creatinine, along with the corresponding serum parameters after an eight hour fast. %TRP is then calculated according to the formula depicted in figure 1. A timed post-fasting urine is requested to avoid fluctuations as a result of variable absorption of phosphate and possible effects of insulin or glucose on phosphate handling. The tubular maximum of reabsorption for phosphate (TmP/GFR) is derived from a nomogram, which was devised by Walton and Bijvoet [41] to correct for the non-linear relationships of %TRP and TmP/GFR when TRP is higher than 80%. TmP/GFR reflects the threshold of the serum phosphate concentration above which phosphate is no longer fully reclaimed from the glomerular filtrate in the kidney. While the TmP/GFR derived from the Walton and Bijvoet nomogram is generally sufficient in adults, the nomogram does not accommodate the higher normal range of serum phosphate values in newborns and toddlers and thus calculation of TP/GFR may be more accurate in this paediatric population [42] (fig. 1). Inappropriately high %TRP in the setting of hyperphosphataemia or inappropriately low %TRP in the setting of hypophosphataemia is suggestive of a proximal renal tubular defect as the underlying cause, which

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ea

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0.9

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1.6 1.8

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0.9 5.0

2.0

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0. 00

1.4

1. 00

Actual plasma phosphate conc. (PO4)

1.0

Renal threshold phosphate conc. (TmPO4/GFR)

1. 00

0

0

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CP

Fig. 1. Calculation of %TRP, TmP/GFR, and TP/GFR. To determine TmP/GRF, first calculate % TRP (% TRP = 1 – (UPO4 × Screat)/(SPO4 × Ucreat) (note that the units for both phosphate and both creatinine measurements must be the same) and then derive TmP/GFR from the Walton and Bijvoet nomogram [41]: The inner axes are mmol/L and the outer axes mg/100 ml. TP/GFR is calculated using the following formula: SPO4 – (UPO4 × Screat/Ucreat), using simultaneous urine and blood creatinine and phosphorus concentrations.

0.0

can subsequently be further classified by determining the patient’s vitamin D status: concordantly (inappropriately) reduced %TRP and 1,25(OH)2D levels suggest excess FGF23 action, while increased %TRP and 1,25(OH)2D levels suggest diminished activity of this hormone. In contrast, discordantly (appropriately) elevated 1,25(OH)2D levels suggest an FGF23-independent, possibly primary, renal tubular defect leading to abnormal serum phosphate levels (table 1). Excess production of 1,25(OH)2D may lead to increased absorption of calcium in the gut, resulting in hypercalciuria and some suppression of PTH production and may, in the setting of hypophosphataemia, be diagnostic for HHRH [43]. It is important to keep in mind that vitamin D deficiency and secondary hyperparathyroidism may mask these findings and need to be corrected before the above testing [44]. Circulating FGF23 levels can be determined from EDTA plasma, which preserves FGF23 relatively well, using several commercially available enzyme-linked immunometric assays [40, 45, 46]. The existing assays permit the diagnosis of FGF23dependent disorders of phosphate homeostasis, when FGF23 levels are elevated above the normal range [47, 48]. None of the currently available assays, however, is sensitive enough to detect suppressed or inappropriately normal FGF23 levels with sufficient confidence, thus limiting their utility for distinguishing FGF23-independent hypophosphataemic disorders such as HHRH from the FGF23-dependent hypophosphataemic disorders [40, 45]. The C-terminal FGF23 assay (Immutopics, Inc., San Clemente, Calif., USA) uses antibodies directed against two distinct epitopes within the C-terminal region of FGF23 and thus could detect intact FGF23 and C-terminal

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Table 1. Serum biochemical findings in disorders of phosphate homeostasis and tissue mineralisation Parameter

Hypophosphataemia

Hyperphosphataemia

Normophosphataemia

FGF23dependent

FGF23independent

FGF23deficient

FGF23resistant

Acquired

TIO, post-renal transplant

Post hepatectomy

NA

NA

NA

Inherited

XLH, ADHR, ARHP, OGD, OSD, FD/MAS, NF1+2

HHRH

HFTC1, HFTC2

HFTC3

pulmonary alveolar microlithiasis, NFTC

S-Ca

NL

NL

NL to high

NL to high

NL

S-PO4

low

low

high

high

NL

S-PTH

NL to high

NL to low

NL to low

NL to high

NL

S-1,25(OH)2D

NL to LOW

HIGH

high

high

NL

S-FGF23

NL to HIGH

low

low

high

NL

U-PO4

HIGH

high

low

low

NL

U-Ca

NL to LOW

high

NL to HIGH

NL to HIGH

NL

Current treatment

phosphate and alfacalcidol or calcitriol

phosphate replacement only

phosphate binders, acetazolamide, PTH

phosphate binders, acetazolamide, PTH

supportive measures

fragments; this assay appears to be particularly helpful for distinguishing hereditary familial tumoral calcinosis (HFTC) 1 and 2, from HFTC3 (see below for details), if genetic testing is unavailable [10, 49, 50].

Clinical Signs of Hyperphosphataemia

Clinical signs of chronic hyperphosphataemia include ectopic tissue mineralisation of juxta-articular muscular and subcutaneous tissues (fig. 5). Patients with tumoral calcinosis often also show dental pulp stones, which may lead to a complete obliteration of the dental pulp cavities (fig. 3a). Other clinical features, which may constitute the only clinical evidence for tumoral calcinosis, can include eyelid calcifications,

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a

e

c

b

f

d

g

Fig. 2. Skeletal findings in XLH. a Femoral bowing in XLH with looser zones (arrow) and proliferative changes along the linea aspera (arrowheads). b Periosteal bone proliferation in the forearm along the interosseous membrane with pseudarthrosis formation (arrowheads) and Looser zones (arrow). c Endosteal bone proliferation (arrows). d Enthesopathic changes at both greater and lesser femoral trochanters. e Diffusely increased lateral lumbar bone density. f coarsened vertebral trabecular pattern. g Loss of most metaphyseal trabeculae in the tibia (from [137], with permission).

a

b

Fig. 3. a Dental findings. Dental radiograph demonstrating sclerotic teeth with blunt roots and obliterated pulp cavity in a patient with HFTC1 (from fig. 1a in [138], with permission). b Orthopantograph showing deciduous molar teeth with stainless steel crowns and dental cyst at the lower canine teeth (fig. 2 from [139], with permission).

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vascular calcifications, and/or nephrocalcinosis (fig. 4). There can also be mineralisation of the juxta-articular bone marrow cavities. However, the remaining skeleton often shows low bone mineral density due to a mineralisation defect, which at the moment is only poorly understood [51, 52]. Pulmonary alveolar microlithiasis (PAM) is diagnostic for patients with homozygous loss-of-function mutations of NaPi-IIb (SLC34A2) and may be due to the local accumulation of phosphate (fig. 7) [53]. PAM is initially asymptomatic and may be an incidental finding on radiographic images, but can lead to slow and potentially fatal deterioration of pulmonary function [54].

Clinical Signs of Hypophosphataemia

Bone pain, bowing and waddling gait is the classical diagnostic triad for hypophosphataemic rickets in growing children; osteomalacia is the corresponding finding in adults. The radiological findings of rickets and osteomalacia include undermineralisation of the osteoid, which leads to a blurring of the microtrabecular architecture (fig. 2f, g). The consequences are bone pain and impaired mechanical properties of the affected bones leading to bowing, and stress fractures (looser zones, fig. 2a, b). Lack of chondrocyte apoptosis in the growing skeleton leads to an expansion of the epiphyses, giving rise to swollen wrists and rachitic rosary [55]. Serum biochemical findings suggesting rickets and osteomalacia include elevated bone specific alkaline phosphatase, osteocalcin, procollagen, pyridinoline cross-links and N- and C-telopeptides [56, 57]. When compared to the effects on the skeleton, the mechanism of muscle weakness caused by hypophosphataemia is less well understood and may be related to the role of phosphate in intracellular signal transduction and synthesis of ATP or creatine phosphate [56, 57]. While rickets or osteomalacia are observed to variable degrees with all hypophosphataemic disorders, subtle but important differences can guide the differential diagnostic and therapeutic decisions. Enthesopathies occur in patients with XLH, ADHR and ARHP. The term refers to painful or indolent mineral deposits near the insertion sites of tendons usually at the lower extremities (fig. 2d, e), which can be identified on radiographs [58]. Patients with XLH may also form dental cysts leading to tooth decay (fig. 3b), craniosynostosis, midfacial hypoplasia, and frontal bossing, which may be related to severity of hypophosphataemia, but often cannot be reversed or prevented by therapy with 1,25(OH)2D and phosphate supplements. Thus, local effects of FGF23 excess and activation of canonical FGF receptor (KL-independent) signalling may play a role [58, 59].

Genetic Testing

Genetic testing for the inherited disorders discussed is generally done by direct sequence analysis of the affected genes. In the UK testing for genes causing

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hypophosphataemic rickets is undertaken by the Clinical Genetics Laboratory at the Royal Devon and Exeter Hospital (http://www.rdehospital.nhs.uk/prof/molecular_ genetics/tests/clinical_genetics/rickets.html). In the USA testing is offered by a number of commercial and non-commercial laboratories to which the website of GeneDx provides a helpful directory (http://www.genedx.com/).

Disorders of Renal Phosphate Excretion

Hyperphosphataemic Disorders Hyperphosphataemic Familial Tumoral Calcinosis, (HFTC) (#211900) Tumoral calcinosis is a clinically and genetically heterogeneous group of disorders first described by [61] and then by [62]. Tumoral calcinosis is characterised by calcium-phosphate deposits in tissues but, distinct from disorders of heterotopic ossification, osteogenic cells and matrix formation are absent. For the purpose of this chapter, the hyperphosphataemic forms of familial tumoral calcinosis are classified as types 1–3 (HFTC1–3), all of which follow an autosomal-recessive mode of inheritance and all, furthermore, show inappropriately enhanced renal tubular absorption of phosphate leading to hyperphosphataemia as a common pathophysiological mechanism. The activity of renal 1α-hydroxylase is increased resulting in elevated serum 1,25(OH)2D levels and thus increased intestinal absorption of calcium (and phosphate), suppression of parathyroid hormone production and hypercalciuria. The increased serum calcium-phosphate product then leads to the characteristic tissue mineralisations observed in tumoral calcinosis (fig. 4, 5). Familial tumoral calcinosis type 1 (HFTC1) is caused by homozygous loss-of-function mutations in the gene encoding GALNT3 [6] (*601756). GALNT3 is responsible for O-glycosylation and proper secretion of intact FGF23 [25]. Patients with HFTC1 characteristically have low or undetectable intact, but increased C-terminal FGF23 levels. HFTC1 is allelic with hyperostosis-hyperphosphataemia syndrome (HSS) (#610233) which, in addition to the serum-biochemical abnormalities, is characterised by recurrent, transient, painful swellings of the long bones associated with the radiographic findings of periosteal reaction and cortical hyperostosis (fig. 6) [63, 64]. HSS can be distinguished from HFTC1 by the presence of bone involvement and the absence of skin involvement. HFTC2 is caused by homozygous loss-of-function mutations in FGF23 (*605380), which presumably interfere with normal O-glycosylation and secretion of bioactive intact FGF23. HFTC2 is characterised by low or undetectable circulating intact FGF23 levels [65, 66]. However, C-terminal FGF23 fragments may be secreted and thus also in patients with HFTC2 normal or high levels of FGF23 are often detected in the circulation [50].

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e a

c

b

d

f

Fig. 4. Tumoral calcinosis. Clinical features of a patient with HFTC2 showing developmental deformity of the forearm. a Periarticular swelling of the knee. b Eye-lid calcifications. c Renal medullary calcifications. d osteopaenia and osteosclerosis. e and aortic and valvular calcifications of the heart (fig. 1 from [140], with permission).

Fig. 5. Clinical features in HFTC1. The left panel shows a large subcutaneous tumour over the left outer thigh. The right panel shows a periarticular calcified mass over the left acetabulum (fig. 1b from [6], with permission).

Recently, a 13-year-old girl with a disorder resembling HFTC1 and HFTC2 was reported who had extremely high circulating levels of intact FGF23 [10]. Radiographs of the patient showed osteopaenia, patchy sclerosis in the hands, feet, long bones, and calvaria, intracranial calcifications, and calcifications of the dura and carotid arteries. Interestingly, and distinct from the first two forms of HFTC, she had elevated PTH levels due to four-gland parathyroid hyperplasia. Nucleotide sequence analysis of the genes encoding FGF23 and GALNT3 revealed no mutation. Very high circulating levels of intact FGF23 were also observed in mice, in which KL (+604824), the co-receptor for FGF23, had been ablated (see above). The authors therefore next decided to analyse the gene encoding KL, which led to the discovery of a homozygous missense mutation in the second putative beta-glycosidase domain. This mutation

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Fig. 6. Hyperostosis hyperphosphataemia syndrome (HHS). Different from patients with HFTC1 tissue calcifications are missing in HHS. Shown here is the characteristic osteosclerosis and mild metaphyseal dilatation of tibia and fibula. Note the bone islands in the left tibia (arrows) (fig. 2 in [63], with permission).

a

b

Fig. 7. Pulmonary alveolar microlithiasis (PAM). a Chest radiograph of a patient affected by PAM, showing fine microliths with a diffuse, uniform spread obscuring the cardiac and diaphragmatic borders (sandstorm lung). b CT scan with diffuse, ground-glass opacities (stony-lung) in all pulmonary fields, and faint calcific densities, sometimes confluent at posterior and inferior subpleural regions (from fig. 1 in [54], with permission).

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presumably inactivates KL, leading to end-organ resistance to FGF23 [10]. We have designated this HFTC3. Heterozygous carriers of the genetic mutations that cause HFTC1–3 are asymptomatic and do not require specific treatment. Treatment of homozygous individuals currently relies on minimising the intestinal absorption of phosphate through appropriate binders such as aluminium hydroxide, or sevelamer [50], and on inhibiting renal phosphate reabsorption with acetazolamide [67]; in one study, treatment with PTH was attempted [68]. Calcilytic agents that reduce the activity of the calciumsensing receptor and thus stimulate endogenous PTH secretion [69] or, once available for human use, recombinant FGF23 may become rational treatments of HFTC in the future.

Hypophosphataemic Disorders Tumour-Induced Osteomalacia (TIO) Tumour-induced osteomalacia (TIO), also referred to as oncogenic osteomalacia (OOM), is an acquired disorder of FGF23 excess [23], or possibly FGF7 excess [22], which are secreted by usually benign mixed connective tissue tumours. Other factors such as matrix extracellular protein (MEPE) [70] or secreted frizzled related protein 4 (sFRP4) [71] were also isolated from TIO tumours and may contribute to abnormal regulation of renal phosphate handling. Drezner [39] reviewed 120 cases of tumourinduced osteomalacia, and he identified four distinct morphologic patterns: • primitive-appearing, mixed connective tissue tumours, • osteoblastoma-like tumours, • nonossifying fibroma-like tumours, and • ossifying fibroma-like tumours. Hypophosphataemia was also described in patients with widespread fibrous dysplasia of bone, neurofibromatosis and linear naevus sebaceous syndrome (see further below) and concurrent with breast carcinoma, prostate carcinoma, oat cell carcinoma, small cell carcinoma, multiple myeloma and chronic lymphocytic leukaemia. Proof of a causal relationship has been that removal of the tumour resulted in appropriate biochemical and radiographic improvements. However, since most cases were reported before the discovery of FGF23, matrix extracellular phosphoglycoprotein (MEPE), or sFRP-4, the phosphaturic factor secreted by these previously reported tumours has not been determined. The tumours are commonly located in the facial skeleton or in the tendons of hands and feet, and may only be a few millimetres large and indolent. They commonly escape detection by physical examination and computed tomography scans, and may require more sensitive techniques for localisation including whole-body octreotide scans [72] or PET-CT scans using [18F]-FDG [73] or [68Ga]-DOTANOC [74] as tracers. Selective vein sampling [75] can permit localisation, particularly in individuals with markedly elevated circulating FGF23 levels

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Table 2. Table of genetically determined hyperphosphataemic and hypophosphataemic disorders with their OMIM numbers, the genes involved together with their OMIM numbers and the chromosomal locations of the genes Disorder

Abbrevia- OMIM tion

Inheritance

Gene

OMIM

Gene location

*601756 2q24-q31

Hyperphosphataemic disorders Hyperphosphataemic familial tumoral calcinosis

HFTC

Hyperphosphataemic familial tumoral calcinosis type 1 Hyperostosishyperphosphataemia syndrome Hyperphosphataemic familial tumoral calcinosis type 2 Hyperphosphataemic familial tumoral calcinosis type 3

HFTC1

#211900

AR

HSS

#610233

AR

GALNT3 UDP-N-acetyl-α-Dgalactosamine:polypeptide N-acetylgalactosaminyltransferease 3

HFTC2

#211900

AR

fibroblast growth factor 23

FGF23

*605380 12p13.3

HFTC3

#211900

AR

Klotho

KL

+604824 13q12

XLD

phosphate-regulating gene with homologies to endopeptidases on the X chromosome

PHEX

*300550 Xp22.2-p22.1

Hypophosphataemic disorders X-linked dominant hypophosphataemic Rickets

XLHR

#307800

Autosomal-dominant hypophosphataemic rickets

ADHR1 ADHR2

#193100 AD %612089 AD

fibroblast growth factor 23 KL

FGF23 KL

*605380 12p13.3 +604824 13q12

Autosomal-recessive hypophosphataemia

ARHP

#241520

AR

dentin matrix acidic phosphoprotein 1

DMP1

*600980 4q21

Hereditary hypophosphataemic rickets with hypercalciuria

HHRH

#241530

AR

solute carrier family 34 (sodium/phosphate cotransporter), member 3

SLC34A3

*609826 9q34

Hypophosphataemia with osteoporosis and nephrolithiasis type I

NPHLOP1

#612286

AD

solute carrier family 34 (sodium/phosphate cotransporter), member 1 (see text p. 148)

SLC34A1?

*182309 5q35

Hypophosphataemia with osteoporosis and nephrolithiasis type II

NPHLOP2

#612287

AD

solute carrier family 9, isoform A3, regulatory factor 1 (see text p. 148)

SLC9A3R1? *604990 17q25.1

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Table 2. Continued Disorder

Abbrevia- OMIM tion

Inheritance

Gene

Osteoglophonic dysplasia

OGD

#166250

AD

fibroblast growth factor receptor 1

Opsismodysplasia

OSD

%258480 AR

not known

%163200 sporadic somatic mutation

fibroblast growth factor receptor 33

FGFR3

#174800

sporadic somatic mutation

GNAS complex locus

GNAS

+139320 20q13.2

#156400

AR

PTH/PTHrP receptor 1

PTHR1

*168468 3p22-p21.1

SchimmelpenningFeuerstein-Mims syndrome McCune-Albright fibrous dysplasia

MAS/FD

Jansen’s metaphyseal chondrodysplasia

OMIM

FGFR1

Gene location

*136350 8p11.2-p11.1

4p16.3

Neurofibromatosis type I

NF1

+162200

AD

neurofibronim

NF1

17q11.2

Neurofibromatosis type II

NF2

#101000

AD

neurofibronim 2 (merlin)

NF2

*607379 22q12.2

#610455

AR

sterile alpha motif domain- SAMD9 containing protein 9

#265100

AR

solute carrier family 34 (sodium/phosphate cotransporter), member 2

Tissue mineralisation disorders Normophosphataemic tumoral calcinosis

NFTC

Pulmonary alveolar microlithiasis

SLC34A2

*610456 7q21 *604217 4p15.31p15.2

[76–78]. Therapy consists of surgical tumour excision, if its location has been revealed, which usually results in normalisation of serum phosphate levels within 24 h. In those patients, where localisation of the tumour is impossible or if resection of the tumour is incomplete, symptomatic therapy is used as will be described in more detail for the inherited forms of hypophosphataemia below.

Other Acquired Syndromes of Renal Phosphate Wasting Another increasingly recognised acquired syndrome of renal phosphate wasting is post-renal transplant hypophosphataemia, which often cannot be attributed to tertiary hyperparathyroidism alone [79, 80]. Bhan et al. [33] and Pande et al. [34] recently showed that post-transplant hypophosphataemia correlated inversely with serum FGF23 levels and coined the term ‘tertiary hyperphosphatonism’ due to persistent production of FGF23, which is longer than would be expected from the half-life of the

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hormone [33, 81]. Hypophosphataemia in the setting of inappropriate renal phosphate excretion has also been recognised with severe burn injuries [82, 83], and after partial hepatectomy [36], although it may be independent of FGF23 in these cases [35]. X-Linked Dominant Hypophosphataemia (XLH) (#307800) XLH, the most common form of hypophosphataemia, was first recognised by Albright et al. [84] in 1937. Lack of male-to-male transmission was observed by Winters et al. [85] in 1958 and suggested X-linked inheritance. Using a positional cloning approach, the genetic defect was ultimately identified in 1995 [2] and a large number of different loss-of-function mutations in PHEX, phosphate-regulating gene with homologies to endopeptidases on the X chromosome (*300550), have since been reported [86]. Details of these mutations are available on a dedicated database (http://www.phexdb. mcgill.ca/). It remains uncertain why males and females are equally affected. Deletion of the Phex gene in hyp mice results in increased FGF23 gene transcription in osteocytes resulting in increased circulating levels of FGF23 and thus renal phosphate wasting [87], which is similar to findings in human XLH patients [58, 59]. It was therefore concluded that PHEX may be involved in the feedback regulation of FGF23 secretion [14]. XLH can be severe, often leading to stunted growth despite treatment with phosphate and active vitamin D analogues, although some patients have normal growth [88]. Additional clinical features include craniosynostosis, frontal bossing and mid-facial hypoplasia as described above [58, 59] (see chapter 15, case 23). Treatment consists of oral phosphate supplementation and active vitamin D analogues, which provides symptomatic relief and improves the bone abnormalities, but is usually unable to normalise serum phosphate levels. Treatment is furthermore complicated by the development of secondary hyperparathyroidism, hypercalciuria and nephrocalcinosis [58, 59]. Thus, treatment needs to be monitored carefully for these complications. We prefer potassium-containing over sodium-containing phosphate supplements since the former seem to induce less sodium-related phosphaturia, although formal studies to support this practice are still missing. Parathyroidectomy may be required to control tertiary hyperparathyroidism and is generally associated with a reduced phosphate requirement and improved control. It is conceivable that the development of this complication may be slowed in the future by the use of calcium-sensing receptor agonists such as cinacalcet, which has been successfully used to normalise parathyroid hormone secretion and to reduce the magnitude of phosphaturia in XLH patients [89, 90]. Thiazide diuretics may be helpful in slowing the progression of nephrocalcinosis [91]. Replacement of the membrane-anchored PHEX with a soluble form of PHEX did not prove effective to reverse hypophosphataemia in hyp mice, while treatment with anti-FGF23 antibodies has been successful in these animals and holds promise to become a therapeutic option for humans with XLH [92]. Growth hormone therapy has been reported to improve linear growth in some patients, although it remains unclear whether the observed improvements have been in part attributable to an increase in tubular reabsorption of phosphate during growth hormone treatment [93].

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Autosomal-Dominant Hypophosphataemic Rickets (ADHR) (#193100) ADHR follows an autosomal-dominant mode of inheritance and is caused by heterozygous ‘gain-of-function’ mutations in FGF23 (*605380) that prevent cleavage at the RXXR site [4]. The two amino acid residues of FGF23, R176 or R179 that are mutated in patients affected by ADHR constitute a site for cleavage by subtilisin/furin-like endopeptidases. Just like O-linked glycosylation of T178 in wild-type FGF23 [25], mutation of either residue protects FGF23 from proteolytic cleavage and degradation [94] resulting in persistent FGF23 activity, with an incompletely understood feedback mechanism that escapes normal regulation by phosphate and 1,25(OH)2D [45]. The clinical course of ADHR is comparable to mild forms of XLH. As a result, phosphate and 1,25(OH)2D supplementation are often only required during skeletal growth in childhood. A sporadic case resembling autosomal-dominant hypophosphataemic rickets (%612089) was recently reported by Brownstein et al. [95], who described a 13-month-old girl, who initially presented with hyperparathyroidism. Her hypophosphataemia persisted after surgical cure of her hyperparathyroidism, and mutations in the known candidates including PHEX, FGF23, FGF-receptor 1 (FGFR1) and DMP1 were excluded. Cytogenetic analysis revealed a de novo chromosomal translocation with breakpoint adjacent to the gene encoding for alpha-KL. Indeed, her plasma alpha KL levels and beta-glucuronidase activity were increased and her FGF23 levels were elevated, which may explain increased proximal tubular action of FGF23 leading to renal phosphate wasting. This condition would appear to be the converse of HFTC3. Autosomal-Recessive Hypophosphataemia (ARHP) (#241520) ARHP is caused by homozygous, presumably loss-of-function mutations in DMP1 [5] (*600980). Intact DMP1 is cleaved into a 35- and a 57-kDa fragment, possibly by bone morphogenic protein 1 (BMP1) [96] which, in turn, is activated by a complex consisting of the endopeptidase SPC2 and the co-activator 7B2 [97]. Transgenic overexpression of 57 kDa DMP1 is both necessary and sufficient to rescue the bone phenotype (and probably the hypophosphataemia resulting from increased FGF23 secretion) of DMP1 null mice [98]. The 57-kDa C-terminal fragment of DMP1 appears to have nuclear effects which, when lost, leads to excess secretion of FGF23 resulting in renal phosphate wasting and hypophosphataemia [98]. Thus, the 57-kDa fragment may be required for suppression and/or feedback regulation of gene transcription and/or secretion of FGF23 [5]. Treatment of ARHP is symptomatic and relies, like XLH and ADHR, on oral phosphate supplementation and repletion of 1,25(OH)2D with either alfacalcidol or calcitriol to suppress development of hyperparathyroidism. Hereditary Hypophosphataemic Rickets with Hypercalciuria (HHRH) (#241530) HHRH is a rare disorder of autosomal-recessive inheritance that was first described in 1985 in a large consanguineous Bedouin kindred [43]. Unlike patients with XLH,

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individuals affected by HHRH do not develop dental abscesses or craniofacial abnormalities (i.e. frontal bossing, scaphocephaly, Chiari I malformation) [99, 100] and, different from patients affected by XLH, ADHR, and ARHR, FGF23 levels of HHRH patients appear to be suppressed [101], contributing to a compensatory increase in the plasma level of 1,25(OH)2D. This appropriate rise in the biologically active form of vitamin D results in absorptive hypercalciuria, the cardinal feature that distinguishes HHRH from most other Mendelian hypophosphataemic disorders. The measurement of 1,25(OH)2D and urinary calcium excretion is thus essential for establishing the diagnosis of HHRH, although both may be normal if vitamin D deficiency is present [44]. HHRH is caused by homozygous or compound heterozygous loss-of-function mutations in NaPi-IIc/SLC34A3 [101, 102] (*609826). Heterozygous mutations can lead to increased urinary calcium excretion, and occasionally some of the above biochemical features of HHRH, while bone changes are missing. Individuals with two mutated SLC34A3 alleles can initially present with renal stones alone even if clinical symptoms of rickets or osteomalacia are missing [44, 103]. In contrast to patients with XLH, ADHR or ARHP, who are usually treated with high doses of alfacalcidol or calcitriol and multiple daily doses of oral phosphate, the effective therapy of individuals affected by HHRH consists of oral phosphate supplementation alone. The prescription of biologically active vitamin D analogues is contraindicated and may lead to hypercalcaemia, hypercalciuria, nephrocalcinosis, and possibly renal insufficiency [44, 103]. Hypophosphataemia with Osteoporosis and Nephrolithiasis Type I (#612286) and Type II (#612287) Prie et al. [104] investigated a heterogeneous group of patients with idiopathic hypercalciuria, osteoporosis and renal stones. Using a candidate gene approach they found 2/20 individuals heterozygous for non-synonymous SNPs in NaPi-IIa/SLC34A1 [104] (*182309) and 7/94 individuals heterozygous for non-synonymous SNPs in NHERF-1/SLC9A3R1 [105] (*604990). Although the authors present experimental in vitro evidence for dominant negative effects of the NaPi-IIa/SLC34A1 alterations on proximal renal tubular phosphate reabsorption, their findings have been challenged by others [106]. Likewise, some of the identified NHERF-1 alterations are listed in the NCBI dbSNP database as low frequency polymorphisms [107]. Further study is thus required to prove that mutations in NaPi-IIa or NHERF-1 are indeed responsible in the described clinical syndromes.

Other Inherited Forms of Renal Phosphate Wasting Osteoglophonic dysplasia (OGD) (#166250) is an autosomal-dominant disorder caused by activating missense mutations in the gene encoding fibroblast growth

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factor receptor-1 (FGFR1) [108, 109] (*136350). Affected individuals have clinical features of the syndromes caused by all three fibroblast growth factor receptors, including craniosynostosis, midfacial hypoplasia, prognathism and rhizomelic chondrodysplasia. In addition, they have symmetrical radiolucent metaphyseal defects, which appear to produce FGF23. Consequently, most affected individuals develop hypophosphataemia due to renal phosphate wasting while their 1,25(OH)2D levels remain inappropriately normal [110, 111]. Opsismodysplasia (OSD) (%258480) [112] is an autosomal-recessive skeletal dysplasia that is characterised by a delay in epiphyseal ossification, platyspondyly and metaphyseal cupping, resulting in brachydactyly with short metacarpals and phalanges. The genetic defect is unknown. Like OGD, opsismodysplasia can go along with FGF23 excess leading to renal phosphate wasting [113]. The Schimmelpenning-Feuerstein-Mims syndrome (%163200) encompasses linear naevus sebaceous syndrome (LNSS), epidermal naevus syndrome (ENS) (see chapter 15, case 24) and phakomatosis pigmentokeratotica. It is characterised by sebaceous naevi, often in the face, abnormalities of the central nervous system, ocular anomalies, including coloboma, and skeletal defects [114–117]. Most patients with LNSS or ENS carry mosaic FGFR3 mutations [118]. LNSS/ENS may go along with hypophosphataemic rickets [119, 120], and some of the naevi were shown to secrete FGF23 thus providing an explanation for the underlying renal phosphate wasting [119–124]. However, it is unknown whether the FGFR3 mutations alone or additional unknown somatic mutations lead to renal phosphate wasting. Fibrous dysplasia (FD)/McCune-Albright syndrome (MAS) (#174800) is caused by somatic activating missense mutations in the alpha subunit of the stimulatory G-protein (encoded by GNAS) [125, 126] (+139320). The classical triad of MAS includes polyostotic FD, café-au-lait spots, typically large and with a ragged ‘coast of Maine’ appearance, and non-central precocious puberty, particularly in girls. However, a number of other endocrine disorders such as thyrotoxicosis, pituitary gigantism, and Cushing syndrome are often present as well [125]. The non-mineralising bone lesions of FD/MAS may secrete FGF23, which can lead to hypophosphataemic rickets or osteomalacia [127–129]. Phosphate levels should always be checked and, if necessary, corrected before the bone lesions of MAS are treated with bisphosphonates. FGF23-mediated hypophosphataemia can also be observed in Jansen’s metaphyseal chondrodysplasia (#156400), which is caused by heterozygous activating PTH/PTHrP receptor mutations (*168468) and may be, as in FD/MAS, a consequence of agonistindependent Gs-alpha activation [130]. Finally, hypophosphataemia leading to osteomalacia has been described in some individuals with neurofibromatosis 1 (+162200) and 2 (#101000) [131, 132], although the mechanism remains to be clarified.

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a

b

c

d Fig. 8. Normocalcaemic tumoral calcinosis. Clinical features of NFTC include erythematous papular eruption (a), these papules later become calcified ulcerating tumours (b), severe gingivitis (c), and calcified material in the upper dermis (d) (fig. 2 in [135], with permission).

Disorders of Tissue Mineralisation

Normophosphataemic Tumoral Calcinosis (NFTC) (#610455) NFTC is an autosomal-recessive disorder caused by homozygous loss-of function mutations in sterile alpha motif domain-containing protein 9 (SAMD9) (*610456), a factor involved in the physiologic responses to tissue injury [133–135]. The genetic defect was identified by homozygosity mapping in five families of Jewish Yemenite origin. Distinct from HFTC, serum biochemical parameters including calcium, phosphate, vitamin D3 metabolites, and PTH levels are normal in this disorder, which resembles the dystrophic calcinosis seen at sites of (chronic) tissue injury. Affected individuals develop reddish-to-hyperpigmented skin lesions over the extremities during the first year of life, which later become calcified. In addition, severe conjunctivitis and gingivitis were observed in most of the affected individuals (fig. 8). Treatment consists of local supportive measures.

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Pulmonary Alveolar Microlithiasis (#265100) Pulmonary alveolar microlithiasis is a rare autosomal-recessive disorder characterised by the deposition of calcium phosphate microliths throughout the lungs [53]. The diagnosis is often made incidentally since most patients are asymptomatic for several years or even for decades. A ‘sandstorm-appearing’ chest X-ray is a typical diagnostic finding. The onset of this potentially lethal disease varies from the neonatal period to old age, and the disease follows a long-term progressive course, resulting in a slow deterioration of lung function. Genetic linkage studies led to the discovery of homozygous loss-of-function mutations in the sodium-phosphate co-transporter NaPi-IIb/SLC34A2 (*604217) [53, 136]. This co-transporter is closely related to NaPi-IIa and NaPi-IIc, but unlike these transporters, it is mainly expressed in the intestine, the lung and testicles. Therapy consists in supportive measures.

Conclusions

In the past few years there have been considerable advances in our understanding of the regulation of phosphate homeostasis, in which the newly discovered hormone, FGF23, plays a particularly important role. Although much remains to be learned about this novel hormonal system, our present understanding has already provided better tools for the diagnosis of a number of genetic disorders of phosphate metabolism, both those associated with hyper- and with hypophosphataemia, and provided a rationale for their treatment. In the future, it may also allow newer drugs to be developed that are more effective in treating these conditions.

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91 Seikaly MG, Baum M: Thiazide diuretics arrest the progression of nephrocalcinosis in children with X-linked hypophosphatemia. Pediatrics 2001;108: E6. 92 Yamazaki Y, Tamada T, Kasai N, et al: Anti-FGF23 neutralizing antibodies show the physiological role and structural features of FGF23. J Bone Miner Res 2008;23:1509–1518. 93 Seikaly MG, Brown R, Baum M: The effect of recombinant human growth hormone in children with X-linked hypophosphatemia. Pediatrics 1997; 100:879–884. 94 White KE, Carn G, Lorenz-Depiereux B, BenetPages A, Strom TM, Econs MJ: Autosomaldominant hypophosphatemic rickets (ADHR) mutations stabilize FGF-23. Kidney Int 2001;60: 2079–2086. 95 Brownstein CA, Adler F, Nelson-Williams C, et al: A translocation causing increased alpha-Klotho level results in hypophosphatemic rickets and hyperparathyroidism. Proc Natl Acad Sci USA 2008;105:3455– 3460. 96 von Marschall Z, Fisher LW: Dentin matrix protein-1 isoforms promote differential cell attachment and migration. J Biol Chem 2008;283:32730–32740. 97 Yuan B, Meudt J: 7B2 protein mediated inhibition of DMP1 cleavage in osteoblasts enhances FGF-23 production in hyp-mice. JBMR 2008;23:s16 (abstract 1053). 98 Lu Y, Qin C, Xie Y, Bonewald LF, Feng JQ: Studies of the DMP1 57-kDa functional domain both in vivo and in vitro. Cells Tissues Organs 2008;189:175– 185. 99 Jones A, Tzenova J, Frappier D, et al: Hereditary hypophosphatemic rickets with hypercalciuria is not caused by mutations in the Na/Pi cotransporter NPT2 gene. J Am Soc Nephrol 2001;12:507–514. 100 Tenenhouse HS, Econs MJ: Mendelian hypophosphatemias; in Scriver CR, Beaudet AL, Valleet D (eds): The Metabolic Basis of Inherited Diseases. New York, McGraw-Hill, 2001, pp 5039–5067. 101 Lorenz-Depiereux B, Benet-Pages A, Eckstein G, et al: Hereditary hypophosphatemic rickets with hypercalciuria is caused by mutations in the sodium-phosphate cotransporter gene SLC34A3. Am J Hum Genet 2006;78:193–201. 102 Bergwitz C, Roslin NM, Tieder M, et al: SLC34A3 mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria predict a key role for the sodium-phosphate cotransporter NaPiIIc in maintaining phosphate homeostasis. Am J Hum Genet 2006;78:179–192.

Phosphate Disorders

103 Jaureguiberry G, Carpenter TO, Forman S, Juppner H, Bergwitz C: A novel missense mutation in SLC34A3 that causes hereditary hypophosphatemic rickets with hypercalciuria in humans identifies threonine 137 as an important determinant of sodium-phosphate cotransport in NaPi-IIc. Am J Physiol Renal Physiol 2008;295:F371-F379. 104 Prie D, Huart V, Bakouh N, et al: Nephrolithiasis and osteoporosis associated with hypophosphatemia caused by mutations in the type 2a sodium-phosphate cotransporter. N Engl J Med 2002;347:983– 991. 105 Karim Z, Gerard B, Bakouh N, et al: NHERF1 mutations and responsiveness of renal parathyroid hormone. N Engl J Med 2008;359:1128–1135. 106 Virkki LV, Forster IC, Hernando N, Biber J, Murer H: Functional characterization of two naturally occurring mutations in the human sodium-phosphate cotransporter type IIa. J Bone Miner Res 2003;18:2135–2141. 107 Bergwitz C, Bastepe M: NHERF1 mutations and responsiveness of renal parathyroid hormone. N Engl J Med 2008;359:2615–2616. 108 Beighton P, Cremin BJ, Kozlowski K: Osteoglophonic dwarfism. Pediatr Radiol 1980;10:46–50. 109 Sklower BS, Kassner G, Qazi Q, Keogh MJ, Gorlin RJ: Osteoglophonic dysplasia: review and further delineation of the syndrome. Am J Med Genet 1996; 66:154–162. 110 Farrow EG, Davis SI, Mooney SD, et al: Extended mutational analyses of FGFR1 in osteoglophonic dysplasia. Am J Med Genet [A] 2006;140:537–539. 111 White KE, Cabral JM, Davis SI, et al: Mutations that cause osteoglophonic dysplasia define novel roles for FGFR1 in bone elongation. Am J Hum Genet 2005;76:361–367. 112 Maroteaux P, Stanescu V, Stanescu R, Le Marec B, Moraine C, Lejarraga H: Opsismodysplasia: a new type of chondrodysplasia with predominant involvement of the bones of the hand and the vertebrae. Am J Med Genet 1984;19:171–182. 113 Zeger MD, Adkins D, Fordham LA, et al: Hypophosphatemic rickets in opsismodysplasia. J Pediatr Endocrinol Metab 2007;20:79–86. 114 Gellis SS, Feingold M: Linear nevus sebaceous syndrome. Am J Dis Child 1970;120:139–140. 115 Menascu S, Donner EJ: Linear nevus sebaceous syndrome: case reports and review of the literature. Pediatr Neurol 2008;38:207–210. 116 Rogers M: Epidermal nevi and the epidermal nevus syndromes: a review of 233 cases. Pediatr Dermatol 1992;9:342–344. 117 Solomon LM, Fretzin DF, Dewald RL: The epidermal nevus syndrome. Arch Dermatol 1968;97:273– 285.

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118 Hafner C, van Oers JM, Vogt T, et al: Mosaicism of activating FGFR3 mutations in human skin causes epidermal nevi. J Clin Invest 2006;116:2201–2207. 119 Skovby F, Svejgaard E, Moller J: Hypophosphatemic rickets in linear sebaceous nevus sequence. J Pediatr 1987;111:855–857. 120 Zutt M, Strutz F, Happle R, et al: SchimmelpenningFeuerstein-Mims syndrome with hypophosphatemic rickets. Dermatology 2003;207:72–76. 121 Chou YY, Chao SC, Shiue CN, Tsai WH, Lin SJ: Hypophosphatemic rickets associated with epidermal nevus syndrome and giant hairy nevus. J Pediatr Endocrinol Metab 2005;18:93–95. 122 Hoffman WH, Jain A, Chen H, Fedarko NS: Matrix extracellular phosphoglycoprotein (MEPE) correlates with serum phosphorus prior to and during octreotide treatment and following excisional surgery in hypophosphatemic linear sebaceous nevus syndrome. Am J Med Genet [A] 2008;146A:2164–2168. 123 Hoffman WH, Jueppner HW, Deyoung BR, O’dorisio MS, Given KS: Elevated fibroblast growth factor-23 in hypophosphatemic linear nevus sebaceous syndrome. Am J Med Genet A 2005;134:233–236. 124 John M, Shah NS: Hypophosphatemic rickets with epidermal nevus syndrome. Indian Pediatr 2005; 42:611–612. 125 Weinstein LS, Yu S, Warner DR, Liu J: Endocrine manifestations of stimulatory G protein alpha-subunit mutations and the role of genomic imprinting. Endocr Rev 2001;22:675–705. 126 Weinstein LS, Shenker A, Gejman PV, Merino MJ, Friedman E, Spiegel AM: Activating mutations of the stimulatory G protein in the McCune-Albright syndrome. N Engl J Med 1991;325:1688–1695. 127 Collins MT, Chebli C, Jones J, et al: Renal phosphate wasting in fibrous dysplasia of bone is part of a generalized renal tubular dysfunction similar to that seen in tumor-induced osteomalacia. J Bone Miner Res 2001;16:806–813. 128 Riminucci M, Collins MT, Fedarko NS, et al: FGF23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting. J Clin Invest 2003;112: 683–692. 129 Yamamoto T, Miyamoto KI, Ozono K, et al: Hypophosphatemic rickets accompanying McCuneAlbright syndrome: evidence that a humoral factor causes hypophosphatemia. J Bone Miner Metab 2001;19:287–295.

130 Brown WW, Juppner H, Langman CB, et al: Hypophosphatemia with elevations in serum FGF23 in a child with Jansen’s metaphyseal chondrodysplasia (FGF23 in Jansen’s syndrome). J Clin Endocrinol Metab 2009;94:17–20. 131 Haviv YS, Silver J: Late onset oncogenic osteomalacia-associated with neurofibromatosis type II. Clin Nephrol 2000;54:429–430. 132 Konishi K, Nakamura M, Yamakawa H, et al: Hypophosphatemic osteomalacia in von Recklinghausen neurofibromatosis. Am J Med Sci 1991; 301:322–328. 133 Chefetz I, Ben Amitai D, Browning S, et al: Normophosphatemic familial tumoral calcinosis is caused by deleterious mutations in SAMD9, encoding a TNF-alpha responsive protein. J Invest Dermatol 2008;128:1423–1429. 134 Li CF, MacDonald JR, Wei RY, et al: Human sterile alpha motif domain 9, a novel gene identified as down-regulated in aggressive fibromatosis, is absent in the mouse. BMC Genomics 2007;8:92. 135 Topaz O, Indelman M, Chefetz I, et al: A deleterious mutation in SAMD9 causes normophosphatemic familial tumoral calcinosis. Am J Hum Genet 2006; 79:759–764. 136 Huqun, Izumi S, Miyazawa H, et al: Mutations in the SLC34A2 gene are associated with pulmonary alveolar microlithiasis. Am J Respir Crit Care Med 2007;175:263–268. 137 Hardy DC, Murphy WA, Siegel BA, Reid IR, Whyte MP: X-linked hypophosphatemia in adults: prevalence of skeletal radiographic and scintigraphic features. Radiology 1989;171:403–414. 138 Specktor P, Cooper JG, Indelman M, Sprecher E: Hyperphosphatemic familial tumoral calcinosis caused by a mutation in GALNT3 in a European kindred. J Hum Genet 2006;51:487–490. 139 Batra P, Tejani Z, Mars M: X-linked hypophosphatemia: dental and histologic findings. J Can Dent Assoc 2006;72:69–72. 140 Chefetz I, Heller R, Galli-Tsinopoulou A, et al: A novel homozygous missense mutation in FGF23 causes familial tumoral calcinosis associated with disseminated visceral calcification. Hum Genet 2005; 118:261–266.

Clemens Bergwitz, MD Endocrine Unit, Massachusetts General Hospital Boston, MA 02114 (USA) Tel. +1 617 726 3966, Fax +1 617 726 7543, E-Mail [email protected]

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Chapter 10 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 157–169

Primary Osteoporosis Nick Bishop Academic Unit of Child Health, University of Sheffield, Sheffield Children’s Hospital, Sheffield, UK

Abstract Primary osteoporosis, as defined by bone loss associated with significant fracture, is most commonly caused in children by one or other of the forms of osteogenesis imperfecta (OI). These are a group of disorders that are characterised by abnormalities in type I collagen synthesis or processing. Other types of primary osteoporosis, such as those caused by abnormalities of osteoblasts, are not discussed in this chapter. There are now eight types of OI described. Most are autosomal dominant and they vary in severity from a slight increase in bone fragility with occasional fractures and no bone deformity to severe forms with poor growth, intrauterine fractures and severe bone deformity. The most seriously affected children may not survive. Diagnosis depends on taking a good personal and family history together with a thorough examination. This may be supported by genetic testing or bone biopsy although neither of these is performed routinely. DXA scanning will usually support the diagnosis. Good management requires a multidisciplinary approach involving paediatricians, surgeons, occupational and physiotherapists, dentists, social workers etc. The mainstay of medical treatment is bisphosphonates which, over the past ten years, have revolutionised the approach to management. Surgery is required in more severe cases to straighten limbs or stabilise the spine. Physiotherapy is particularly Copyright © 2009 S. Karger AG, Basel important to try to maintain mobility.

Osteoporosis in childhood is defined as reduced bone mass for body size and the presence of significant fracture. The primary forms range in severity from mild to severe, with accompanying growth retardation and bony deformity reflecting the severity of the bone disease. Most are inherited, although the genetic origin is not known in some. The most common cause of primary osteoporosis is the group of disorders known collectively as osteogenesis imperfecta (OI). The majority of these cases result from defects in the synthesis or metabolism of type I collagen. It is only these conditions that will be discussed in this chapter. Other causes of primary osteoporosis, such as idiopathic juvenile osteoporosis and the osteoporosis pseudoglioma syndrome are discussed in chapter 12. OI is a group of rare heritable disorders characterised by low bone mass and bone fragility resulting in fractures, often accompanied by chronic bone pain. The severity

of the condition ranges from mild to lethal. A Danish study of a geographically defined population observed that the point prevalence at birth was 21.8 per 100,000 with the population prevalence 10.6 per 100,000. Overall OI has an incidence of between 1 in 10,000 and 1 in 20,000 [1, 2]. Approximately two thirds of those surviving infancy are at the mild end of the spectrum.

Classification

A widely accepted classification system that distinguishes between four clinical types (I-IV) was originally proposed and modified by Sillence and colleagues [3, 4]. In essence, type I OI is mild, type II is lethal, type III is severe with progressive bony deformity and type IV is intermediate between types I and III. The clinical phenotype within each of these types of OI is, to a degree, variable in terms of other features such as ligamentous laxity, presence of Wormian bones, dentinogenesis imperfecta and hearing loss. Scleral hue typically remains blue in types I and III, but fades over time in type IV. During the last decade, careful clinical phenotyping and study of bone biopsy material in Montreal identified two new types of OI (type V and type VI) [5, 6] and a further rhizomelic type (type VII) [7, 8], restricted to a group of First People in northern Canada, was added. Many OI sufferers have been found to have mutations in one of the two genes encoding type I collagen. Recently, genetic analysis of the rhizomelic group, together with data from a mouse model, identified mutations in the gene CRTAP [9] as being responsible for both this form of OI and some lethal cases. Further study of the protein complex incorporating the crtap protein has shown that abnormalities in another partner protein, prolyl-3-hydroxylase, are also associated with a severe OI phenotype (type VIII) [10, 11]. Additional forms of OI are the Cole-Carpenter [12–14] and Bruck [15, 16] syndromes. There is no known genetic origin for Cole-Carpenter; some cases of Bruck syndrome are due to defects in the PLOD2 gene that encodes bone-specific telopeptide lysyl hydroxylase. The clinical, radiological, pathological and genetic features are documented in table 1.

Clinical Aspects of Care

History Specific aspects of the medical history that should be sought in cases of suspected OI are as follows: • Fractures and dislocations – number, site, causation, degree of trauma, age at first fracture, timing of most recent fracture.

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• Orthopaedic interventions – e.g. manipulation, insertion of wires or rods, scoliosis correction, spinal fusion. • Back pain (often described by the child as ‘stiffness’) – timing, relationship to exercise and sleep, severity (particularly night waking). • Exercise: – Ambulant children – duration of walking before stopping for a rest; can they run and keep up with their friends? – Non-ambulant children – amount of care-giver assistance for activities of daily living • Use of aids to mobility. • Features of basilar invagination and hydrocephalus: – increasing clumsiness – difficulty with fine motor tasks suggesting loss of finger-tip sensation – swallowing problems – symptoms of cranial nerve palsies – headaches worse with straining, sneezing, or on waking. • Diet – amount and type of dairy produce, details of any supplements of calcium and vitamin D. • Use of therapy services, orthotics. • Family history – approximately half of all mildly and moderately affected individuals (types I and IV) will have a family history. Enquire specifically about: – hearing loss – recurrent fractures or dislocations – reducing height in older family members – teeth that chip or crack – hernias. • Motor development – age at walking even in mild OI can be >18 months. • School – are adaptations currently in place or needed?

Initial Examination From the top down look for: • Brachycephaly and occipital droop suggesting altered cranio-cervical junction anatomy and basilar invagination. • Large anterior fontanelle and sutural diastasis. • Blue scleral hue [17]. • Dentinogenesis imperfecta. • Ligamentous laxity – use the Beighton scale [18] for consistency, but also check for flat feet and genu valgum. • Muscle strength is also reduced in OI. This can be assessed clinically or may be quantified by the measurement of grip strength using a dynamometer.

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• Skin elasticity – may be reduced; note cross-over features with Ehlers-Danlos syndrome, some of whom have mutations in COL1A1 or 2 [19–21]. • Limb deformity – the degree of shortening and bowing reflects disease severity – forearm pronation and supination are reduced in OI V – check legs for discrepancy in length – assess mechanical axis – consider coax vara. • The back should be assessed standing (where possible) and bending forward for scoliosis and kyphosis. A ‘flat spot’ in the otherwise smooth curve of the spinous processes may be associated with underlying crush fracture, but is not an invariable sign. • Range of movement and strength are major determinants of both activities of daily living and the need for care-giver assistance and require expert assessment (usually by specialised therapists rather than doctors).

Differential Diagnosis In infancy, other causes of bone fragility leading to unexplained fractures include metabolic bone disease of prematurity, rare inherited metabolic diseases (e.g. I-cell disease) and non-accidental injury (NAI). Severe bone disease is radiologically apparent. The most difficult differentiation is between mild OI and NAI [22]. In older children, once malignancy, endocrine and inflammatory conditions are excluded, the diagnosis of idiopathic juvenile osteoporosis can be considered. Some of these children present in the classical fashion described by Dent et al. [23], but some present without the metaphyseal fractures and neo-osseous ossification features. Children with recurrent fractures and a bone mass that is more than two standard deviations below that predicted for body size are now defined as having osteoporosis by the ISCD criteria [24].

Investigation There are no definitive biochemical or imaging biomarkers for OI. However, bone turnover is typically elevated in the untreated state even in the absence of recent fracture. Plain X-rays may be used to assess the mechanical axis, the degree of bowing deformity and any coxa vara, the presence of vertebral crush fractures, scoliosis and spondylolisthesis in the spine. Wormian bones in the lambdoid suture may be normal; in the sagittal suture they are a strong indicator of skeletal abnormality. DXA scans can quantify the amount of mineralised bone at a given skeletal site – typically the spine, hip or whole body – and provide a means to assess some aspects of

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Table 1. Different types of OI and related conditions, together with the principal features, gene mutations (where known) and modes of inheritance: OMIM number for each condition and its associated gene is also shown OI type

OMIM

Phenotype (during childhood)

Genetic origin

Inheritance

OMIM

Chromosome location

I

#166200

Mild motor delay. Bowing of long bones. Vertebral crush fractures. Ligamentous laxity, hernias, mixed conductive/ sensorineural deafness, blue sclerae. Subdivided on the basis of the presence or absence of dentinogenesis imperfecta (A = absent, B = present).

Typically null allele of COL1A1, resulting from stop, frameshift or splice site mutations

AD

+120150

17q21.31-q22

IIA

#166210

Lethal. Subdivided by appearance of ribs.

Missense mutations in COL1A1 or COL1A2

AD +120150 *120160

17q21.31-q22 7q22.1

Complete loss of CRTAP

AR

*605497

3p22

+120150 *120160

17q21.31-q22 7q22.1

IIB

#610854

Lethal. Similar to type IIA.

III

#259420

Severe, progressively Missense mutations in deforming. Typically COL1A1, COL1A2; null have fractures in utero, allele of COL1A2 very poor post-natal growth. Characteristic facies with small mid face and pointed chin. Triangular facial appearance less noticeable with bisphosphonate treatment. Very delayed motor development. Almost all need intramedullary rodding. Blue sclerae remain. All have dentinogenesis imperfecta.

Primary Osteoporosis

AD usually new mutation or parental mosaicism

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Table 1. Continued OI type

OMIM

Phenotype (during childhood)

IV

#166220

V

Inheritance

OMIM

Chromosome location

Moderately severe. Missense mutations in May have fractures in COL1A1, COL1A2 utero, but better postnatal growth than type III. Blue sclerae fade with age; may have dentinogenesis imperfecta

AD

+120150 *120160

17q21.31-q22 7q22.1

%610967

Moderately severe. Unknown Metaphyseal sclerosis in early life, followed by calcification of interosseous membranes in the forearm and lower leg. Characteristic bowing of the forearms. Hypertrophic callus formation following fractures and surgery.

AD

?

?

VI

%610968

Severe, progressively Unknown deforming. Osteomalacic on bone biopsy, possibly as a result of abnormal matrix deposition – normal lamellar structure is disrupted.

Possibly AR

?

?

VII

#610682

Moderately severe. Rhizomelic in both arms and legs; femurs and humeri are very bowed. White sclerae

Cryptic splice site in intron 1 of CRTAP

AR

*605497

3p22

VIII

#610915

Very severe/lethal. Round face, white sclerae, thin ribs (may be beaded). Most cases are reported in children whose families originate from West Africa, Pakistan and Ireland.

Deletion of LEPRE1

AR

*610339

1p34

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Genetic origin

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Table 1. Continued OI type

OMIM

Phenotype (during childhood)

Genetic origin

Inheritance

OMIM

Chromosome location

Bruck 1

%259450

Contractures. White sclerae, mild DI. Moderately-severe bone disease.

Maps to 17p12

AR

?

17p12

Bruck 2

#609220

Clinical phenotype as for Bruck 1.

PLOD2; bone-specific telopeptide lysyl hydroxylase

AR

*601865

3q23-q24

Normal at birth; develop craniosynostosis, ocular proptosis, hydrocephalus and diaphyseal fractures.

Unknown

Unknown

?

?

112240 ColeCarpenter

therapeutic intervention. However, it is clear that the scans also measure calcified cartilage and, as such, may not accurately represent the bony response to therapy. QCT both of the vertebrae and at peripheral sites has been reported but remains a research tool at present (see chapter 5 for further details). MRI scans may be useful to detect crush fractured vertebrae in younger children whose vertebrae are difficult to visualise accurately using plain X-rays. MRI and CT may be required to visualise cranio-cervical junction anatomy in cases of basilar invagination, and to define any associated syrinx. Bone biopsy is undertaken either where there is difficulty in determining the underlying diagnosis [25] or, in some centres, before starting bisphosphonate therapy. If performed, double-labelling of bone using two brief (two day) courses of a tetracycline ten days apart will provide the opportunity to evaluate dynamic as well as static parameters of bone activity [26] (see chapter 4 for a more detailed discussion). Where mild OI is the differential diagnosis in unexplained fractures in infancy, a court may request genetic testing. Such testing is available at a number of centres worldwide. In the UK it comprises direct DNA sequencing with multiple ligationdependent probe amplification to detect large scale deletions and duplications. The detection rate for COL1A1 and COL1A2 mutations using this approach is thought to be >99%. However, it is important to inform those requesting the test that a significant proportion (10%) of individuals with the OI phenotype are negative on testing of COL1A1/2.

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Management The key to effective management in OI is the multidisciplinary approach. The initial consultation includes not only an assessment of severity and the need for medical therapy such as a bisphosphonate, but also of the functional and social needs of the child and their family. A comprehensive team should include specialist nursing, physiotherapy and occupational therapists, pain management, and input from orthopaedic and dental colleagues. Input from therapists is highly individualised according to patient need, as is nursing input. There is typically an important need for education of families concerning the pathology of the disease, the effects of intervention and likely medium to long term outcomes. The whole team is involved in this process, and families should also be directed to national (in the UK the Brittle Bone Society) and international organisations (the OI Foundation in the USA/Canada and OIFE in Europe). Managing severely affected infants can be particularly challenging. Such infants may have compromised respiratory function at birth because rib fractures in utero lead to lack of chest expansion and pulmonary hypoplasia, in addition to the impaired mechanics of the rib cage post-natally. In some severely affected infants, respiratory support, ranging from nasal oxygen to mechanical ventilation, is required. Infants with pre-existing respiratory compromise may deteriorate when infused with pamidronate [27] and should be carefully monitored in a setting where there is access to high dependency or intensive care. We have seen one infant in highoutput cardiac failure, probably secondary to extensive healing fractures. Frequent physiotherapy and occupational therapy input is required in order to enable the child to progress safely through their gross motor milestones. Side-lying initially of the head and subsequently the whole body may help maintain head shape and prevent severe brachycephaly. This in turn will help maintain normal cranio-cervical junction anatomy and normal orbital shape. Infants should be turned regularly and enabled to spend time prone where tolerated. For transportation the ideal car seat is one that allows maximum recline and can fit into the chassis of a pushchair in order to minimise handling. Careful management of positioning is required in severely affected infants in order to enable head control, sitting and crawling to proceed safely. Aids to mobility include orthoses, braces and appropriate wheelchair support. Therapists often start at the foot and work up in terms of providing aids to mobility. Even mildly affected children may benefit from insoles and supportive footwear. In more severely affected children, hip-knee-ankle-foot orthoses (HKAFOs) are often considered initially for children under five years of age who are ready to stand and walk. Once some pelvic control is achieved then a walking aid may be introduced. In the school setting particularly, joint hypermobility can impair the development of fine motor skills. Modified pencil holders for writing and wrist splints to reduce fatigue can be helpful.

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As with many chronic diseases, OI has a significant impact on daily life. How one is treated by others means that the psychosocial aspects to management are important to address, especially during adolescence. Our observation has been that, despite their often severe disability, children and adolescents with OI are often bright and sparky individuals with a large number of friends. Nevertheless, support and advice from the multidisciplinary team along with physical adaptations and other aids may be required to ensure integration into school. Concern may be expressed by schools about participation in sports and individual advice is often required. On-going monitoring for all children is required in a number of areas. Our practice is to obtain annual DXA and lateral spine films to evaluate bone accretion and check for occult vertebral crush fracture in mildly affected children not currently receiving bisphosphonate therapy. The frequency of review is dictated by the severity of the disease and the age of the child. We review severely affected infants six times in the first year; older severely affected children are typically seen four times each year, coinciding with their intravenous bisphosphonate therapy. For those on therapy, DXA is undertaken six monthly to evaluate the response to treatment. Therapy input may be required between clinic visits, tailored to individual need. All children with dentinogenesis imperfecta need annual dental review; some are seen more frequently if they have accelerated carious deterioration, or need specific intervention such as crowns, veneers or cosmetic work. Hearing should be evaluated every three years from age ten years so as to identify the minority of children (<10%) who develop early hearing loss.

Surgery and Anaesthesia Ensure surgical and anaesthetic staff are aware of bone and tooth fragility so as to avoid problems during induction of anaesthesia and moving of the child. Drugs that cause muscle fasciculation, e.g. succinylcholine, should be avoided. Abnormal bleeding can occur in OI. Although there is an increased intraoperative risk of hyperthermia [28], malignant hyperthermia appears no more common than in the general population.

Intramedullary Rodding The indications for rodding include correction of deformity and prevention of recurrent fracture. Rodding is typically undertaken in the lower limbs and less often in the humerus. Various rods are available, including expanding or telescopic forms (Sheffield, Bailey-Dubow) and straight (Rush) rods and (Nancy) nails [29–35]. Straight rods are used more often when growth potential is limited, when there are issues of access at the ankle, and in the humerus. Overlapping Nancy nails have been

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used as an alternative to telescopic rods. Telescopic rods need replacing less often but are more prone to bend when exposed to three-point bending forces – further extension is then impossible. Distraction osteogenesis has been demonstrated to be effective and safe in achieving the correction of both limb-length discrepancy and bowing deformity [36].

Spinal Surgery The indications for spine surgery include the prevention of progression of scoliosis and, in some cases, a full correction can be achieved. The choice of approach in terms of halo traction, posterior fusion and instrumentation depends on the severity of the scoliosis and the age of the patient. Post-surgical care is often provided on a highdependency or intensive care unit. Spondylolisthesis may require fusion of adjacent vertebrae.

Basilar Invagination Basilar invagination is more frequent in patients with dentinogenesis imperfecta and can be complicated by communicating hydrocephalus and brain stem compression [37–39]. The initial detection can be made using plain lateral X-rays of the craniocervical junction. Management consists of either posterior fossa or anterior approaches prior to occipito-cervical fusion with or without removal of the odontoid peg and stabilisation of the skull with regard to the thorax. Surgery is difficult and progression has been reported in up to 80% of patients [39].

Medical Therapy Using Bisphosphonates The initial observational reports documenting the significant benefits of cyclical intravenous bisphosphonates appeared in 1987 [40], with larger case series following in the early to mid-1990s [41–43]. Randomised controlled trials of both intravenous [44, 45] and oral bisphosphonates [46, 47] have now been published, largely focusing on children with moderate to severe forms of the disease. The use of bisphosphonates is covered in detail elsewhere in this book (see chapter 12). In summary, the beneficial effects observed in the RCTs have been reduction in fracture rate and increased bone mass. In the observational case series, reduction in bone pain, improved mobility and increased size of previously crush-fractured vertebrae have also been reported. The rapidity of response, particularly of vertebral shape, is greatest in infancy [48–51]. The administration of intravenous pamidronate is often associated with an acute phase reaction that resembles the onset of an influenza-like episode. It generally lasts two

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to four days and does not usually recur when a dose of 2–3 mg pamidronate is used over the first cycle of therapy. Bisphosphonate therapy does not change the quality of bone in OI; rather it increases the total amount of mineralised bone within the skeletal envelope, and reduces fracture risk by improved architecture – wider bones are stronger bones. The increase in the amount of heavily mineralised bone may cause problems for orthopaedic surgeons; the bone is certainly brittle and needs careful handling. There is no evidence that fracture healing is delayed following treatment with bisphosphonates (although callus remodelling is slower), but osteotomy healing may be slower if the bone ends have been cauterised [52]. Osteonecrosis of the jaw has not been reported in any child with OI who has received bisphosphonate therapy.

Other Therapies Growth hormone has been shown to increase markers of bone formation and bone volume (measured on biopsy); spine bone mass increased in those who also increased their rate of linear growth [53]. Calcitonin was used for a time in the 1980s, but has been superseded by bisphosphonates. Bone marrow transplantation has been undertaken in seven severely affected children, but did not show any lasting benefit and these children are now all receiving bisphosphonates [54, 55]

Conclusions

The clinical diagnosis of OI encompasses a range of genetically determined disorders, with the majority having defects in the production of normal type I collagen fibres. This results in an imbalance of mineral and matrix components with excess mineral for fibre resulting in bone that is brittle. There is no treatment available at present that directly addresses this issue. Bisphosphonates have radically altered the course of disease in severely affected infants and children, but successful management of OI requires a multidisciplinary approach. Despite major advances in many areas, much remains to be done.

References 1 Andersen PE Jr, Hauge M: Osteogenesis imperfecta: a genetic, radiological, and epidemiological study. Clin Genet 1989;36:250–255. 2 Kuurila K, Grenman R, Johansson R, Kaitila I: Hearing loss in children with osteogenesis imperfecta. Eur J Pediatr 2000;159:515–519. 3 Sillence DO: Osteogenesis imperfecta nosology and genetics. Ann NY Acad Sci 1988;543:1–15.

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4 Sillence DO, Senn A, Danks DM: Genetic heterogeneity in osteogenesis imperfecta. J Med Genet 1979; 16:101–116. 5 Glorieux FH, Ward LM, Rauch F, Lalic L, Roughley PJ, Travers R: Osteogenesis imperfecta type VI: a form of brittle bone disease with a mineralization defect. J Bone Miner Res 2002;17:30–38.

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6 Glorieux FH, Rauch F, Plotkin H, Ward L, Travers R, Roughley P et al.: Type V osteogenesis imperfecta: a new form of brittle bone disease. J Bone Miner Res 2000;15:1650–1658. 7 Glorieux FH: Genomic localisation of a new variant of osteogenesis imperfecta to chromosome 3p22– 24.1. J Bone Miner Res 1996;11(suppl):S99. 8 Ward LM, Rauch F, Travers R, et al: Osteogenesis imperfecta type VII: an autosomal recessive form of brittle bone disease. Bone 2002;31:12–18. 9 Morello R, Bertin TK, Chen Y, et al: CRTAP is required for prolyl 3- hydroxylation and mutations cause recessive osteogenesis imperfecta. Cell 2006; 127:291–304. 10 Baldridge D, Schwarze U, Morello R, et al: CRTAP and LEPRE1 mutations in recessive osteogenesis imperfecta. Hum Mutat 2008;29:1435–1442. 11 Cabral WA, Chang W, Barnes AM, et al: Prolyl 3-hydroxylase 1 deficiency causes a recessive metabolic bone disorder resembling lethal/severe osteogenesis imperfecta. Nat Genet 2007;39:359–365. 12 Amor DJ, Savarirayan R, Schneider AS, Bankier A: New case of Cole-Carpenter syndrome. Am J Med Genet 2000;92:273–277. 13 Cohen MM Jr: Craniosynostosis update 1987. Am J Med Genet Suppl 1988;4:99–148. 14 MacDermot KD, Buckley B, Van S, V: Osteopenia, abnormal dentition, hydrops fetalis and communicating hydrocephalus. Clin Genet 1995;48:217–220. 15 Breslau-Siderius EJ, Engelbert RH, Pals G, van der Sluijs JA: Bruck syndrome: a rare combination of bone fragility and multiple congenital joint contractures. J Pediatr Orthop B 1998;7:35–38. 16 Ha-Vinh R, Alanay Y, Bank RA, et al: Phenotypic and molecular characterization of Bruck syndrome (osteogenesis imperfecta with contractures of the large joints) caused by a recessive mutation in PLOD2. Am J Med Genet [A] 2004;131:115–120. 17 Zack P, Zack LR, Surtees R, Neville BG: A standardized tool to measure and describe scleral colour in osteogenesis imperfecta. Ophthalmic Physiol Opt 2007;27:174–178. 18 Beighton P, Horan F: Orthopaedic aspects of the Ehlers-Danlos syndrome. J Bone Joint Surg Br 1969; 51:444–453. 19 Malfait F, Symoens S, De Backer J, et al: Three arginine to cysteine substitutions in the pro-alpha (I)-collagen chain cause Ehlers-Danlos syndrome with a propensity to arterial rupture in early adulthood. Hum Mutat 2007;28:387–395. 20 Schwarze U, Hata R, McKusick VA, et al: Rare autosomal recessive cardiac valvular form of EhlersDanlos syndrome results from mutations in the COL1A2 gene that activate the nonsense-mediated RNA decay pathway. Am J Hum Genet 2004;74:917– 930.

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21 Vasan NS, Kuivaniemi H, Vogel BE, et al: A mutation in the pro alpha 2(I) gene (COL1A2) for type I procollagen in Ehlers-Danlos syndrome type VII: evidence suggesting that skipping of exon 6 in RNA splicing may be a common cause of the phenotype. Am J Hum Genet 1991;48:305–317. 22 Bishop N, Sprigg A, Dalton A: Unexplained fractures in infancy: looking for fragile bones. Arch Dis Child 2007;92:251–256. 23 Dent CE, Friedman M: Idiopathic juvenile osteoporosis. Q J Med 1965;34:177–210. 24 Bishop N, Braillon P, Burnham J, et al: Dual-energy X-ray aborptiometry assessment in children and adolescents with diseases that may affect the skeleton: the 2007 ISCD Pediatric Official Positions. J Clin Densitom 2008;11:29–42. 25 Rauch F, Travers R, Norman ME, Taylor A, Parfitt AM, Glorieux FH: Deficient bone formation in idiopathic juvenile osteoporosis: a histomorphometric study of cancellous iliac bone. J Bone Miner Res 2000;15:957–963. 26 Rauch F, Travers R, Parfitt AM, Glorieux FH: Static and dynamic bone histomorphometry in children with osteogenesis imperfecta. Bone 2000;26:581– 589. 27 Munns CF, Rauch F, Mier RJ, Glorieux FH: Respiratory distress with pamidronate treatment in infants with severe osteogenesis imperfecta. Bone 2004;35:231–234. 28 Cole WG: Surgery in osteogenesis imperfecta. Connect Tissue Res 1995;31:S27-S29. 29 Brunelli PC, Novati P: Complications of elongating intramedullary rodding in osteogenesis imperfecta. Am J Med Genet 1993;45:275. 30 Chockalingam S, Bell MJ: Technique of exchange of Sheffield telescopic rod system. J Pediatr Orthop 2002;22:117–119. 31 Engelbert RH, Helders PJ, Keessen W, Pruijs HE, Gooskens RH: Intramedullary rodding in type III osteogenesis imperfecta: effects on neuromotor development in 10 children. Acta Orthop Scand 1995;66:361–364. 32 Karbowski A, Schwitalle M, Brenner R, Lehmann H, Pontz B, Worsdorfer O: Experience with BaileyDubow rodding in children with osteogenesis imperfecta. Eur J Pediatr Surg 2000;10:119–124. 33 Karbowski A, Schwitalle M, Eckardt A: Experiences with different telescope nails in treatment of pediatric osteogenesis imperfecta. Zentralbl Chir 1998;123: 1252–1256. 34 Mulpuri K, Joseph B: Intramedullary rodding in osteogenesis imperfecta. J Pediatr Orthop 2000;20: 267–273. 35 Stockley I, Bell MJ, Sharrard WJ: The role of expanding intramedullary rods in osteogenesis imperfecta. J Bone Joint Surg Br 1989;71:422–427.

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36 Saldanha KA, Saleh M, Bell MJ, Fernandes JA: Limb lengthening and correction of deformity in the lower limbs of children with osteogenesis imperfecta. J Bone Joint Surg Br 2004;86:259–265. 37 Charnas LR, Marini JC: Communicating hydrocephalus, basilar invagination, and other neurologic features in osteogenesis imperfecta. Neurology 1993; 43:2603–2608. 38 Engelbert RH, Gerver WJ, Breslau-Siderius LJ, et al: Spinal complications in osteogenesis imperfecta: 47 patients 1–16 years of age. Acta Orthop Scand 1998; 69:283–286. 39 Sawin PD, Menezes AH: Basilar invagination in osteogenesis imperfecta and related osteochondrodysplasias: medical and surgical management. J Neurosurg 1997;86:950–960. 40 Devogelaer JP, Malghem J, Maldague B, Nagant DD: Radiological manifestations of bisphosphonate treatment with APD in a child suffering from osteogenesis imperfecta. Skeletal Radiol 1987;16:360– 363. 41 Astrom E, Soderhall S: Beneficial effect of bisphosphonate during five years of treatment of severe osteogenesis imperfecta. Am J Hum Genet 1998;87: 758–763. 42 Bembi B, Parma A, Bottega M, et al: Intravenous pamidronate treatment in osteogenesis imperfecta. J Pediatr 1997;131:622–625. 43 Glorieux FH, Bishop NJ, Plotkin H, Chabot G, Lanoue G, Travers R: Cyclic administration of pamidronate in children with severe osteogenesis imperfecta. N Engl J Med 1998;339:947–952. 44 Gatti D, Antoniazzi F, Prizzi R, et al: Intravenous neridronate in children with osteogenesis imperfecta: a randomized controlled study. J Bone Miner Res 2005;20:758–763. 45 Letocha AD, Cintas HL, Troendle JF, et al: Controlled trial of pamidronate in children with types III and IV osteogenesis imperfecta confirms vertebral gains but not short-term functional improvement. J Bone Miner Res 2005;20:977–986. 46 DiMeglio LA, Peacock M: Two-year clinical trial of oral alendronate versus intravenous pamidronate in children with osteogenesis imperfecta. J Bone Miner Res 2006;21:132–140.

47 Sakkers R, Kok D, Engelbert R, et al: Skeletal effects and functional outcome with olpadronate in children with osteogenesis imperfecta: a 2-year randomised placebo-controlled study. Lancet 2004;363: 1427–1431. 48 Astrom E, Jorulf H, Soderhall S: Intravenous pamidronate treatment of infants with severe osteogenesis imperfecta. Arch Dis Child 2007;92:332– 338. 49 DiMeglio LA, Ford L, McClintock C, Peacock M: Intravenous pamidronate treatment of children under 36 months of age with osteogenesis imperfecta. Bone 2004;35:1038–1045. 50 Plotkin H, Rauch F, Bishop NJ, et al: Pamidronate treatment of severe osteogenesis imperfecta in children under 3 years of age. J Clin Endocrinol Metab 2000;85:1846–1850. 51 Senthilnathan S, Walker E, Bishop NJ: Two doses of pamidronate in infants with osteogenesis imperfecta. Arch Dis Child 2008;93:398–400. 52 Munns CF, Rauch F, Zeitlin L, Fassier F, Glorieux FH: Delayed osteotomy but not fracture healing in pediatric osteogenesis imperfecta patients receiving pamidronate. J Bone Miner Res 2004;19:1779–1786. 53 Marini JC, Hopkins E, Glorieux FH, et al: Positive linear growth and bone responses to growth hormone treatment in children with types III and IV osteogenesis imperfecta: high predictive value of the carboxyterminal propeptide of type I procollagen. J Bone Miner Res 2003;18:237–243. 54 Horwitz EM, Prockop DJ, Fitzpatrick LA, et al: Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309–313. 55 Horwitz EM, Prockop DJ, Gordon PL, et al: Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood 2001;97:1227–1231.

Prof. Nick Bishop Academic Unit of Child Health, University of Sheffield Sheffield Children’s Hospital, Western Bank Sheffield S10 2TH (UK) Tel. +44 114 271 7228, Fax +44 114 275 5364, E-Mail [email protected]

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Chapter 11 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 170–190

Secondary Osteoporosis S.F. Ahmed ⭈ M. Elmantaser Bone and Endocrine Research Group, University of Glasgow, Royal Hospital for Sick Children, Yorkhill, Glasgow, UK

Abstract Secondary osteoporosis is more difficult to define in children than in adults but clearer definitions have recently been provided by the International Society for Clinical Densitometry. Whereas in adults, osteoporosis is defined on the basis of reduced bone density on scanning, in children the definition requires additional clinical parameters to be fulfilled. Secondary osteoporosis may arise either as a result of the effects of underlying disease or as a result of the treatment of such diseases (e.g. with glucocorticoids). The normal balance of bone formation and removal, both by bone modelling and remodelling, is disturbed in such a way as to alter the normal accumulation of bone that occurs during childhood. A multitude of intrinsic and extrinsic factors contributes to these processes. The underlying principles of treatment of secondary osteoporosis is, where possible, to remove the underlying cause. Where this is not possible, minimising the effects of treatment with drugs that adversely effect bone may be sufficient to eliminate any deterioration in bone quality. If this is not possible, the use of bone sparing drugs such as the bisphosphonates may be necessary whilst ensuring that attention is paid to optimising calcium and vitamin D intake and encouraging exercise and mobility. Copyright © 2009 S. Karger AG, Basel

Increased bone fragility in children may either be due to an intrinsic skeletal abnormality (primary osteoporosis) or as a result of a range of chronic systemic diseases and their management (secondary osteoporosis). With improvements in health care and technology, increasing numbers of children and adolescents with chronic debilitating inflammatory and non-inflammatory conditions can look forward to a fuller and longer life. However, this improved survival may be associated with an increased likelihood of unwanted effects on the developing skeleton. This chapter will first define childhood osteoporosis, discuss the underlying pathogenesis of secondary osteoporosis, provide some examples of associated clinical conditions and finally discuss possible management strategies.

Definition

Osteoporosis is a condition characterised by a reduction in bone mass and bone strength leading to an increased risk of fractures. In children, osteoporosis may arise from either an intrinsic skeletal abnormality (primary osteoporosis) or as a result of consequences of systemic diseases and their treatments (secondary osteoporosis). Whilst in adults the definition of osteoporosis is dependent on a T score, which is the standard deviation (SD) score of the observed areal bone mineral density (aBMD) using dual energy X-ray absortiometry (DXA) compared with that of a normal young adult (a T score of < –1 SD indicates osteopaenia whereas a T score of < –2.5 SD indicates osteoporosis), this is not appropriate in children. Even age- and sex-matched standards may not be sufficient in children with chronic disease who often suffer from growth retardation and pubertal delay [1]. These difficulties of interpretation of DXA are discussed in more detail elsewhere (see chapter 5). Other forms of densitometry, such as peripheral quantitative computerised tomography (pQCT) and quantitative ultrasound (QUS), have been utilised in children but neither has been universally adopted as a means of assessing bone health in children with chronic disease. A one-off assessment of biochemical markers of bone turnover does not have much utility given the wide reference for these parameters across the age range. The invasive nature of bone biopsies has precluded them from being performed in most cases of suspected osteoporosis. Recently, the International Society for Clinical Densitometry (ISCD) stated that osteoporosis should not be diagnosed in children based solely on DXA bone mineral density (BMD) [2]. The Society’s position is that the diagnosis of osteoporosis in children necessitates the co-existence of a significant history of fracture and a low BMD or bone mineral content (BMC). A long bone fracture of the lower limbs, compression fractures of vertebrae or two or more long bone fractures of upper limbs are considered as a significant clinical history of fracture. Low BMC or aBMD is defined as a BMC or aBMD Z-score that is less than or equal to –2.0, adjusted for age, gender and body size, as appropriate. Whilst it is prudent not to diagnose children with a sole abnormality of a low BMC or aBMD with osteoporosis, it is increasingly becoming clear that children with chronic diseases can suffer fractures without necessarily having a particularly low size-adjusted BMC or aBMD [3]. Perhaps a fall in bone mass maybe a better indicator of fractures in children with chronic disease and requires further investigation [4].

Pathophysiology

At a general level, osteoporosis results from a failure in the physiological balance of bone remodelling (bone formation and bone resorption) and bone modelling (bone formation) (fig. 1). In children and adolescents, it is normal for bone formation

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Bone-reducing factors PTH 1,25OHD TNF␣ GC

TNF-␣ GC IL-6 Activation

IL-1␣Ⲑ␤ Resorption

Resting phase

RANK RANKL

Reversal phase

GC Formation

Bone-enhancing factors IFN-␥ TGF-␤ TGF-␤ BMP IGF-I, II FGF PDGF E2

Activation Resorption

RANK Reversal phase

Resting phase OPG

PTH

Formation

Fig. 1. Bone remodelling compromises five phases (1) resting, (2) activation, (3) resorption, (4) reversal and (5) formation. GC, IL-1α/β, TNF-α, 1,25(OH)2D and PTH enhance bone resorption (phase 2) by activation osteoclastogenesis and increased RANKL expression. On the other hand, TGF and IFN-γ suppress bone resorption (phase 2) by inhibiting RANKL expression. TNF-α, GC and IL-6 stop transformation of resorption phase into reversal whereas, TGF-β, BMP, PGE2, FGF stimulate bone formation by enhancing osteoclast apoptosis. The interaction of RANKL and RANK is opposed by the decoy receptor osteoprotegerin (OPG), which can bind RANKL and prevent osteoclast activation.

greatly to exceed bone resorption, with the net effect of bone accretion. In early adulthood, there is a steady-state balance between bone formation and resorption and then, in late adulthood, the rate of bone resorption increases resulting in age-related osteoporosis [5].

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Osteoclast Activity A balance between the activation and inhibitory factors that control osteoclastogenesis is important in dictating the level of bone resorption. Osteoclastogenesis and osteoclast survival are promoted by receptor activator of nuclear factor kappa-β ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), which are released from osteoblasts or stromal cells. RANK-RANKL interaction can be blocked by the decoy receptor osteoprotegerin (OPG) and RANKL expression can be upregulated by cytokines such as TNFα and IL-1 (see chapter 3 for further details). PTH and glucocorticoids increase RANKL and decrease OPG. IL-1, IL-6 and PGE2 have a positive impact on the release of RANKL [6]. RANKL can also be promoted by 1,25-dihydroxyvitamin D (1,25(OH)2D). On the other hand, osteoclastogenesis can be inhibited by TGF-ß and oestrogen. Oestrogen also downregulates IL-1, IL-6, M-CSF, RANKL and TNF-α [7], thus inhibiting the activation of osteoclast production. Besides proinflammatory cytokines, activated T cells can also secrete a number of the above factors (OPG, RANKL and M-CSF) that are involved in regulating osteoclast activity [8]. Thyroid hormone (T3) is essential for osteoclast differentiation but T3-induced osteoclastogenesis is not regulated by RANKL/OPG interaction. T3 can induce osteoclast formation in the absence of osteoblasts and its effects on osteoclasts are likely to be mediated by other mechanisms such as increased expression of c-Fos and Fra-1 [9]. The effect of TSH on bone is independent of the circulating T3. The TSH receptor is expressed on the surface of both osteoclast and osteoblasts. TSH inhibits bone cell differentiation and reduces bone turnover by suppressing LRP-5 and FLK-1 in osteoblast and by downregulating RANKL in osteoclasts. An absence of TSH receptor signalling results in increased bone turnover in favour of bone resorption rather than bone formation [10].

Osteoblast Activity Among the many local and systemic substances that control bone formation, IGF-1 and IGF-2 (insulin-like growth factors) have an anabolic effect on bone formation. IGFs release collagen proteins from osteoblasts and reduce bone matrix degradation by inhibiting collagenase [11]. They have a key role in bone turnover and bone growth [12]. IGFs combine with a group of six secreted IGF-binding proteins (IGFBPs 1–6) with the most abundant in bone being IGFBP-4 and IGFBP-5. IGFBP4 has the ability to block the action of IGFs and inhibit bone formation. IGFBP-5 is a polypeptide chain and the amino terminal is attached to IGFs. This protein is released during endochondral bone formation and global knockout of IGFBP-5 in mice is associated with osteoporosis. On the other hand, a recent study has shown that IGFBP-5 inhibits bone morphogenic protein 2 (BMP-2)-induced osteoblast differentiation and function and blocks bone growth. According to this finding, IGFBP-5 inhibits IGF actions in bone

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cells [13]. Vanderschueren et al. [14] demonstrated that androgens increase the rate of periosteal bone formation in males at puberty, whereas oestrogens decrease this rate, but stimulate endosteal bone apposition. Furthermore, oestrogens increase expression of Fas ligand in osteoblasts. Fas ligand pathways induce the apoptosis of pre-osteoclasts [15]. There are many factors that promote osteoblast differentiation including bone morphogenetic proteins (BMPs), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), PTH, PTH-related protein (PTHrP), vascular endothelial growth factor (VEGF) and peptides such as activin, inhibin, and amylin [5, 16].

Osteocyte Activity In children, the modelling process increases cross-sectional bone strength and bone mass, whereas, in adults, modelling does not increase bone accretion, but it can strengthen trabecular bone throughout life. According to the mechanostat theory, bone modelling is initiated when bone strains exceed 1,000 microstrains. If strain remains below that threshold, then the modelling process is turned off. The bone remodelling threshold is felt to be about 50–100 microstrains. If strain stays below this threshold, then bone remodelling by BMU will be activated in favour of bone resorption (disuse mode) resulting in net bone loss. Therefore, physical inactivity, reduced muscle strength and lack of weight bearing activities may result in osteoporosis [17]. With the recent reports of increased bone fragility and osteoblastic dysfunction in osteocyte-ablated mice, the role of osteocytes in maintaining bone health has become increasingly important [18]. Mechanical stimuli can be translated into intracellular signals by osteocytes through extracellular transmembrane receptors such as Integrins and CD44, which stimulate bone remodelling through the production of secondary intracellular messengers including prostaglandin (PGE2), cyclooxygenase-2 (COX-2) and nitric oxide (NO) [19]. Proinflammatory cytokines such as TNF-α can induce osteocyte apoptosis and in vitro studies suggest that mechanical loading can reduce TNF-α-induced apoptosis in osteocytes [20]. On the other hand, mechanical unloading, which occurs under conditions of microgravity or a long bed rest, result in canaliculi fluid stasis and induce osteocyte apoptosis [20, 21].

Glucocorticoids Glucocorticoids (GC) can affect bone health through numerous skeletal and extraskeletal mechanisms (fig. 2). They decrease bone formation by stimulating osteoclastogenesis and inhibiting osteoblastogenesis and it seems that the response of the skeleton to GC exposure is biphasic; the initial bone loss may be due to increased survival of osteoclasts [22], whereas the second phase may be due to osteocyte and osteoblast apoptosis [23]. This might provide an explanation for a higher rate of bone

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Growth plate Vascular development Paracrine IGF-I in growth plate Somatostatin

GH IGF-I

Proliferation and hypertrophy of chondroctyes

Primary disease

Bone LH/FSH Glucocorticoids

Estrogens Testosterone

Osteoblast activity Osteoclast activity

Altered bone remodelling in favour of resorption

Bone loss and growth retardation

Matrix synthesis Renal and GI calcium loss

Nutrition and VitD

PTH Muscle bulk

Skeletal load

Inflammatory cytokines

Fig. 2. Mechanisms of GC-induced bone loss and growth retardation.

loss during the first few months of therapy, followed by a slower rate of bone loss thereafter. Mesenchymal stem cells give rise not only to osteoblasts, but also to chondrocytes, adipocytes, fibroblasts and myoblasts. GCs may lead to a shift in mesenchymal cell differentiation toward adipocytes and away from osteoblasts and inhibit terminal cellular differentiation of osteoblasts. Other possible indirect effects on bone include decreased calcium and phosphate absorption and increased renal calcium excretion leading to secondary hyperparathyroidism. GC may also induce a myopathy and muscle weakness which may lead to a reduction in bone strain and mechanical stimuli. They may lead to a reduction in synthesis of constituents of bone such as type 1 collagen and other non-collagenous proteins [24]. Finally, whilst most reports have concentrated on the adverse effect of exogenous GC, it is likely that disease itself may alter the balance between active and inactive forms of endogenous GC at the level of bone tissue itself in favour of the active form, thus increasing the overall GC exposure [25]. For a detailed review of the effects of GC on the skeleton in children, the reader is directed to Mushtaq and Ahmed [26] and Ward [27].

Aetiology of Secondary Osteoporosis There are many chronic childhood conditions which may be associated with secondary osteoporosis (table 1). In most cases, more than one factor contributes to the

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Table 1. Conditions that may be associated with secondary osteoporosis in children and the overlap between conditions that may affect bone health and conditions that are associated with fractures in children Conditions that may affect bone health in children

Conditions that may affect bone health in children and conditions that are actually associated with published reports of fractures in children

Inflammation Juvenile idiopathic arthritis Systemic lupus erythematosis Inflammatory bowel disease Dermatomyositis

+ + + +

Glucocorticoids Acute lymphoblastic leukaemia Nephrotic syndrome Cushing’s syndrome/disease Cystic fibrosis

+ + + +

Mechanical Cerebral palsy Duchenne muscular dystrophy Spinal cord injury

+ + +

Endocrine Hypogonadism Hyperthyroidism Hyperprolactinaemia Glucocorticoid excess Diabetes mellitus Klinefelter syndrome Turner syndrome Growth hormone deficiency

– – – + – – – –

Nutrition Hypocalcaemia Vitamin D deficiency Anorexia nervosa Coeliac disease Malabsorption Parenteral nutrition

+ + + + + +

Iatrogenic Anticonvulsants Heparin and Warfarin Methotrexate Radiotherapy Medroxyprogesterone acetate Thyroxine Glucocorticoids Gonadotropin-releasing hormone agonists

+ – + + – – + –

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Table 1. Continued Conditions that may affect bone health in children

Conditions that may affect bone health in children and conditions that are actually associated with published reports of fractures in children

Inborn errors of metabolism Lysinuric protein Intolerance Glycogen storage disease Galactosaemia Gaucher disease Homocystinuria

+ + – + _

Haematological disorders Sickle cell disease Thalassaemia Haemophilia

+ + –

Renal Chronic metabolic acidosis Chronic renal failure Hypercalciuria Isolated hypophosphataemia

+ + + –

Miscellaneous Idiopathic scoliosis Organ transplantation

– +

+ = Fracture; – = no fracture.

aetiology and will include chronic inflammation, glucocorticoid exposure, lack of physical activity, malnutrition, drugs that alter bone metabolism and hypogonadism. In many situations, the clinician who is primarily managing the original disease has to achieve a difficult balance between controlling the disease and using drugs such as glucocorticoids.

Chronic Inflammation Osteoporotic fractures are associated with several chronic inflammatory conditions such as inflammatory bowel disease (IBD), juvenile idiopathic arthritis (JIA), systemic lupus erythematosis (SLE) and Crohn’s disease (CD). In IBD, fractures may even be the presenting feature before the introduction of glucocorticoids and in vitro culture models of rodent calvarial bones have shown a clear adverse effect of serum from children with IBD which can be blocked by IL-6 antibodies, thus highlighting the importance of the proinflammatory cytokines themselves [28]. Whilst these

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Table 2. Bone densitometry in children with IBD Study

n

Method

Site

Result

Issenman,1993 [85]

17

BMC for age BMC for height

LS

low for height

Semeao, 1997 [86]

5

BMD SDS

LS

–2.3, –5.1, fractures

Cowan, 1997 [87]

32

pBMC for bone area

TB, Fem

97%, 93%

Boot, 1998 [88]

55

BMD SDS

TB, LS

–0.75, –0.95

Gokhale, 1998 [89]

99

BMD SDS

LS, Fem

< –1–60%

Herzog, 1998 [90]

43

BMD SDS BMD for height age

LS

< –2–44% < –2–14%

Semeao, 1999 [91]

119

BMD SDS

LS

< –1–70%, < –2–32%

Mauras, 2002 [92]

10

BMD SDS

LS

–0.7, –4.1

Ahmed, 2004 [1]

73

pBMC for bone area

TB, LS

101%, 98%

conditions may be associated with reduced bone mass due to several factors, including active inflammation, reduced lean mass and the use of drugs such as GCs, doubts do remain about the prevalence of true low bone mass when this is corrected for size. For instance, table 2 summarises a number of studies that have reported BMC or BMD in children with IBD and the studies that have reported size-adjusted data show a relatively lower incidence of low BMC.

Glucocorticoid-Induced Osteoporosis Approximately 5–10% of children may use glucocorticoids at sometime during childhood [29]. In adults, a rapid loss of BMD is observed in glucocorticoid therapy, particularly in the fist year of treatment. Whilst this loss may persist throughout the duration of treatment, it may reverse partially on cessation of GCs. Studies in children receiving chemotherapy for leukaemia show an imbalance between markers of bone formation and bone resorption and show reversibility [30]. This is particularly marked during periods of high-dose GC therapy. However, markers of bone formation remain low even during maintenance chemotherapy when

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children received relatively low doses of GC [29]. Trabecular bone seems to be more sensitive than cortical bone to the catabolic effects of GC [31] but this is not universally reflected in fractures in children where, in some conditions such as ALL children receiving GC therapy, appendicular fractures are commoner than fractures of the axial skeleton [32–34]. The largest study to evaluate the incidence of fractures among paediatric GC users was a case-control study involving over 37,000 children treated with four or more courses of oral GCs for a mean duration of 6.4 days [35]. Compared to controls, GC-treated children had an adjusted odds ratio for fracture of 1.32 (95% CI 1.03–1.69). Moreover, the risk of fracture may depend on the dose of GC, with an incidence of about 2.6 versus 1.6% in the lowdose group [36]. For the treatment of some conditions such as ALL, dexamethasone is preferred to prednisolone for its greater lymphocytotoxic effect, and our group’s preliminary data confirm a previous observation of higher bone morbidity in those children receiving dexamethasone compared to prednisolone [32, 34]. In comparison with prednisolone, dexamethasone may be almost ten times more potent at suppressing bone turnover [29]. Besides GC, other chemotherapy and even the disease process itself might affect bony morbidity [33, 36]. For example, methotrexate, a folate antagonist, induces bone resorption and decreases bone formation [37]. However, until now, long-term follow-up of children with ALL who have just received chemotherapy have not revealed any abnormalities of bone mineral content [38].

Impaired Mobility Osteoporosis may occur in children with conditions that are associated with impaired mobility, such as cerebral palsy, spinal cord injury, head injury, muscular dystrophy and spinal muscular atrophy [39]. Many children will suffer from additional skeletal morbidity including scoliosis, fixed flexion deformities as well as joint subluxation and dislocation. In children with cerebral palsy, pathological fractures are commoner at the femoral shaft and supracondylar region and this may be due to abnormalities of growth and presence of contractures in the major joints, particularly knees and hips [40, 41]. In addition, there are several contributory factors, including muscle weakness, malnutrition and use of anticonvulsants. In Duchenne muscular dystrophy (DMD), the use of GC as therapy for slowing down the progression of the muscular dystrophy may also contribute to the pathogenesis of secondary osteoporosis in DMD boys. In a longitudinal study of the use of deflazacort in 79 children with DMD who had regular spine X-rays, the incidence of lower limb fractures was about 25% and similar in the treated and untreated group [42]. Despite this, vertebral fractures only occurred in 20% of the treated group. Abnormalities in markers of bone turnover and lower BMD have also been described in children with DMD who are receiving GC [43]. However, lumbar spine BMD is often preserved

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in children with impaired mobility and should not be the sole means of assessing bone fragility [39].

Hypogonadism Hypogonadism may exist in children due to a variety of reasons. Primary hypogonadism includes syndromes such as the Turner and Klinefelter syndromes. Secondary causes of hypogonadism may result from congenital or acquired abnormalities of the hypothalamic-pituitary axis. The association of low bone mass with rare genetic abnormalities of sex steroid production or action such as androgen insensitivity, defective aromatase activity and oestrogen receptor alpha defect have emphasised the important role of oestrogens and androgens in maintaining bone health. It is unclear whether low bone mass is commoner in adolescents with temporary physiological hypogonadism that is encountered during constitutional delay of growth and puberty. Only one study in young adults has shown an association between low bone mass and a past history of delayed puberty [44] which was not confirmed in a subsequent study in this group where adjustment of bone density for height was made [45]. In adolescents and young adults with cystic fibrosis, hypogonadism has been reported as a pivotal condition that may contribute to the occurrence of fractures [46]. It is likely that, in children, hypogonadism by itself may not predispose an otherwise healthy child to fractures but the state of hypogonadism in a child with coexisting chronic illness may be more detrimental.

Growth Hormone Deficiency Whilst GH and IGF-1 are important regulators of growth plate chondrocytes and longitudinal growth, there is a relative lack of clarity regarding the importance of their role in changing bone mass in children. GH and IGF-1 may exert direct effects on osteoblasts as these cells do express the receptors for these hormones [12]. The two hormones may also alter lean mass which may exert an indirect effect on bone mass. Finally, GH may alter PTH sensitivity, thus indirectly influencing bone turnover. Bouillon et al. [47] reported that the fracture rates in GHD adults with multiple pituitary hormone deficits (closed plate) was three times that in control population and ten times higher if they have open plates. Many children with GHD have multiple pituitary hormone deficits and it may be difficult to tease out the contributory role on bone mass of each replacement drug, particularly corticosteroids and sex steroids [48], as well as the low TSH levels that may be present due to central hypothyroidism. There is currently no evidence that children with GHD suffer higher rates of fracture than other children.

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Thyroid Disease Hypothyroidism in children when treated with thyroxine replacement has not been reported to be associated with any abnormalities of bone health as assessed by DXA or pQCT [49, 50]. However, pQCT assessed cortical bone density was significantly lower in a group of children and adolescents with untreated hyperthyroidism [51].

Diabetes Several contributory factors can cause low bone mass in patients with diabetes, including high urinary calcium excretion, high PTH level and low serum levels of insulin and IGF-1 [37]. Low BMD may be dependent on the duration of illness as well as adequacy of control [52, 53]. However, in practice, low BMD is not of clinical importance in diabetes. Calcium and Vitamin D Whilst population studies often show that a low calcium intake may be related to the risk of fractures in healthy school children, overt osteoporosis that is linked to a profound lack of calcium and the vitamin D is rarely described. However, there is a sufficient number of reported cases to suggest that highly selective diets that avoid milk may lead to a severe lack of calcium and multiple fractures [54]. In addition, vitamin D deficiency rickets may also be associated with fractures [55]. Anorexia Nervosa Anorexia nervosa (AN) is a common disorder among girls in developed countries, with a prevalence of 0.2–1.0% [56]. AN is frequently associated with low bone mass and may be severe enough to result in increased bone fragility and an elevated fracture risk throughout life. Low BMD in patients with AN is associated with a reduced state of bone turnover, an increase in bone resorption markers and decreased bone formation markers. Several factors are responsible for low BMD in teenagers with AN, including decreased lean mass, a low fat mass, hypogonadism, low IGF-1 and a high level of cortisol. In addition to the low caloric intake, these individuals have a restricted intake of calcium, vitamin D and protein. Anticonvulsants Bone diseases are frequently associated with the use of anticonvulsants including diphenylhydantoin, phenobarbital, sodium valproate, and carbamazepine. The risk factors for developing low bone mass are dependent on the dose and duration of treatment, treatment combination, concomitant vitamin D therapy and sun exposure. The cytochrome P450 enzyme system is involved in the metabolism of vitamin D

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(see chapter 2), and antiepileptic drugs (AED) that induce cytochrome P450 activity are most commonly associated with a detrimental effect on bone through functional vitamin D deficiency [57]. However, the mineralisation rate in anticonvulsant druginduced bone disease is normal, but there is an increased frequency of bone remodelling and bone turnover [58]. Heparin Heparin induces bone resorption and reduces bone formation in in vitro studies through direct inhibitory effects on DNA and collagen synthesis in bone and by reducing PGE2 levels [59]. Risk factors for heparin-induced osteoporosis include duration of therapy (more than 6 months) and heparin dose (more than 15,000 U) [37]. Walton et al. [60] reported that long-term heparin therapy caused a subclinical reduction in BMD in about a third of patients. Recently, Ariyoshi et al. [61] found that heparin has an inhibitory effect on osteoclastogenesis by RANKL due to its binding to RANK. Chemotherapy Fractures in children receiving chemotherapy, particularly for ALL, is a well-recognised occurrence. Besides glucocorticoids, there is a possibility that other drugs used in the chemotherapy protocol may also contribute to this increased predisposition. Repeated administration of methotrexate, as a single agent, to children with ALL in remission is accompanied by bony morbidity in the form of pain, osteoporosis, and fractures [62]. High-dose methotrexate is more likely to have an adverse effect on bone turnover in favour of bone resorption [63]. At concentrations achievable in vivo, methotrexate inhibits the proliferation of human osteoblasts in vitro [64]. With the addition of 1,25(OH)2D to these cultures, osteoblast maturation is increased. In similar experiments, it was shown that vincristine, daunorubicin, etoposide, and asparaginase caused a reduction in type 1 collagen synthesis [65]. In addition, alkylating therapy, such as ifosfamide, may be associated with renal phosphate loss, and some drugs such as vincristine may contribute to poor mobility.

Diagnostic Approaches to Secondary Osteoporosis

Clinical history is very important in children with recurrent fractures and should include the age of onset, the nature of the fractures and the force involved. The management of previous fractures, including documentary and radiological evidence of the fractures, is often useful to confirm a history of multiple fractures. Birth weight and gestation and a detailed neonatal history are very useful in cases of prematurity. The age of onset and the severity of any chronic condition and its therapy should be assessed carefully. In children with chronic inflammatory diseases and malignancies details of glucocorticoids, chemotherapy and

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radiotherapy is necessary. A history of the extent of physical activities undertaken by the child and the child’s nutrition and use of any vitamin supplements needs to be documented. Detailed physical assessment of the child should include the measurement of height, sitting height, weight and an assessment of the child’s nutritional status. The skeleton should be examined for any deformity and abnormalities such as kyphosis, scoliosis and localised vertebral tenderness. The possibility of a genetic disorder that predisposes to fractures should not be ignored. Whilst there is no need for a skeletal survey, lateral radiographs of the spine may be of diagnostic value. First-line biochemical and haematological investigations should include a basic biochemistry screen that checks renal and liver function, calcium, phosphate, 25OHD and PTH, a full blood count and an ESR. Subsequent investigations shall be guided by the results of these initial tests as well as any specific pointers in the history. Measurement of markers of bone turnover have not been conclusively shown to have a diagnostic value in children with unexplained osteoporosis but they may be useful in monitoring therapy. The use of dual energy X-ray absorptiometry (DXA) measurement of BMC and BMD has rapidly gained popularity in children and adolescents [66]. Whilst it is a relatively quick procedure, non-invasive and involves a low radiation dose, DXA cannot measure true volumetric BMD and cannot differentiate between cortical and trabecular bone [67]. However, the recent ISCD definition of osteoporosis in children is based on DXA measurements. The largest DXA reference ranges exist for total body and lumbar spine regions. In some neuromuscular conditions with regional abnormalities of bone mass such as cerebral palsy or DMD, the DXA scan may be technically difficult to perform, analyse and interpret, particularly if there are no reference ranges for the area studied. However, the value of a one-off DXA BMC is limited. In otherwise healthy youth, fracture risk at the radius has been reported to increase by approximately twofold for every 1 SD reduction in spine BMC or BMD [68]. However, in children with chronic disease, DXA BMC does not seem to relate clearly to fracture risk [3]. This has also been observed in adult women receiving post-menopausal glucocorticoid therapy who are at risk of fractures at a higher BMD than untreated women [69]. A diagnostic algorithm based on the mechanostat theory of bone-muscle development has been proposed for the characterisation of bone disease in children with chronic illness [70] and it is possible that lean mass adjusted BMC may be a better predictor of fractures [3]. Quantitative ultrasound (QUS) is another technique that was initially developed to assess calcaneal bone status in adults. Two measures are important in QUS; speed of sound (SOS) and broadband attenuation (BUA). SOS is referred to as the ratio between the traversed distance and the transit time (m/s) and it is dependent on several factors; including bone density, bone structure and elasticity. The major advantages of QUS are low cost, accessibility and no radiation. QUS may be particularly suitable for those children who are relatively immobile such as preterm babies [71] or

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children with neurodisability [72] but its role in predicting fractures in children with chronic disease is unclear. Peripheral quantitative computed tomography (pQCT) can measure true vBMD rather than aBMD and may be a useful technique in children with chronic disease and growth retardation and can provide detailed assessment of bone size and geometry which can be used to calculate bone strength. On the other hand, pQCT underestimates cortical vBMD when the cortical thickness is below 2 mm [73]. Longitudinal measurements may be difficult to interpret because of concerns about the site of measurement which may change due to longitudinal bone growth [67].

Prevention and Treatment

General Approach Avoidance of risk factors is the most important step to take for management of secondary osteoporosis. Whilst there is good evidence that a number of the factors above are associated with an increased fracture risk, particularly when in combination, there is relatively poor evidence that the interventions outlined below have the potential to reduce fractures in children with secondary osteoporosis. A comprehensive assessment of all risk factors should be undertaken before initiating a treatment plan (fig. 3). For children on GC therapy, avoidance of prolonged systemic use and preference for targeted and shorter duration courses may be beneficial. GC therapy can be administered intra-articularly, rectally, topically or directly to the lungs rather than systemically and these routes should be preferred to oral or intravenous, depending on the disease itself. The use of GC sparing immunomodulator therapy is increasingly being used to avoid GC exposure. In addition, the promise of selective GC receptor modulators that do not exert the harmful skeletal effects of GC in the future would allow clinicians to continue to avail themselves of the beneficial effects of GC [74]. Hypogonadism, inadequate nutrition (including calcium and vitamin D deficiency), excess thyroxine or GC replacement, malabsorption, and impaired mobility should also be addressed as part of the overall management plan. Whilst administration of vitamin D and calcium can improve BMD in children with CP and who were treated with AEDs compared to untreated patients [75, 76], there is no clear evidence that the use of vitamin D supplements in vitamin D-sufficient children with chronic disease prevents or treats secondary osteoporosis.

Physical Activity Recently, the effect of load-bearing exercise to maintain bone density and avoid osteoporosis has shown some acceptable results [77]. Chad et al. [78] reported an increase

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Annual visit Ht, Wt, puberty UE, 25OHD, ALP, PTH, DXA

Boys >13 years old with testes <4 ml Girls >12 years with no breast development

Ht <-2 SDS or Ht <-1 SDS and Ht Vel <-1 SDS

Endocrine/metabolic bone review

25O HD <30 nmol/l PTH-normal

Review diet and supplements

25O HD <30 nmol/l PTH-raised

Vitamin D therapy

pBMC <-1.5 SDS

pBMC <-0.5 SDS

pBMC >-0.5 SDS

DXA – after 1 year

DXA – after 2 years

Non-traumatic fractures may alter referral pathway

Fig. 3. Practical recommendations for growth and bone health assessment in children with chronic inflammatory disease; pBMC-predicted BMC for bone area; alternately, BMC can also be adjusted for height age.

in femoral neck density by 5.6% in children with CP who were treated with load bearing exercise, in comparison with a decrease by 6.3% in the control group. Several studies have suggested that low level, high frequent mechanical stimuli in the form of whole body vibration (WBV) may increase the quantity and quality of trabecular bone [77]. Bone and muscle mass can be increased in women (15–20 years) with low BMD by a short daily exposure of WBV [79]. This has also been shown in a group of children with CP [80]. However, it is unclear whether this intervention is associated with a change in fracture frequency in those children who are at a high risk of osteoporosis.

Bisphosphonates Bisphosphonates (BP) are the commonest drugs used in the treatment of osteoporosis. In children with osteogenesis imperfecta, pamidronate therapy is associated with improvements in vertebral BMD, muscle mass and skeletal morbidity [81]. Over the past two decades, a variety of oral and parenteral bisphosphonates has been used in children to treat osteogenesis imperfecta as well as a number of conditions associated with secondary osteoporosis. A recent review of bisphosphonate therapy for children and adolescents with secondary osteoporosis concluded that whilst there was a poor

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level of evidence to recommend the use of bisphosphonates as a primary prevention measure, there were sufficient short-term safety and efficacy data to support their use for a limited period of less than three years in cases of fractures and pain [82]. A number of different doses and preparations have been used until now and it is unclear whether they have a differential effect on functional outcomes such as pain and fractures [83]. Bisphosphonate therapy in children may initially be associated with an acute reaction such as fever, muscle pain, headache and vomiting and hypocalcaemia when administered intravenously. Some animal trials have reported that high doses of bisphosphonates have a negative impact on growth but this has not been observed in children. Bisphosphonates should not be used during pregnancy and all women of reproductive age should have a pregnancy test because of concerns about teratogenicity, although these have not yet been confirmed in humans [38]. Skeletal complications such as osteonecrosis of the jaw have been described in adults but not in children or adolescents. Whilst a safe upper limit for bisphosphonate therapy has not been established, the adverse effect of greatest concern in children is over-suppression of bone modelling and remodelling. Iatrogenic osteopetrosis and pathologic fractures have been described in a child treated for over 2 years with a relatively high dose of pamidronate [84].

Conclusions

Secondary osteoporosis may occur as the result of a number of conditions. In some cases this is the unavoidable consequence of treatment of an underlying condition. In others it results from the underlying condition itself. Assessment of clinical significance of secondary osteoporosis requires a thorough understanding of the mechanisms involved and interpretation of the results of investigations, particularly of methods of measuring bone density, should be done by someone experienced in their use. Treatment should initially be directed towards removing the cause of the osteoporosis or, if that is not possible, minimising the effects of treatment. If osteoporosis persists and causes significant clinical problems, it may be necessary to institute specific measures directed towards counteracting the effects of that treatment. These measures should preferably be undertaken by an experienced bone specialist.

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91 Semeao EJ, Jawad AF, Zemel BS, Neiswender KM, Piccoli DA, Stallings VA: Bone mineral density in children and young adults with Crohn’s disease. Inflamm Bowel Dis 1999;5:161–166. 92 Mauras N, George D, Evans J, et al: Growth hormone has anabolic effects in glucocorticosteroiddependent children with inflammatory bowel disease: a pilot study. Metabolism 2002;51:127–135.

Dr. S. Faisal Ahmed, MD, FRCPCH Bone and Endocrine Research Group, Department of Child Health University of Glasgow, Royal Hospital for Sick Children Yorkhill, Glasgow G3 8SJ (UK) Tel. +44 0141 201 0571, Fax +44 0141 201 0837, E-Mail [email protected]

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Chapter 12 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 191–217

Miscellaneous Bone Disorders M. Zulf Mughal Saint Mary’s Hospital for Women and Children, Manchester, UK

Abstract This chapter deals with a few of the important childhood bone disorders associated with high bone mass as well as conditions associated with fragility fractures and limb deformities, which have not been dealt with in previous chapters. A couple of skeletal dysplasias that can be sometimes be confused with rickets are also dealt with in this chapter. The principal features of the conditions described here are shown in a table. However, a comprehensive review of skeletal dysplasias is beyond the Copyright © 2009 S. Karger AG, Basel scope of this chapter.

Disorders Associated with Excessive Amounts of Bone in the Skeleton

Osteopetrosis Osteopetrosis is a heterogeneous group of inherited bone disorders caused by an inability to produce mature osteoclasts from haematopoietic stem cells, or due to defects in acidification of the extracellular space between the osteoclast ruffled border and the bone surface, which is responsible for resorption of bone matrix (see chapter 3 for details of osteoclast development and function). This leads to progressive osteosclerosis with thickened but fragile bones. Based on the pattern of inheritance, age of onset and severity, osteopetrosis has been classified into four major clinical types. The principal causes are summarised in figure 1.

Autosomal-Recessive (Malignant) Osteopetrosis The autosomal-recessive (malignant) form of osteopetrosis (OPTB) is itself a heterogeneous group of genetic disorders caused by defects in production of mature osteoclasts, or due to molecular defects that lead to non-functional osteoclasts. The former group, which is characterised by paucity of mature osteoclasts, is referred to as the

‘osteoclast-poor OPTB’, whereas the later group, which is associated with normal or elevated numbers of non-functional osteoclasts as the ‘osteoclast-rich OPTB’.

Osteoclast-Rich Autosomal-Recessive Infantile Osteopetrosis Over 50% of infants with ‘osteoclast-rich OPT’ have been shown to be caused by mutations in the TCIRG1 gene (*604592) (T cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 protein a isoform 3) (OPTB1) (#259700), which is responsible for the transport of hydrogen ions into the resorption lacunae, where a low pH is necessary for bone resorption [1]. Approximately 10% of cases of OPTB (OPTB4) (#611490) are caused by mutations in the CLCN7 gene (chloride channel 7) (*602727), a gene encoding for a chloride channel in the osteoclast-ruffled border membrane, where it compensates for active proton extrusion by chloride release [2]. A particularly severe form of osteoclast-rich OPTB (OPTB5) (#259720) caused by mutations in the OSTM1 gene (osteopetrosis-associated transmembrane protein 1) (#259720) has been described in a handful of infants [3]. Affected infants present in the first 3 months of life [4], although they may also present in the neonatal period with symptomatic hypocalcaemia due to a failure of osteoclastic mediated bone resorption, which plays an important role in maintaining plasma calcium concentration within the normal limits. Symptoms are often nonspecific and include: • feeding problems and failure to thrive due to bulbar nerve involvement, • recurrent infections due to defective neutrophil superoxide function, • leucoerythroblastic anaemia and thombocytopaenia due to obliteration of bone marrow cavity, • deafness and roving nystagmus due to visual impairment. Clinical features include macrocephaly, a square shaped head, frontal bossing, cranial nerve palsies (ptosis, squint, facial nerve palsy) and hepatosplenomegaly. Dental eruption is often delayed. In spite of having dense bones children with osteopetrosis are prone to fragility fractures. Neurological problems (blindness, deafness and facial palsy) in OPTB due to mutations in TCIRG1 gene arise from progressive entrapment of cranial nerves caused by osteosclerosis of the base of the skull. In contrast, infants with OPTB due to CLCN7 mutations often develop a primary encephalopathy and retinopathy [5], whereas those due OSTM1 mutations are associated with a progressive neurodegenerative disorder, characterised by profound hypotonia and seizures [3].

Management Left untreated, OPTB is fatal during infancy or early childhood. Since osteoclasts are derived from haematopoietic cell origin, bone marrow transplantation (BMT) is the

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RANK

RANKL

H2O + CO2

HCO–3

CAII H+ + HCO–3

Cl–

Cl– PLEKHM CTSK Ruffled border

Fig. 1. Diagrammatic representation of the mechanisms of the different forms of osteopetrosis. See appendix 1 for an explanation of the abbreviations.

H+ Cl– TCIRG1

H+

Bone

OSTM1 CLCN7

Sealing zone

Cl–

Resorption lacuna

only effective treatment for infants with the ‘osteoclast-rich’ form of OPTB secondary to TCIRG1 mutations. Ideally, this treatment should be undertaken as early as possible and preferably before 3 months of age. In patients with OPTB secondary to mutations in CLCN7, the primary encephalopathy and retinopathy does not regress after bone marrow transplantation [5]. Due to the severity of neurological manifestations and the aggressive nature of the bone disease, BMT has not been considered in the few reported infants with in OPTB due to OSTM1 mutations [3]. Supportive treatment includes nutritional support, appropriate orthopaedic treatment of fractures, blood transfusions for treatment of anaemia, prompt treatment of infections with appropriate antibiotics and optic nerve decompression where it is appropriate [4].

Osteoclast-Poor Autosomal-Recessive Infantile Osteopetrosis (OPTB2) (#259710) Children with ‘osteoclast-poor’ OPT tend to have severe disease which tends to progress more slowly than in those with ‘osteoclast-rich’ OPT [6]. These patients have been shown to have paucity of mature osteoclasts on bone biopsies [6] and, unlike infants with ‘osteoclast-rich OPTB’, they cannot be cured by BMT. Recently, ‘osteoclast-poor’ OPT has been shown to be due to mutations in the gene encoding for receptor activator of nuclear factor-κ B ligand (RANKL) (*602642), a key cytokine necessary for formation and activation of osteoclasts (see chapter 3 for details of osteoclast differentiation) [6]. Therefore, in the future it may be possible to treat such patients with soluble RANKL.

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Table 1. Table of genetic conditions discussed in this chapter Condition

OMIM

Location

Gene

Gene product

Autosomal-recessive 1 (OPTB1)

#259700

11q13.4q13.5

TCIRG1 subunit T cell immune regulator subunit of the vacuolar proton pump

Autosomal-recessive 2 (OPTB2)

#259710

13q14

TNFSF11

tumour necrosis factor superfamily 11

Autosomal-recessive 3 (OPTB3)

#259730

8q22

CA2

carbonic anhydrase II

Autosomal-recessive 4 (OPTB4)

#611490

16p13

CLCN7

chloride channel-7 protein

Autosomal-recessive 5 (OPTB5)

#259720

6q21

OSTM1

osteopetrosisassociated transmembrane protein-1

Autosomal-recessive 6 (OPTB6)

#611497

17q21.3

PLEKHM1

plekstrin homology domain-containing protein, family M member 1

Autosomal-recessive 7 (OPTB7)

#612301

18q22.1

TNFRSF11A (RANK)

tumour necrosis factor receptor superfamily, member 11a

Autosomal-dominant 2 (OPTA2)

#166600

16p13

CLCN7

chloride channel-7 protein

Pyknodysostosis

#265800

1q21

CTSK

cathepsin K

Hyperostosis corticalis generalisata (van Buchem disease)

#239100

17q12-q21

SOST

sclerostin

Sclerosteosis (SOST)

#269500

17q12-q21

SOST

sclerostin

Activating LRP5 mutations (OPTA1)

#607634

11q13.4

LRP5

low-density lipoprotein receptor protein 5

Osteopetroses

Other bone sclerosing conditions

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OMIM

Inheritance

Osteoclasts

Severity

Cause

604592

AR

osteoclast-rich

malignant

failure of acidification

602642

AR

osteoclast-poor

benign

failure of osteoclast transformation

611492

AR

osteoclast-rich

intermediate

failure of acidification; RTA

602727

AR

osteoclast-rich

malignant/ intermediate

failure of acidification

607649

AR

osteoclast-rich

malignant

failure of acidification

611466

AR

osteoclast-rich

intermediate

abnormal vesicular transport

*603499

AR

osteoclast-poor

malignant

failure of osteoclast transformation

602727

AD

osteoclast-rich

benign

failure of acidification

601105

AR

osteoclast-rich

failure of bone matrix resorption

605740

AR

osteoclast-rich

increased osteoblast signalling

605740

AR

osteoclast-rich

increased osteoblast signalling

*603506

AD

osteoclast-rich

Miscellaneous Disorders

benign

increased osteoblast signalling

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Table 1. Continued Condition

OMIM

Location

Gene

Gene product

Camurati-Engelmann disease (PDD)

#131300

19q13.1

TGFB1

transforming growth factor-β1

Fibrodysplasia ossificans progressiva (FOP)

#135100

2q23-q24

ACVR1/ALK2

activin receptor I/ activin-like kinase 2

Progressive osseous heteroplasia (POH)

#166350

20q13.2

GNAS

GNAS complex – paternally inherited

Caffey’s disease

#114000

17q21.31q22

COL1A1

collagen type 1, alpha 1

Osteoporosis pseudoglioma syndrome (OPPG)

#259770

11q13.4

LRP5

low-density lipoprotein receptor protein 5

Idiopathic juvenile osteoporosis (IJO)

259750

Juvenile Paget’s disease (JPD)

#239000

8q24

TNFRSF11B (OPG)

tumour necrosis factor receptor superfamily, member 11b (osteoprotegerin)

Geroderma osteodysplasticum (GO)

%231070

1q24

?

?

McCune-Albright/fibrous dysplasia (MAS/FD)

#174800

20q13.2

GNAS

somatic mutation GNAS complex

Perinatal lethal

#241500

1p36.1-34

TNSALP

liver alkaline phosphatase (tissue non-specific alkaline phosphatase)

Infantile

#241500

1p36.1-34

TNSALP

liver alkaline phosphatase (tissue non-specific alkaline phosphatase)

Childhood

#241510

1p36.1-34

TNSALP

liver alkaline phosphatase (tissue non-specific alkaline phosphatase)

Skeletal fragility and low bone mass

Hypophosphatasia

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OMIM

Inheritance

*190180

AD

*102576

AD

+139320

AD

+120150

AD

*603506

AR

*602643

AR

?

AR

*171760

AR

*171760

AR

*171760

AR/AD

Miscellaneous Disorders

Osteoclasts

Severity

Cause

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Table 1. Continued Condition

OMIM

Location

Gene

Gene product

Adult

#146300

1p36.1-34

TNSALP

liver alkaline phosphatase (tissue non-specific alkaline phosphatase)

1p36.1-34

TNSALP

liver alkaline phosphatase (tissue non-specific alkaline phosphatase)

Odontohypophosphatasia

Other miscellaneous conditions Generalised arterial calcification

#241510

6q22

ENPP1

ecto-nucleotide pyrophosphate/ phosphodiesterase 1

Jansen’s metaphyseal chondrodysplasia

#156400

3p22-p21.1

PTH1R

parathyroid hormone type 1 receptor

Cleidocranial dysplasia

#119600

*600211

CBFA1 (RUNX2) core-binding factor, runt domain, alpha subunit 1

The conditions themselves are shown together with the genes responsible for them. The OMIM numbers of the conditions and those of the genes responsible are shown in the adjacent columns.

Recently, a second form of ‘osteoclast-poor’ OPT has been described (OPTB7) (#612301) in which the cause is not within the RANKL but within its receptor, RANK (TNFRSF11A) (*603499) [7]. Its clinical course is similar to that of OPTB2 but is characterised by the addition of low plasma immunoglobulins. It is allelic with both familial expansile osteolysis (FEO) (#174810) and one form of Paget disease of bone (#602080).

Intermediate Form of Autosomal-Recessive Infantile Osteopetrosis The commonest intermediate form of autosomal-recessive osteopetrosis (OPTB4) (#611490) usually presents in the first few years of life with pathological fractures, short stature, mandibular prognathism, proptosis, mild anaemia, hepatosplenomegaly and propensity to mandibular osteomyelitis. Visual impairment may become evident during early infancy. Like some forms of OPT and autosomal dominant osteopetrosis, this condition has been shown to be caused by mutations in the CLCN7 gene (*602727) [2]. It has been successfully treated with bone marrow transplantation [8].

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OMIM

Inheritance

*171760

AD

*171760

AD

*173335

AR

*168468

AD

*600211

AD

Osteoclasts

Severity

Cause

Another rare form of intermediate autosomal-recessive OPT (OPTB6) (#611497) is caused by mutations in the plekstrin homology domain-containing protein, family M member 1 (PLEKHM1) gene (*611466). This is involved in osteoclast vesicular transport and results in an intermediate form of OPT. Late tooth eruption is a feature and the radiological appearances of an ‘Ehrlenmeyer flask’ are characteristic [9].

Autosomal-Dominant Osteopetrosis Autosomal-dominant osteopetrosis (OPTA2) (#607634) or the Albers-Schönberg disease, is caused by heterozygous mutations of the CLCN7 gene (*602727) [2, 10]. It is a less severe condition, which is often diagnosed serendipitously on a radiograph performed for another clinical reason, or through a low trauma fracture of a long-bone occurring during childhood or adolescence. Fractures of posterior arch of the vertebrae may lead to spondylolisthesis. Patients with OPTA2 are prone to developing osteomyelitis of the jaw, which can occur after dental extraction or treatment (fig. 2).

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Fig. 2. A 15-year-old girl with autosomal-dominant osteopetrosis developed chronic osteomyelitis of the jaw following dental surgery. Photograph of the lower face region showing chronic sinuses.

Osteopetrosis with Renal Tubular Acidosis and Cerebral Calcification (OPTB3) (#259730) These patients have a mild form of osteopetrosis, which is associated with renal tubular acidosis and cerebral calcification. Other clinical manifestations include short stature and mental retardation. Biochemical changes include hyperchloraemic acidosis and increased urinary pH, due to renal tubular acidosis. It is inherited as an autosomal-recessive trait and is caused by an inactivating mutation of the gene encoding carbonic anhydrase II (CA2) (*611492) [11]. As in OPTA2, patients with this condition have an increased propensity to pathological fractures (fig. 3) (see chapter 8).

Iatrogenic Osteopetrosis Whyte et al. [12] described a 12-year-old boy who developed radiological and histological changes of osteopetrosis due to prolonged treatment with high doses of cyclical intravenous aminobisphosphonate pamidronate.

Radiological Findings in Osteopetrosis Radiological changes depend on severity of the disease but there is generalised osteclerosis of all bones, with lack of differentiation between cortex and medullary cavity. Even though the bones are dense, they are prone to fragility fractures (fig. 4).

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Fig. 3. a Transverse fracture of the proximal femoral shaft which occurred after a trivial fall in a girl with osteopetrosis secondary to carbonic anhydrase deficiency II. b CT scan of the head showing extensive intracranial calcification in the same patient.

a

b

Fig. 4. Bilateral femoral neck fractures in a 15-year-old girl with autosomal-dominant osteopetrosis.

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Fig. 5. Hand radiograph of boy with pyknodysostosis showing generalised osteosclerosis and partial aplasia of terminal phalanges.

Failure of normal bone remodelling leads to persistence of calcified primary spongiosa within bone (bone within a bone appearance), vertebral endplate thickening giving rise to the ‘rugger jersey spine’, and clubbed appearance of metaphyses. Other features include transverse radiolucent metaphyseal bands and radiological changes of rickets superimposed on the generalised osteosclerosis.

Pycnodysostosis (#265800) This is an autosomal-recessive disorder characterised by disproportionate short stature, characteristic head and facial features (relatively large head, wide cranial sutures, delayed closure of the anterior fontanelle, prominent forehead, proptosis, receding chin and increased angle of the mandible), dental mal-occlusion, hypoplastic claviculae, total or partial aplasia of terminal phalanges (fig. 5) and hypoplastic fingernails. These patients have generalised increase in bone density and predisposition to fragility fractures. Many children suffer from chronic upper airway obstruction resulting from mid-facial hypoplasia and micrognathia. Pycnodysostosis has been shown to be caused by inactivating mutations in the cathepsin K (CTSK) gene (*601105), located on chromosome 1q21 [13]. Cathepsin K is a protease secreted by osteoclasts which plays an important part in bone resorption through degrading of collagen type 1 in the bone matrix (fig. 1). Therefore, in this condition, the osteoclasts resorb mineral but not the matrix. Radiographs of long-

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bones show generalised osteosclerosis with fractures (fig. 6), while those of the skull show delayed closure of cranial sutures and fontanelles. Management of pycnodysostosis involves dealing with secondary orthopaedic and upper air way problems. There is no effective medical therapy.

Van Buchem Disease (VBCH) (#239100) and Sclerosteosis (SOST) (#269500) Van Buchem disease and sclerostosis are autosomal-recessive disorders characterised by progressive osteosclerosis of the skeleton, especially affecting the skull, facial bones and the mandible. Other skeletal sites affected include ribs, clavicles, long bone diaphyses, neural arches and spinous processes of vertebrae. Entrapment of cranial nerves leads to facial palsy, deafness, blindness and loss of smell. Compared to Van Buchem disease, patients with sclerosteosis have a more aggressive disease which is often associated with hand malformations (syndactyly, radial deviation of the terminal phalanges and absent or dysplastic nails). Sclerostosis results from loss of function mutations of the SOST gene (*605740) [14], which encodes for sclerostin, a protein produced by osteocytes. Van Buchem disease results from a deletion downstream to the SOST gene, which is thought to result in a decreased gene expression [15]. Sclerostin is responsible for inhibition of bone formation. Therefore, deficiency of this protein leads to increased bone formation that is not associated with increased bone resorption. This imbalance between bone formation and bone resorption results in a net increase in bone mass (see chapter 3).

High Bone Mass Conditions Caused by Activating Mutation in the LRP5 Gene (OPTA1) (#607634) This condition, which used to be called autosomal-dominant osteopetrosis type 1, is characterised by generalised high bone mass. It is no longer classified as a form of OPT as the defect does not lie within the osteoclasts. Some cases are associated with a large and square jaw and the presence of torus platinus (lump on the hard palate). Radiographs show generalised osteosclerosis with thick cortices and narrow medullary cavities of the long bones. There is usually marked thickening of the calvarium and base of the skull. This condition has been shown to be caused by activating mutations in the gene that encodes the low-density lipoprotein receptorrelated protein 5 (LRP5) (*603506) [16] (see chapter 3 for details). It is therefore the opposite condition to those cases of idiopathic juvenile osteoporosis (IJO) who have been described in association with heterozygous inactivation mutations of LRP5 (see below).

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Fig. 6. Radiograph of the tibia and fibula of a boy with pyknodysostosis showing generalised osteosclerosis and healed fracture of the mid-tibia.

Fig. 7. Radiograph of the tibia and fibula in a boy with Camurati-Engelmann syndrome showing patch cortical hyperostosis.

Camurati-Engelmann Disease (Progressive Diaphyseal Dysplasia) (PDD) (#131300) This condition is characterised by progressive cortical hyperostosis affecting both the periosteal and endosteal surfaces of the diaphysis of tubular bones, resulting in narrowing of medullary cavities (fig. 7). The ribs, pelvis, skull, and vertebrae may also be involved. It usually presents with diffuse musculoskeletal pain, muscle weakness and a waddling gait. It is a dominantly inherited condition caused by mutations in

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Fig. 8. Radiograph of the left foot of in a boy with fibrodysplasia ossificans progressiva showing congenital deformity of the big toe.

the transforming growth factor beta 1 (TGF-β1) gene (TGFB1) (*190180) [17] (see chapter 3). Symptoms can be improved with non-steroidal anti-inflammatory drugs and short courses of oral corticosteroids. Treatment with aminobisphosphonates has been found to be ineffective [18].

Fibrodysplasia Ossificans Progressiva (FOP) (#135100) Fibrodysplasia ossificans progressiva is a rare genetic disorder characterised by congenital malformation of big toes with hallux valgus (fig. 8) and progressive ossification of connective tissues, such as skeletal muscle, ligaments and tendons. Recurrent flareups, which are often precipitated by soft tissue injury, lead to rapidly appearing soft tissue swellings. The sudden appearance of these pre-osseous swellings, usually around the neck or back often results in patients undergoing investigations for inflammatory

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205

or malignant soft tissue diseases. The disease often runs waxing and waning course with long quiescent periods. FOP is caused by a common mutation in activin receptor I/activin-like kinase II (ACVR1/ALK2) (*102576), which is a bone morphogenetic protein type I receptor [19]. It is a dominantly inherited condition but most cases of FOP are due to new mutations. At the present time, there are no specific BMP receptor antagonists or BMP pathway signal transduction inhibitors that could be used to ‘cure’ FOP or modify its course. Acute flare-ups can be treated with short courses of oral corticosteroids, non-steroidal anti-inflammatory drugs and possibly aminobisphosphonates [20]. Early diagnosis of the condition is important in order to avoid iatrogenic damage to tissues, including intramuscular injections, surgery and unnecessary dental treatment, which can trigger heterotopic ossification. In the future, it may be possible to treat this condition through blocking ACVR1/ALK2 signalling.

Progressive Osseous Heteroplasia (POH) (#166350) Progressive osseous heteroplasia (POH) is caused by paternally acquired inactivating mutations of the heterotrimeric G protein α-subunit (GNAS1) gene (+139320) [21]. Patients with this condition have extensive ossification of cutaneous and subcutaneous tissues as well as skeletal muscle. Most patients with POH do not have phenotypic or biochemical features of Albright hereditary osteodystrophy (see chapter 6). POH is easily distinguished from fibrodysplasia ossificans progressive by the presence of cutaneous ossification and the absence of congenital malformation of big toes.

Caffey’s Disease (#114000) Caffey’s disease, or infantile cortical hyperostosis, was first described in 1945 [41]. Symptoms usually appear before the age of 5 months. Perinatal lethal disease has also been described. Symptoms consist of acute inflammation of bones, particularly the mandible, ribs and tibiae, which cause considerable pain and distress. It is an autosomal-dominant condition that, unusually, is caused by mutations in the COL1A1 gene (+120150) [42] that is also responsible for some forms of osteogenesis imperfecta. However, these patients have no predisposition to fracture although some patients display joint hypermobility as seen in some types of Ehlers-Danlos syndrome. The precise cause of the bone inflammation is not certain but may be related to raised levels of prostaglandins. A similar condition is occasionally seen in infants with congenital cyanotic heart disease who have been treated with prostaglandins for long periods to maintain the patency of a ductus arteriosus. There may be some response to non-steroidal anti-inflammatory drugs such as indometacin or naproxen (see chapter 15, case 35). Symptoms usually settle spontaneously within two years but can occasionally recur intermittently thereafter.

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Disorders Associated with Skeletal Fragility and Low Bone Mass Other than Osteogenesis Imperfecta

Osteoporosis-Pseudoglioma Syndrome (OPPG) (#259770) This is a rare autosomal-recessive inherited condition characterised by fragility fractures of long bones and vertebrae and blindness caused by impaired retinal blood vessel regression during infancy. Other features reported in patients with osteoporosis-pseudoglioma syndrome include learning difficulties, muscular hypotonia, and ligamentous laxity. It arises from homozygous inactivating mutations in the LRP5 gene (see chapter 3) [22]. There are anecdotal reports that intravenous aminobisphosphonate pamidronate therapy improves bone density and may prevent progressive vertebral deformity (see chapter 15, case 28).

Idiopathic Juvenile Osteoporosis (IJO) (259750) This a rare disorder in which a previously healthy boy or a girl presents in the prepubertal period with insidiously occurring vertebral fractures and sub-metaphyseal fragility fractures of long bones. Children with IJO may also present with a history of difficulty in walking caused by proximal muscle weakness and chronic pain in the back, hip and lower limbs. Clinical examination may disclose thoracolumbar kyphosis or a pigeon-chest deformity caused by vertebral fractures and an abnormal walking gait. Radiological features of IJO include fractures of vertebral bodies, generalised osteopaenia with thin cortices and areas of sclerosis and or radiolucent bands at the metaphyses of the long bones. Bone mineral density is reduced at both the axial and appendicular skeletal sites. Detailed histomorphometry on bone biopsy specimens shows changes consistent with reduced osteoblast numbers with low bone remodelling activity [23]. IJO tends to remit spontaneously after the onset of puberty. However, many patients are left with residual skeletal deformities such as kyphoscoliosis. There are anecdotal reports of improvement in areal bone mineral density and symptoms following cyclical intravenous treatment with the aminobisphosphonate pamidronate. Recently, heterozygous inactivating mutations in the gene encoding the low-density lipoprotein receptor-related protein 5 (LRP5) (*603506) have been described in a proportion of patients with IJO [24].

Juvenile Paget’s Disease (JPD) (#239000) JPD, which is also known as familial idiopathic hyperphosphatasia, is a rare generalised bone disorder characterised by increased bone turnover. It is an autosomal-recessive disease caused by inactivating mutations in the osteoprotegerin (OPG; TNFRSF11B)

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(*602643) gene [25]. In addition to producing the cytokine RANKL, which binds to the RANK on preosteoclasts causing them to differentiate into active bone-resorbing cells, ostoblasts also secrete osteoprotegerin, a member of the tumour necrosis factor superfamily. Osteoprotegerin acts as an endogenous antagonist that prevents binding of RANKL to RANK, thereby inhibiting osteoclast activity (see chapter 3). Children with JPD are relatively normal at birth and present during early childhood with debilitating bone pain, short stature, long-bone and vertebral fractures, progressive limb deformities and deafness. The biochemical hallmark of JPD is markedly elevated serum alkaline phosphatase (ALP) activity. Serum concentrations of immunoreactive osteoprotegerin are undetectable in patients with homozygous complete deletion of TNFRSF11B gene, while those of RANKL are elevated. Radiographs show widened and deformed long bones with irregular thickening of the cortices and loss of cortico-medullary differentiation. Bone biopsy shows a characteristic pattern of trabeculae arranged in parallel plates with little connectivity, rather than the normal ‘honeycomb’ appearance. Treatment with intravenous aminobisphosphonates has been shown to suppress bone turnover, prevent deformity and improve hearing in children with JPD [26]. There is an encouraging report of reduction in bone turnover and improvement in bone mineral density in JPD patients treated with recombinant osteoprotegerin [27].

Geroderma Osteodysplasticum (GO) (%231070) GO is a rare disorder characterised by generalised lax but non-stretchy wrinkly skin, which is more marked over the extremities and abdomen [28]. Patients with GO have a distinctive aged appearance due to the wrinkly skin (fig. 9), mid-facial hypoplasia and mandibular prognathism. Patients with GO also have variable degrees of axial and appendicular osteoporosis, which can lead to vertebral and long-bone fractures, respectively. Other musculoskeletal problems include generalised hypermobility of joints, congenital dislocation of hips and kyphoscoliosis. It is an autosomal-recessively inherited condition and, recently, the gene for this condition has been mapped to chromosome 1q24 [29]. Recently, it has been shown that this condition is caused by inactivating mutations in SCYL1BP1, which is highly expressed in skin and osteoblasts [30]. The author has used intravenous aminobisphosphonate pamidronate therapy in 2 children with history of long-bone and vertebral compression fractures.

McCune-Albright Syndrome and Fibrous Dysplasia (MAS/FD) (#174800) Fibrous dysplasia (FD) is a skeletal disorder caused by replacement of normal bone with benign fibrous connective tissue containing immature non-lamellar bone. It may affect a single skeletal site (monostotic FD) or multiple sites (polyostotic FD,

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Fig. 9. Characteristic wrinkly skin in a toddler with geroderma osteodysplasticum.

McCune-Albright syndrome, MAS), usually in variable combinations with characteristic irregular pigmented café-au-lait skin lesions (fig. 10) and endocrine abnormalities (thyrotoxicosis, precocious puberty, acromegaly and Cushing syndrome). Both FD and MAS are caused by somatic post-zygotic activating mutations of the gene encoding for the heterotrimeric G protein α-subunit (GNAS1). Skeletal manifestations include debilitating pain, deformity, or pathological fracture through a weakened fibrous dysplasia lesion of the bone (fig. 11). The pelvis, lower limbs and facial bones are among the most frequently involved. Cranial bone involvement can lead to orbital or optic nerve compression. Progressive scoliosis may develop in some patients. Approximately half the patients with FD/MAS also have hypophosphataemic rickets/ osteomalacia, caused by excessive synthesis of FGF-23, which causes increased renal phosphate loss and reduced 1,25(OH)2D synthesis (see chapter 9). Treatment with cyclical intravenous aminobisphosphonate pamidronate decreases pain and biochemical markers of bone turnover but no reduction in fracture rates [31]. Multiple osteotomies and intramedullary nailing are often necessary to correct deformities of lower limb bones. Scoliosis will require treatment with bracing, insertion of metal rods and posterior spinal fusion procedures. Hypophosphataemic

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Fig. 10. Characteristic irregular pigmented café-au-lait skin lesions in a boy with McCuneAlbright syndrome.

rickets/osteomalacia is treated along the same lines as X-linked hypophosphataemic rickets (see chapter 9).

Hypophosphatasia Hypophosphatasia (HPP) is a group of inherited disorders characterised by impaired mineralisation of bone and tooth [32, 33]. It is caused by deficiency of tissue nonspecific alkaline phosphatase (TNSALP) (*171760) activity, which leads to accumulation of several TNSALP substrates, such as pyridoxal 5⬘-phosphate and inorganic pyrophosphate (PPi). Extracellular accumulation of PPi inhibits the development and growth of hydroxyapatite crystals. It also impairs skeletal mineralisation leading to rickets in a growing child and osteomalacia in adults.

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Fig. 11. Radiograph showing pathological fracture through a weakened area of the femoral shaft in a boy with McCuneAlbright syndrome.

The biochemical hallmark of HPP is low plasma alkaline phosphatase activity for age of the child and increased urinary phosphoethanolamine (PEA)/creatinine (Cr) ratio. However, the urinary PEA/Cr ratio may be normal in milder cases of HPP. Serum inorganic phosphate concentration is often raised or at the upper end of the reference range for age of the child. Infants with severe (perinatal and infantile) forms of HPP can have symptomatic hypercalcaemia, which in turn may lead to hypercalciuria, nephrocalcinosis and renal impairment. HPP is inherited as either an autosomal dominant or recessive trait; all patients have inactivating mutations of the gene that encodes TNSALP, located on chromosome 1p36.1–34. The severity of HPP is related to the age of presentation and on the degree of TNSALP deficiency. Based on the age of the patient when the diagnosis is made and severity of the disease, five forms of HPP have been described. For a discussion of the genetics, see chapter 2. The perinatal lethal form of HPP (#241500) is characterised by severely demineralised skeleton so that the affected infant is either stillborn, or dies within few days of birth due to respiratory failure arising from hypoplastic lungs. Those with the infantile form of HPP (#241500) appear healthy at birth. However, by 6 months of age they develop symptoms of poor feeding, failure to thrive and some go on to have pyridoxine (vitamin B6)-dependent seizures. Clinical features include hypotonia, rachitic deformities of long bones, large fontanelles and craniosynostosis. These infants usually die of pneumonia, which arises secondary to their soft rib-

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cage (see chapter 15, case 32). However, spontaneous improvement can also occur. Radiographs show severely demineralised skeleton with rachitic changes and pathological fractures. Premature exfoliation of primary teeth with intact roots before the age of 5 years is the hallmark of the childhood form of HPP (#241510). These children are often of short stature, have mild swelling of metaphyses of long bones and some may have a dolichocephalic skull. Craniosynostosis may cause raised intracranial pressure. They often walk with a waddling gait and may complain of non-specific bone pain. Radiographs of their long bones show mild rachitic changes with ‘tongue like’ areas of lucency projecting from growth plates into metaphyses (fig. 12, 13) (see chapter 15, case 33). The adult form of HPP (#146300) may come to light through osteomalacia causing recurrent and slowly healing metatarsal stress fractures. They may also develop pseudogout and chondrocalcinosis, caused by precipitation of inorganic PPi crystals. Odontohypophosphatasia is the form of HPP that is associated with premature loss of fully rooted primary teeth, without skeletal involvement. There is no established effective therapy for HPP. In an 8-month-old girl with lifethreatening infantile form of HPP, bone marrow transplantation resulted in a transient clinical and radiological but no biochemical improvement [34]. Seven years after treatment of a similarly affected 9-month-old infant with donor bone fragments (inserted intraperitoneally and subcutaneously) and cultured TNSALP-replete osteoblasts, the clinical phenotype changed from the infantile to childhood form of HPP [35]. Enzyme replacement using a bone-targeted recombinant TNALP was found to be effective in the treatment of the TNSALP knockout mice, mimicking the infantile form of HPP [36], and a clinical trial is currently underway in human infants. In a middle aged lady with the adult form of HPP, treatment with subcutaneous injections of recombinant human parathyroid hormone 1–34 (teriparatide, which stimulates TNSALP synthesis by osteoblasts) resulted in healing of metatarsal stress fractures [37]. However, the use of teriparatide has been restricted in children because in rats this preparation causes a dose-dependent increase in the risk of osteosarcoma.

Generalised Arterial Calcification of Infancy Generalised arterial calcification of infancy (GACI) (#208000) is a rare disease characterised by calcification of medium and large arteries. Most patients die within the first year of life due to myocardial infarction and progressive ischaemia of vital organs. It is an autosomal-recessively inherited condition caused by homozygous or compound heterozygous inactivating mutations in the ecto-nucleotide pyrophosphate/phosphodiesterase 1 (ENPP1) (*173335) gene [38], located on chromosome 6q22. Patients with GACI have low levels of the ENPP1 enzyme. This cell surface enzyme regulates soft-tissue calcification by generating inorganic pyrophosphate, a solute that serves as an essential physiologic inhibitor of hydroxyapatite deposition. Some children do survive beyond

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Fig. 12. At the age of 3 years, a previously healthy child started shedding her teeth spontaneously. The figure shows her teeth with intact roots, which is a characteristic feature of childhood form of hypophosphatasia or odontohypophosphatasia. Her serum alkaline phosphates activity was 123 IU/l (230–700) and genetic testing showed that she had compound heterozygous mutations of the TNSALP gene (c.350A>G,p.Y117C and c.400_401AC>CA, p.T134H). As she had clinical and radiological changes (see figure 13), the diagnosis of childhood hypophosphatasia was made.

infancy and into young adulthood, and there is some evidence to suggest that survival is associated with early treatment with bisphosphonates or later development of hypophosophataemia and phosphaturia [39]. Apart from aminobisphosphonate pamidronate, management is directed at treatment of complications, such as hypertension. In a patient with GACI in whom the diagnosis of secondary hypophosphataemic rickets was made at the age of 11 years, treatment, which involved the use of alphacalcidiol (see chapter 9), was undertaken by the author with regular monitoring (high-resolution spiral CT scans of the heart, echocardiography and ultrasound scans of the liver and spleen) to ensure that it did not result in progression of the pre-existing arterial calcification.

Jansen’s Metaphyseal Chondrodysplasia Jansen’s metaphyseal chondrodysplasia (JMC) (#156400) is a rare autosomal-dominant form of short-limbed dwarfism. It is caused by heterozygous activating mutations of the gene encoding for the parathyroid hormone-parathyroid hormone-related peptide (PTH-PTHrP) receptor, located on the short arm of chromosome 3p22-p21.1 [40]. The abnormal chondrocyte differentiation in growth plates leads to characteristic short-limbed dwarfism. The activation of the PTH-PTHrP receptor in the kidney

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Fig. 13. Radiograph of the knee of a girl with childhood form of hypophosphatasia showing mild widening, fraying and patchy sclerosis of metaphyses. The characteristic ‘tongue’ of radiolucency is visible in the proximal tibial metaphyses.

and bone leads to high plasma 1,25(OH)2vitamin D concentration, hypercalcaemia, hypercalciuria and hyperphosphatemia, despite normal or low plasma concentrations of PTH and PTHrP. Apart from short and bowed legs with swollen ends of the long bones, patients with JMC have characteristic cranio-facial features including high skull vault, wide cranial sutures, hypertelorism, high arched palate, prominent cheeks and micognathia. Radiographs show classical rachitic changes at metaphyses of long bones with generalised osteopaenia, subperiosteal bone resorption and pathological fractures. Currently, there is no specific treatment for this condition although the author is aware of one patient who is currently being treated with intravenous pamidronate which has had some effect in reducing the very rapid rate of bone turnover that occurs in these patients (see chapter 15 case 34). The associated, often intermittent, hypercalcaemia is managed as previously described (see chapter 7).

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Cleidocranial Dysplasia Cleidocranial dysplasia (CCD) (#119600) is an autosomal-dominant generalised skeletal dysplasia caused by mutations in the CBFA1 (RUNX2) gene (*600211) [43] (see chapter 3). Aplasia or absence of the clavicles, resulting in an ability to appose the shoulders, persistence of an open fontanelle, late erupting teeth and short stature are typical features. Scoliosis may become a problem. A notable radiological feature is that of Wormian bones in the skull, a feature also seen in osteogenesis imperfecta, although there is no particular predisposition to fracture despite the fact that the bones appear somewhat undermineralised on radiographs (see chapter 15, case 36).

Conclusions

There are many conditions that give rise to abnormalities of bone mineralisation. Some of these are described in previous chapters. These conditions result in disorders that cause either an increase or a decrease in bone mineral content. They are all rare but important in providing insight into the mechanisms of bone mineralisation since each has a genetic basis. Although there is currently no established treatment for many of these conditions, an understanding of the genetic abnormalities may allow development of specific treatments in the future.

References 1 Frattini A, Orchard PJ, Sobacchi C, et al: Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis. Nat Genet 2000;25:343–346. 2 Frattini A, Pangrazio A, Susani L, et al: Chloride channel ClCN7 mutations are responsible for severe recessive, dominant, and intermediate osteopetrosis. J Bone Miner Res 2003;18:1740–1747. 3 Pangrazio A, Poliani PL, Megarbane A, et al: Mutations in OSTM1 (grey lethal) define a particularly severe form of autosomal recessive osteopetrosis with neural involvement. J Bone Miner Res 2006;21:1098–1105. 4 Wilson CJ, Vellodi A: Autosomal recessive osteopetrosis: diagnosis, management, and outcome. Arch Dis Child 2000;83:449–452. 5 Kasper D, Planells-Cases R, Fuhrmann JC, et al: Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration. EMBO J 2005;24:1079–1091. 6 Sobacchi C, Frattini A, Guerrini MM, et al: Osteoclast-poor human osteopetrosis due to mutations in the gene encoding RANKL. Nat Genet 2007; 39:960–962.

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7 Guerrini MM, Sobacchi C, Cassani B, et al: Human osteoclast-poor osteopetrosis with hypogammaglobulinemia due to TNFRSF11A (RANK) mutations. Am J Hum Genet 2008;83:64–76. 8 Dini G, Floris R, Garaventa A, et al: Long-term follow-up of two children with a variant of mild autosomal recessive osteopetrosis undergoing bone marrow transplantation. Bone Marrow Transplant 2000;26:219–224. 9 Van Wesenbeeck L, Odgren PR, Coxon FP, et al: Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans. J Clin Invest 2007;117:919–930. 10 Waguespack SG, Hui SL, Dimeglio LA, Econs MJ: Autosomal dominant osteopetrosis: clinical severity and natural history of 94 subjects with a chloride channel 7 gene mutation. J Clin Endocrinol Metab 2007;92:771–778. 11 Sly WS, Hewett-Emmett D, Whyte MP, Yu YS, Tashian RE: Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Proc Natl Acad Sci USA 1983;80:2752–2756.

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12 Whyte MP, Wenkert D, Clements KL, McAlister WH, Mumm S: Bisphosphonate-induced osteopetrosis. N Engl J Med 2003;349:457–463. 13 Gelb BD, Shi GP, Chapman HA, Desnick RJ: Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency. Science 1996;273:1236– 1238. 14 Brunkow ME, Gardner JC, Van Ness J, et al: Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein. Am J Hum Genet 2001;68:577–589. 15 Balemans W, Patel N, Ebeling M, et al: Identification of a 52 kb deletion downstream of the SOST gene in patients with van Buchem disease. J Med Genet 2002;39:91–97. 16 Boyden LM, Mao J, Belsky J, et al: High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med 2002;346:1513–1521. 17 Kinoshita A, Saito T, Tomita H, et al: Domainspecific mutations in TGFB1 result in CamuratiEngelmann disease. Nat Genet 2000;26:19–20. 18 Castro GR, Appenzeller S, Marques-Neto JF, Bertolo MB, Samara AM, Coimbra I: Camurati-Engelmann disease: failure of response to bisphosphonates: report of two cases. Clin Rheumatol 2005;24:398– 401. 19 Shore EM, Xu M, Feldman GJ, et al: A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva. Nat Genet 2006;38:525–527. 20 Kaplan F, International Clinical Consortium on Fibrodysplasia Ossificans Progressiva: The medical management of fibrodysplasia ossificans progressiva: current treatment considerations.3:182 http:/ www.ifopa.org/pdf/FOPRXGuidelinesFinal_8–27– 08.pdf 21 Adegbite NS, Xu M, Kaplan FS, Shore EM, Pignolo RJ: Diagnostic and mutational spectrum of progressive osseous heteroplasia (POH) and other forms of GNAS-based heterotopic ossification. Am J Med Genet [A] 2008;146A:1788–1796. 22 Ai M, Heeger S, Bartels CF, Schelling DK: Clinical and molecular findings in osteoporosis-pseudoglioma syndrome. Am J Hum Genet 2005;77:741– 753. 23 Rauch F, Travers R, Norman ME, Taylor A, Parfitt AM, Glorieux FH: Deficient bone formation in idiopathic juvenile osteoporosis: a histomorphometric study of cancellous iliac bone. J Bone Miner Res 2000;15:957–963. 24 Hartikka H, Makitie O, Mannikko M, et al: Heterozygous mutations in the LDL receptor-related protein 5 (LRP5) gene are associated with primary osteoporosis in children. J Bone Miner Res 2005;20: 783–789.

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25 Cundy T, Hegde M, Naot D, et al: A mutation in the gene TNFRSF11B encoding osteoprotegerin causes an idiopathic hyperphosphatasia phenotype. Hum Mol Genet 2002;11:2119–2127. 26 Cundy T, Wheadon L, King A: Treatment of idiopathic hyperphosphatasia with intensive bisphosphonate therapy. J Bone Miner Res 2004;19: 703–711. 27 Cundy T, Davidson J, Rutland MD, Stewart C, DePaoli AM: Recombinant osteoprotegerin for juvenile Paget’s disease. N Engl J Med 2005;353:918– 923. 28 Hunter AG, Martsolf JT, Baker CG, Reed MH: Geroderma osteodysplastica: a report of two affected families. Hum Genet 1978;40:311–324. 29 Newman WG, Clayton-Smith J, Metcalfe K, et al: Geroderma osteodysplastica maps to a 4 Mb locus on chromosome 1q24. Am J Med Genet [A] 2008; 146A:3034–3037. 30 Hennies HC, Kornak U, Zhang H, et al: Gerodermia osteodysplastica is caused by mutations in SCYL1BP1, a Rab-6 interacting golgin. Nat Genet 2008;40:1410– 1412. 31 Plotkin H, Rauch F, Zeitlin L, Munns C, Travers R, Glorieux FH: Effect of pamidronate treatment in children with polyostotic fibrous dysplasia of bone. J Clin Endocrinol Metab 2003;88:4569–4575. 32 Mornet E: Hypophosphatasia. Best Pract Res Clin Rheumatol 2008;22:113–127. 33 Whyte MP: Enzyme Defects and the Skeleton; in Rosen CJ (ed): Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, ed 7. Washington, American Society for Bone and Mineral Research, 2008, pp 454–458. 34 Whyte MP, Kurtzberg J, McAlister WH, et al: Marrow cell transplantation for infantile hypophosphatasia. J Bone Miner Res 2003;18:624–636. 35 Cahill RA, Wenkert D, Perlman SA, et al: Infantile hypophosphatasia: transplantation therapy trial using bone fragments and cultured osteoblasts. J Clin Endocrinol Metab 2007;92:2923–2930. 36 Millan JL, Narisawa S, Lemire I, et al: Enzyme replacement therapy for murine hypophosphatasia. J Bone Miner Res 2008;23:777–787. 37 Whyte MP, Mumm S, Deal C: Adult hypophosphatasia treated with teriparatide. J Clin Endocrinol Metab 2007;92:1203–1208. 38 Rutsch F, Ruf N, Vaingankar S, et al: Mutations in ENPP1 are associated with ‘idiopathic’ infantile arterial calcification. Nat Genet 2003;34:379–381. 39 Rutsch F, Böyer P, Nitschke Y, et al: Hypophosphatemia, hyperphosphaturia and bisphosphonate treatment are associated with survival beyond infancy in generalized arterial calcification of infancy (GACI). Circulation Cardiovasc Genet 2008;1:133–140.

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40 Schipani E, Langman CB, Parfitt AM, et al: Constitutively activated receptors for parathyroid hormone and parathyroid hormone-related peptide in Jansen’s metaphyseal chondrodysplasia. N Engl J Med 1996;335:708–714. 41 Caffey J, Silverman W: Infantile cortical hyperostosis, preliminary report on new syndrome. Am J Roentg 1945;54:1–16.

42 Gensure RC, Makitie O, Barclay C, et al: A novel COL1A1 mutation in infantile cortical hyperostosis (Caffey disease) expands the spectrum of collagenrelated disorders. J Clin Invest 2005;115:1250– 1257. 43 Otto F, Kanegane H, Mundlos S: Mutations in the RUNX2 gene in patients with cleidocranial dysplasia. Hum Mutat 2002;19:209–216.

M. Zulf Mughal, MBChB, FRCPCH, FRCP, DCH Saint Mary’s Hospital for Women and Children Hathersage Road Manchester, M13 0JH (UK) Tel.+44 161 276 6501, Fax +44 161 276 6907, E-Mail [email protected]

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Chapter 13 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 218–232

Drugs Used in Paediatric Bone and Calcium Disorders Moira Cheung Imperial College London, MRC Clinical Sciences Centre, Hammersmith Hospital, London, UK

Abstract Calcium and bone disorders in paediatrics are treated with a variety of drugs, many of which, although licensed for use in adults, are not so in children but are nevertheless used on the basis of accepted practice. The mainstay of drug treatment for osteoporosis is the bisphosphonates which alter the balance between bone accretion and reabsorption mainly by temporarily reducing the activity of osteoclasts. Vitamin D and its metabolites are used for the treatment of various forms of rickets and vitamin D deficiency and the active metabolites are also employed when hypoparathyroidism causes hypocalcaemia. Phosphate supplements may also be required in some forms of rickets. Hypercalcaemia is treated initially with hyperhydration and diuretics but may require more specific treatment with either calcitonin or bisphosphonates. Several newer drugs have either recently been introduced or are under consideration. These include the calcimimetics (cinacalcet), rank ligand inhibitors (osteoprotegerin and denusomab), cathepsin K inhibitor, sclerostin, bone morphogenic protein 2, and calciolytic drugs. More recently, recombinant alkaline phosphatase and PTH have been used to treat hypophosphatasia and hypoparathyroidism respectively. These developments promise to direct treatment more specifically to targeting individual conditions as our Copyright © 2009 S. Karger AG, Basel understanding of these conditions increases.

The skeleton of a newborn differs vastly from that of an adult and is transformed through a process of site-specific bone accrual, modelling and remodelling. Medications that modify this process cannot only affect longitudinal growth but also the final morphology of the skeleton. Despite the availability of medications that help to maintain the function of bone, it is important to remember the contribution of nutrition and exercise in the management of bone and calcium disorders (see chapter 11). A holistic approach is particularly important when managing secondary osteoporosis where control of the primary disease process, where possible, is the ultimate treatment for the bone disorder. Unfortunately, as for many drugs used in paediatrics, there are few good randomised trials of drugs used in bone and calcium disorders on which to base management decisions.

The route of administration and therapeutic half-life for medications in children can present a challenge. Non-compliance due to reasons such as difficult access, needle phobia or multiple daily doses is an important factor to consider when selecting treatment regimes.

Drugs Used in the Treatment of Paediatric Osteoporosis

The choice of drugs available to children with osteoporosis is largely limited to bisphosphonates. Teriparatide, a recombinant DNA version of parathyroid hormone, is currently being used in postmenopausal osteoporosis; however, the finding that it causes osteosarcomas in growing rats has curtailed its use in children [1].

Bisphosphonates The use of bisphosphonates has been well established for many decades in adult patients with postmenopausal osteoporosis, Paget disease of the bone, myeloma and bone metastases. However, its use in children with osteoporosis only became widespread after a publication in 1998 [2] in which treatment with cyclical pamidronate was demonstrated to improve bone mineral density and reduce both fracture rate and pain in children with osteogenesis imperfecta (OI). Bisphosphonates are structural analogues of naturally occurring inorganic pyrophosphates. They act to inhibit osteoclast function. Bisphosphonates have a high affinity to bone mineral which is selectively internalised by osteoclasts resulting in inhibition of an enzyme, farnesyl pyrophosphate synthase [3]. Without farnesyl pyrophosphate synthase, the cytoskeleton is disrupted causing osteoclast dysfunction. Osteoclasts are involved in renewing (remodelling) and shaping (modelling) bone by a process known as resorption (removing bone) [4]. When osteoclast action is disrupted (decreasing bone turnover), less bone is removed leaving a greater amount of bone in the skeleton. The aim is that, by increasing the amount of bone, the resistance to fractures also increases. Pamidronate, the most commonly used bisphosphonate in children, has a halflife of about 28 h. Although the majority is excreted by the kidneys during treatment cycles, small amounts of bisphosphonates are buried in the bone and accumulate there. The active drug can be re-released during remodelling and this re-release may increase during periods of high bone turnover such as around the time of childbirth [5].

Indications • Bisphosphonates should be administered under the guidance of a specialist centre which has experience of its use in children.

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• Osteoporosis – inappropriately low bone mineral density with associated significant fractures or deformity. • Hypercalcaemia. • Soft tissue calcification. • Pain management in conditions such as polyostotic fibrous dysplasia.

Treatment Outcomes Benefits The majority of the literature on bisphosphonate use in children derives from the treatment of patients with OI with intravenous cyclical pamidronate. There have been very few placebo-controlled trials or trials comparing different dosing and treatment regimes. However, most centres now consider that cyclical bisphosphonates are the standard of care to patients who are affected with moderate-to-severe OI [6]. Increasingly, bisphosphonates are also being used for patients with secondary osteoporosis such as in cerebral palsy, burns patients, glucocorticoid-induced osteopaenia, connective tissue disorders, anorexia nervosa and leukaemia [7]. Studies have reported a treatment induced rapid increase in well-being and decrease in chronic bone pain. This has been of great benefit to patients with both OI and polyostotic fibrous dysplasia. Mobility of severely affected patients with OI, when compared with historical controls, can be significantly improved due to a decrease in fracture rate, especially if treatment is started early on in life. However, decreased pain levels and increased mobility can also put patients at an increased risk of fractures. Growth has been shown to have significantly increased Z scores after 4 years of treatment in patients with OI [6]. These clinical benefits are coupled with an improvement in bone mineral density as measured using bone densitometry. A double-blind randomised placebo-controlled trial in non-ambulatory children with cerebral palsy showed that lumbar and distal femur bone mineral density was significantly increased in patients treated with pamidronate compared to controls [8]. Furthermore OI patients with collapsed vertebral bodies regained an improved size and shape with treatment [9]. Adverse Effects The first infusion cycle of bisphosphonates is frequently accompanied by an acute phase reaction which may include fever, vomiting and a rash. The symptoms typically occur in the first 12–24 h and can be controlled by simple antipyretics. They do not usually recur with subsequent infusions. Small children with severe disease need more careful monitoring as respiratory compromise has also been reported [10]. Hypocalcaemia, accompanied by a counter regulatory rise in parathyroid hormone and 1,25-dihydroxyvitamin D (1,25(OH)2D), usually occurs with the first infusion and can be most pronounced on day three. This has been reported to be more severe with

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Fig. 1. Hand radiograph of child with osteoporosis treated with regular infusions of pamidronate. Each transverse line represents one cycle of treatment with pamidronate and is seen on the metaphyseal side of the growth plate. This child’s bone age is now mature. Note how the lines get closer together and eventually fuse as growth ceases.

the more potent compounds such as zoledronate which is given as a 1-day infusion [11]. In light of these initial adverse effects it is prudent that patients are admitted for three days after the first infusion. It is also important that pre-treatment calcium and vitamin D levels in patients are optimised to minimise the impact of hypocalcaemia. Each cycle of treatment leaves its mark as a sclerotic band which can be identified on radiographs. These are areas of high density which become progressively less marked due to remodelling further away from the growth plate. Untreated new bone that is added at the growth plate is of a lower density so progressive bands can be seen which correlate with the number of treatment cycles (fig. 1). These transverse lines may contribute to mechanical resistance. However, the interface between both the first treatment line and the last, with untreated bone, are areas of vulnerability due to the large difference in bone density. Fractures at these points have been reported [12]. It is therefore usually recommended that, once treatment has been commenced, it should continue until growth has ceased [13].

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Suppression of resorption interferes with the normal remodelling process that occurs to renew and repair bone. There are concerns that build up of microcracks and calcified cartilage could itself lead to increased bone fragility and poor fracture healing. Osteonecrosis of the jaw has been reported as a complication of bisphosphonate therapy in the adult population. However, to date there have not been any reports of children who have developed this complication despite a number of surveys having been conducted specifically looking for this complication [14]. Bisphosphonate treatment is associated with poor healing of osteotomies (but not fractures) so it is recommended that treatment after an osteotomy is delayed for 4–6 months and until adequate callus formation is confirmed [15]. As bisphosphonates remain buried in the skeleton for many years, concerns remain about children born to mothers who have been previously treated with bisphosphonates. Although small case reports have not yet reported any adverse effects, this cannot be discounted [5]. Inhibition of osteoclast activity also affects its role in modelling. An extreme example of this is shown by a case report of a boy given excessive doses of pamidronate who consequently developed club-shaped metaphyses [16]. Other reported adverse effects of bisphosphonates include iritis and nephritis, which are unusual in children. Oral bisphosphonates are known to cause oesophagitis and must be taken in an upright position and with a full glass of water. Furthermore, gastrointestinal absorption is reduced by the presence of food which should be avoided for 2 h before and after administration. When to Start, Not to Start and When to Stop The greatest effect of bisphosphonates on the skeleton is during growth. Once growth has been completed the gains are, by comparison, small. In patients who are severely affected with osteoporosis, fractures and/or deformities, it would seem sensible to commence therapy as early as possible to reduce fractures, control bone pain and therefore optimise mobility and development. However, the long-term effects on patients who have been treated throughout childhood are still unknown. To avoid areas of weakness between treatment naive bone, which is of lower density, and the higher density treated bone, sudden discontinuation of treatment before growth has finished is generally now not recommended. Instead, some centres advocate a lower maintenance dose after the initial two to four years until the growth plates have fused. It is currently unclear if patients with low bone mass but who are otherwise asymptomatic would benefit from treatment. These patients could potentially suffer from the adverse effects of treatment and optimising nutrition and exercise and pubertal status should be prioritised. Choice of Bisphosphonate and Dosing Regime The most frequent bisphosphonate used in the treatment of childhood osteoporosis is intravenous cyclical pamidronate. The annual dose of pamidronate (commonly

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9–12 mg/kg/year, dependent on centre) remains constant but the regime varies with age (table 1). Zoledronate, due to its greater potency, can be given as a one day infusion. However, in children the dose and treatment intervals have yet to be adequately established. Ideally, bisphosphonates could be given to children in an oral form. However bisphosphonates have low bioavailability. A double-blind placebo-controlled trial involving 139 children and adolescents found that two years of oral alendronate had no significant effect on pain, functional status or fracture incidence despite significantly decreased bone turnover and increased spinal bone mineral density [17]. Studies using oral risedronate in children are still being evaluated.

Drugs Used in Vitamin D Disorders

Vitamin D Vitamin D is a fat-soluble vitamin which initially undergoes hydroxylation at position 25 in the liver. The second hydroxylation of position 1 occurs in the kidneys to produce the active metabolite 1,25-dihydoxyvitamin D (calcitriol). This final step is tightly regulated by parathyroid hormone, calcium and phosphate levels (see chapter 2 for further details). The best indication of stored vitamin D levels in the body is 25OHD which has a circulating half-life of fifteen days. Calcitriol has a short half-life of 5–8 h and levels usually remain high until vitamin D deficiency is severe. The main action of vitamin D is to maintain serum calcium in the normal range. It acts to increase absorption of calcium in the gut and acts on bone cells to release calcium reserves from the skeleton [18]. Studies also suggest that vitamin D has many other roles such as in immune system regulation and protection against cancer [19]. About 80% of the body’s vitamin D comes from the action of UVB (290–320 nm) on skin. Patients who have darker skin pigmentation or regularly cover their skin with sunscreen or clothing may obtain less from this source. A small amount is also derived from the diet as vitamin D2 (ergocalciferol) or D3 (cholecalciferol). Vitamin D is found in fish oils and eggs and baby formula milk and breakfast cereals are usually supplemented. Many countries supplement cow’s milk with vitamin D. In the UK, cow’s milk is now not fortified as over enthusiastic fortification of milk and cereals in the 1940s led to infants developing secondary hypercalcaemia and hypercalciuria. Vitamin D is the treatment of choice for primary vitamin D deficiency: one microgram is equivalent to forty units. Ideally vitamin D deficiency is prevented from before birth by first ensuring expectant mothers have adequate levels. Recommendations for daily requirements in children vary according to countries and between organisations (table 2).

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Table 1. Protocol for pamidronate administration Age

Dose, mg/kg per day for 3 days

Cycle interval

<2 years 2–3 years >3 years

0.5 0.75 1.0

2-monthly 3-monthly 3- to 4-monthly

Maximum dose is 60 mg/day.

In the presence of deficiency, vitamin D can be given as a daily supplement (1,500–10,000 units daily depending on age) and should be given for a total of three months initially. A large single dose, known as stosstherapy, can also be given and can be of particular benefit in cases of poor compliance or malabsorption. 150,000–600,000 units are given every 3 months, depending on age, as divided oral doses over one day or as a single intramuscular injection. Vitamin D toxicity can cause non specific symptoms such as nausea, vomiting, anorexia and constipation. Of greater concern is that toxicity leads to hypercalcaemia and hypercalciuria with their associated consequences [18]. Other drugs that interact with vitamin D include steroids, which can reduce calcium absorption and impair vitamin D metabolism, and phenobarbital and phenytoin which increase hepatic metabolism of vitamin D.

Alfacalcidol (1α-Hydroxycholecalciferol) Alfacalcidol is vitamin D3 which has been hydroxylated in the 1 position but not the 25 position. Taken orally, it needs to be hydroxylated in the liver to become the active metabolite 1,25(OH)2D. The usual dose is 30–50 ng/kg/day. It has a half life of about 30–35 h and, where available, is given once daily in preference to calcitriol. Alfacalcidol is indicated in situations where there is impairment of hydroxylation of vitamin D. This includes hepatic and renal disorders, 1α-hydroxylase deficiency, hypophosphataemic rickets and persistent hypocalcaemia due to hypoparathyroidism or pseudohypoparathyroidism. It is not available in all countries (including the USA) but, where it is available, it is the treatment of choice unless there is a problem with 25-hydroxylation such as in severe liver disease or neonatal hepatitis. 1α-Hydroxyergocalciferol (1αOHD2, Hectorol) is more widely available than alfacalcidol in some countries. It is a similar compound but has only one half to two thirds the potency of alfacalcidol. It is licensed for use in chronic renal disease in the USA.

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Table 2. Recommendations for the daily intake of vitamin D in children Organisation

Daily vitamin D dose

Population

Committee on Medical Aspects of Food and Nutrition Policy (COMA), UK [34]

8.5 μg (340 IU)

infants under 6 months children aged 6 months to 3 years

7 μg (280 IU) Department of Health, UK [35]

7 μg (280 IU)

children under 5 years of age

Institute of Medicine of The National Academies, USA [36]

5 μg (200 IU)

birth to 18 years of age

American Academy of Pediatrics, USA [37]

10 μg (400 IU)

birth to 18 years of age

Calcitriol (1,25(OH)2D3) Calcitriol is the active metabolite of vitamin D. It has a half-life of 5–8 h and cannot be stored in the body. Calcitriol is given orally (usually 15–30 ng/kg/day) or, if need be, as an infusion. Due to its short half-life, it needs to be given 2–3 times a day. Both alfacalcidol and calcitriol need to be given in higher than physiological doses if they are being used to heal rickets (e.g. in 1α-hydroxylase deficiency) (see chapter 8). Due to its short half-life, a short trial of calcitriol may be considered in cases of vitamin D deficiency where there are also concerns about hypocalcaemia. The adverse effects are the same as those of vitamin D toxicity. This medication needs close monitoring for hypercalcaemia and hypercalciuria with annual renal ultrasounds. Paricalcitol (Zemplar®) is a synthetic analogue of calcitriol known as 19-nor calcitriol. As the use of calcitriol increases plasma calcium and phosphate, which is undesirable in chronic renal failure, paricalcitol is designed to have a preferential action on the kidney [20].

Drugs Used in Calcium Imbalance

Hypercalcaemia Loop Diuretics Drugs such as furosemide are used in combination with fluid therapy to lower serum calcium levels in the acute setting (forced diuresis). Monitoring for fluid and electrolyte imbalance, particularly hyponatraemia, hypokalaemia and hypomagnesaemia, is very important. If used for prolonged periods, loop diuretics may induce

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nephrocalcinosis. Thiazide diuretics must not be used under these circumstances as they will exacerbate hypercalcaemia by reducing urinary calcium excretion. Bisphosphonates These can also be used in the acute setting to lower serum calcium levels and infusions can be repeated as required. The usual dose is half of that used in osteoporotic conditions (0.5 mg/kg/dose). Since bone turnover is usually very rapid in hypercalcaemic states, patients may be very sensitive to bisphosphonates and they may only require one or two doses to achieve normocalcaemia. Calcitonin In situations where hypercalcemia does not respond to forced diuresis, calcitonin can be used. It is a synthetic (or recombinant) analogue of the hormone which acts to inhibit calcium absorption from the gut, osteoclast activity in the bones and calcium and phosphate absorption from the kidneys. Calcitonin has a rapid initial effect which only lasts for a few days to a few weeks before losing its effect. It is given by subcutaneous, intramuscular (5–10 units/kg/12 h) or intravenous injection (5–10 units/ kg/6 h). Its use in children is limited as bisphosphonates have superseded it. Corticosteroids Hydrocortisone or prednisolone is used in the treatment of hypercalcaemia due to vitamin D intoxication, subcutaneous fat necrosis or sarcoidosis. They decrease calcium absorption from the gut and increase calcium excretion from the kidneys. Phosphate Supplements Oral phosphate supplementation can be used to decrease calcium absorption from the gut, block renal synthesis of vitamin D and inhibit bone resorption. Cinacalcet Cinacalcet is a calcimetic (imitates the presence of calcium) and acts by increasing the sensitivity of the calcium receptor [30]. It has been primarily used in the treatment of secondary hyperparathyroidism associated with chronic renal failure. It has also been used in familial benign hypocalciuric hypercalcaemia (FBHH) (#145980) [21] and X-linked hypophosphataemic rickets (XLH) (#307800) [22]. It causes a drop in serum calcium and can lead to some nausea and vomiting. It is given as an oral medication twice a day.

Hypocalcaemia Hypocalcaemia presenting in the acute situation can be treated with slow infusion of intravenous calcium preparations such as calcium gluconate (0.3 ml/kg of 10%

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calcium gluconate over 5–10 min). If given in situations where hypocalcaemia has been present for some time, intravenous infusion of calcium may offer almost instant relief from the pain of muscle spasm etc resulting from chronic hypocalcaemia. Rarely, continuous calcium infusions have been required in cases of hereditary vitamin D resistant rickets. This usually needs to be given via a central venous line. The most widely available preparation of calcium for intravenous use is a 10% solution of calcium gluconate. This contains 0.225 mmol/ml. The initial dose followed by a more continuous infusion if necessary, the dose being adjusted according to the plasma calcium level. Oral calcium supplementation is usually only required when there is inadequate dietary intake of calcium. This may occur during periods of greater requirements for calcium such as prematurity, rapid growth, pregnancy or lactation. Mild asymptomatic hypocalcaemia can be managed with simple oral calcium supplementation, of which there are numerous preparations, in conjunction with a vitamin D preparation where appropriate. Hypomagnesaemia is a cause of secondary hypocalcaemia and needs to be corrected with an intravenous (or intramuscular) injection of magnesium sulphate (0.5–1 mmol/kg) followed by oral magnesium (0.2–0.4 mmol/kg) daily. The most widely available solution of magnesium for parenteral use is a 50% solution of MgSO4.7H2O which contains 2 mmol/ml of elemental magnesium. Oral supplementation may cause diarrhoea if given in large quantities. Magnesium glycerophosphate causes fewer side effects and may be used in preference. Renal failure can impair excretion of magnesium and in this event the dose will need to be reduced.

Drugs Used in Phosphate Imbalance

Hyperphosphataemia The mainstay of management for hyperphosphataemia is to treat the underlying disorder, restrict intake and enhance renal excretion of phosphate. Phosphate Binders These are needed in chronic renal failure where there is limited phosphate excretion. Aluminium hydroxide is now rarely used. Calcium carbonate or calcium acetate are the preferred binders of choice. These compounds complex with dietary phosphate to limit its absorption. If hypercalcaemia or extraskeletal calcification becomes a problem, sevelamer (Renegal®) is a good alternative. Sevelamer is also a dietary phosphate binder which is not absorbed. Non-compliance can be an issue as the tablets are large and need to be taken with each meal and cause gastrointestinal upset [23]. Sevelamer is considered to be preferable to calcium salts in chronic renal disease since the latter have a tendency to render bone atonic.

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Loop Diuretics Forced diuresis in patients with normally functioning kidneys is an effective acute management for hyperphosphataemia.

Hypophosphataemia Phosphate Supplements Phosphate supplements form the mainstay of treatment for patients with hypophosphataemic conditions. They are readily absorbed from the gastrointestinal tract but are then often quickly excreted via the kidney in many hypophosphataemic conditions [24]. For this reason, they should ideally be given five times a day at regular intervals from when a child starts mobilising until the end of growth. Unfortunately, the large quantities of phosphate required are associated with significant diarrhoea and doses need to be built up slowly to be tolerated. The gastrointestinal adverse effect profile and the dosing regime often result in non compliance and may limit the dose that can be administered. This can be particularly concerning during the adolescence growth spurt. However, comfort can be taken that with adequate consistent supplementation, normal height can be attained and, once growth has finished, medication can often be stopped. Dose is dependent on the size of the child and their rate of growth [25]. Calcitriol or Alphacalcidol Due to the effect of phosphate reducing calcium absorption and the often abnormal metabolism of vitamin D in hypophosphataemic rickets, calcitriol or alphacalcidol is given in conjunction with phosphate supplementation unless the hypophosphataemia is associated with hypercalciuria such as occurs in hereditary hypophosphataemic rickets with hypercalciuria (HHRH) (#241530) which is caused by a mutation in the sodium/phosphate co-transporter of renal tubules. In any case, serum and urinary calcium levels need to be monitored carefully and with annual renal ultrasounds to assess for renal calcium deposition.

New Drug Therapies In Calcium and Bone Disorders

Several therapeutic targets in modifying bone disorders have been identified and are currently undergoing trials in the adult population. It may be some time before these are available to be used in children.

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RANK Ligand Inhibitors RANK ligand (RANKL) is produced by osteoblasts and is essential for the differentiation, activation and survival of osteoclasts (see chapter 3). Inhibition of RANKL leads to osteoclast inactivation which in turn reduces bone resorption, much like the action of bisphosphonates. Osteoprotegerin (OPG) is a natural competitive antagonist of RANKL and has been used in phase I trials of patients with osteoporosis [26]. It seemed to have few adverse effects. It has also been used successfully to treat juvenile Paget disease (JPD) (#239000) [27] in which mutations in OPG lead to a rickets-like condition with grossly elevated alkaline phosphatase. Denosumab, a human antibody against RANKL, has a greater potency than OPG and is currently undergoing phase III clinical trials.

Cathepsin K Inhibitor Cathepsin K is a lysosomal enzyme found in osteoclasts. This enzyme is needed by the osteoclast to break down the organic matrix of bone after the inorganic mineral component has been dissolved by acid. It acts most importantly on type I collagen in bone but also has the ability to cleave type II collagen in cartilage. Inhibiting these properties would lead to reduction of resorption similar to the action of bisphosphonates and may also have some actions to decrease cartilage destruction. Specific antibody inhibition of cathepsin K has been shown to reduce resorption by 40–50%; however, developing tissue specificity has been problematic [28].

Sclerostin (SOST) Sclerostin is a glycoprotein produced by osteocytes. Osteocytes are sensing osteoblasts that have been buried into bone and which provide feedback information to the osteoblasts helping to regulate bone production. Sclerostin is a competitive inhibitor of the Wnt pathway and, to a lesser degree, the BMP pathway, both pathways being important in bone formation. A neutralising antibody to sclerostin has been developed and is currently undergoing phase I trials. By blocking the action of sclerostin the aim is to increase bone formation and decrease bone resorption [29].

Bone Morphogenic Protein 2 (BMP2) Bone morphogenic proteins (BMP) are a group of glycoproteins important in tissue repair and regeneration. Human recombinant BMP 2 has been used in orthopaedic

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surgery to stimulate new bone formation and has been especially useful in spinal surgery and cases of non-union [29].

Calcilytic Drugs Calcilytic drugs have the property of reducing the sensitivity of the calcium sensing receptor (CaSR) to calcium. As a consequence, they reverse the tendency to hypoparathyroidism in situations where CaSR response to calcium is increased. They are currently being developed as a possible treatment of osteoporosis (as an alternative to PTH), but are also being evaluated for their effect in autosomal-dominant hypoparathyroidism (#146200) in which activating mutations of the CaSR are present [30]. Theoretically, they might be of value in those mutations which cause a left shift of the PTH responsiveness curve (see chapter 2) but are unlikely to be of help where the mutation causes a permanent constitutive activation of the receptor.

Recombinant Tissue Non-Specific Alkaline Phosphatase (TNSALP) Mutations in the gene for tissue non-specific alkaline phosphatase cause a variety of conditions ranging from a severe neonatal form to milder adult manifestations (see chapter 12). The severe neonatal form is usually fatal within a few days to a few weeks and, although various treatment options have been tried, none has proved successful. However, there has recently become available recombinant TNSALP which is currently under investigation as a therapeutic option in the severe neonatal form of the condition. The results are still pending.

Recombinant Parathyroid Hormone (PTH) Two preparations of PTH are currently available although neither is licensed for use in children. PTH 1–84 (natural sequence PTH) has been used for the treatment of osteoporosis in adults but there are currently no reports of its use in children. PTH 1–34 (teriparatide) has been used occasionally for treating hypoparathyroidism. In most instances this has been caused by activating mutations of the CaSR (ADH) (#146200). In the first report, teriparatide was used in the acute situation and demonstrated a rapid response [31] whilst in the second report, a patient was treated for more than a year with a good sustained response [32]. In the UK, 1 patient has been treated so far. The principal responses are an increase in plasma calcium and a reduction in urinary calcium excretion. Teriparatide is more effective if given twice daily by subcutaneous injection [33]. Side-effects include gastrointestinal disturbances, palpitations and headache but it is

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not known if these are a problem in children. There have also been concerns about the possibility of developing osteosarcomas as this occurred in rat studies in which they were administered large doses but it is not known if this will prove to be a potential problem in children given physiological doses.

Conclusions

There are many drugs used in the treatment of bone and calcium disorders in children. Some of them, such as alfacalcidol, are used to overcome a deficiency that results from inborn errors of metabolism but are also used to reverse the effects of hypoparathyroidism. Many of the drugs used, in common with those used in other branches of paediatric practice, are not licensed for use in children but are nevertheless used by extrapolation of their effects in adults. Of these, the bisphosphonates are the ones which have particularly acquired the status of ‘accepted clinical practice’ without having been subjected to formal double blind trials. It is now too late to undertake these trials with the current batch of drugs that are used. However, there are under trial a number of other drugs that are more specifically targeted to individual problems that may, in the future, prove more effective at dealing with these problems than the present armamentarium allows. The results of these trials are awaited.

References 1 Vahle JL, Sato M, Long GG, et al: Skeletal changes in rats given daily subcutaneous injections of recombinant human parathyroid hormone (1–34) for 2 years and relevance to human safety. Toxicol Pathol 2002;30:312–321. 2 Glorieux FH, Bishop NJ, Plotkin H, Chabot G, Lanoue G, Travers R: Cyclic administration of pamidronate in children with severe osteogenesis imperfecta. N Engl J Med 1998;339:947–952. 3 Roelofs AJ, Thompson K, Gordon S, Rogers MJ: Molecular mechanisms of action of bisphosphonates: current status. Clin Cancer Res 2006;12: 6222s-6230s. 4 Russell RG, Xia Z, Dunford JE, et al: Bisphosphonates: an update on mechanisms of action and how these relate to clinical efficacy. Ann NY Acad Sci 2007; 1117:209–257. 5 Munns CF, Rauch F, Ward L, Glorieux FH: Effect of prenatal pamidronate therapy on maternal and fetal outcome: a report of two cases. J Bone Miner Res 2004;19:1742–1745. 6 Rauch F, Glorieux FH: Osteogenesis imperfecta. Lancet 2004;363:1377–1385.

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7 Ward L, Tricco AC, Phuong P, et al: Bisphosphonate therapy for children and adolescents with secondary osteoporosis. Cochrane Database Syst Rev 2007;CD005324. 8 Henderson RC, Lark RK, Kecskemethy HH, Miller F, Harcke HT, Bachrach SJ: Bisphosphonates to treat osteopenia in children with quadriplegic cerebral palsy: a randomized, placebo-controlled clinical trial. J Pediatr 2002;141:644–651. 9 Land C, Rauch F, Munns CF, Sahebjam S, Glorieux FH: Vertebral morphometry in children and adolescents with osteogenesis imperfecta: effect of intravenous pamidronate treatment. Bone 2006;39: 901–906. 10 Munns CF, Rauch F, Travers R, Glorieux FH: Effects of intravenous pamidronate treatment in infants with osteogenesis imperfecta: clinical and histomorphometric outcome. J Bone Miner Res 2005;20: 1235–1243. 11 Munns CF, Rajab MH, Hong J, et al: Acute phase response and mineral status following low dose intravenous zoledronic acid in children. Bone 2007; 41:366–370.

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12 Rauch F, Land C, Cornibert S, Schoenau E, Glorieux FH: High and low density in the same bone: a study on children and adolescents with mild osteogenesis imperfecta. Bone 2005;37:634–641. 13 Rauch F, Cornibert S, Cheung M, Glorieux FH: Long-bone changes after pamidronate discontinuation in children and adolescents with osteogenesis imperfecta. Bone 2007;40:821–827. 14 Chahine C, Cheung MS, Head TW, Schwartz S, Glorieux FH, Rauch F: Tooth extraction socket healing in pediatric patients treated with intravenous pamidronate. J Pediatr 2008;153:719–720. 15 Munns CF, Rauch F, Zeitlin L, Fassier F, Glorieux FH: Delayed osteotomy but not fracture healing in pediatric osteogenesis imperfecta patients receiving pamidronate. J Bone Miner Res 2004;19:1779– 1786. 16 Whyte MP, Wenkert D, Clements KL, McAlister WH, Mumm S: Bisphosphonate-induced osteopetrosis. N Engl J Med 2003;349:457–463. 17 Glorieux FH, Rauch F, Ward LM, et al: Alendronate in the treatment of pediatric osteogenesis imperfecta. J Bone Miner Res 2004;19:S12. 18 Misra M, Pacaud D, Petryk A, Collett-Solberg PF, Kappy M: Vitamin D deficiency in children and its management: review of current knowledge and recommendations. Pediatrics 2008;122:398–417. 19 Bikle D: Nonclassic actions of vitamin D. J Clin Endocrinol Metab 2009;94:26–34. 20 Robinson DM, Scott LJ: Paricalcitol: a review of its use in the management of secondary hyperparathyroidism. Drugs 2005;65:559–576. 21 Timmers HJ, Karperien M, Hamdy NA, de Boer H, Hermus AR: Normalization of serum calcium by cinacalcet in a patient with hypercalcaemia due to a de novo inactivating mutation of the calcium-sensing receptor. J Intern Med 2006;260:177–182. 22 Raeder H, Bjerknes R, Shaw N, Netelenbos C: A case of X-linked hypophosphatemic rickets (XLH): complications and the therapeutic use of cinacalcet. Eur J Endocrinol 2008;(suppl 1):S101–S105. 23 Komaba H, Tanaka M, Fukagawa M: Treatment of chronic kidney disease-mineral and bone disorder (CKD-MBD). Intern Med 2008;47:989–994. 24 Bastepe M, Juppner H: Inherited hypophosphatemic disorders in children and the evolving mechanisms of phosphate regulation. Rev Endocr Metab Disord 2008;9:171–180.

25 Ward LM: Renal phosphate-wasting disorders in childhood. Pediatr Endocrinol Rev 2005;2 Suppl 3:342–350. 26 Schwarz EM, Ritchlin CT: Clinical development of anti-RANKL therapy. Arthritis Res Ther 2007;9 Suppl 1:S7. 27 Cundy T, Davidson J, Rutland MD, Stewart C, DePaoli AM: Recombinant osteoprotegerin for juvenile Paget’s disease. N Engl J Med 2005;353:918– 923. 28 Reid IR: Anti-resorptive therapies for osteoporosis. Semin Cell Dev Biol 2008;19:473–478. 29 Khosla S, Westendorf JJ, Oursler MJ: Building bone to reverse osteoporosis and repair fractures. J Clin Invest 2008;118:421–428. 30 Egbuna OI, Brown EM: Hypercalcaemic and hypocalcaemic conditions due to calcium-sensing receptor mutations. Best Pract Res Clin Rheumatol 2008;22:129–148. 31 Shiohara M, Shiozawa R, Kurata K, et al: Effect of parathyroid hormone administration in a patient with severe hypoparathyroidism caused by gain-offunction mutation of calcium-sensing receptor. Endocr J 2006;53:797–802. 32 Mittelman SD, Hendy GN, Fefferman RA, et al: A hypocalcemic child with a novel activating mutation of the calcium-sensing receptor gene: successful treatment with recombinant human parathyroid hormone. J Clin Endocrinol Metab 2006;91:2474– 2479. 33 Winer KK, Sinaii N, Peterson D, Sainz B Jr, Cutler GB Jr: Effects of once versus twice-daily parathyroid hormone 1–34 therapy in children with hypoparathyroidism. J Clin Endocrinol Metab 2008;93:3389– 3395. 34 49 Nutrition and bone health: with particular reference to calcium and vitamin D. Report of the subgroup on bone health, working group on the nutritional status of the population of the Commitee on Medical Aspects of Food and Nutrition Policy, 1998. 35 Meeting the need for vitamin D. CMO Update 2005;42. 36 Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D and fluoride, 1997. 37 Wagner CL: Prevention of rickets and vitamin D deficiency in infants, children, and adolescents. Pediatrics 2008;122:1142–1152.

Dr. Moira Cheung Imperial College London, MRC Clinical Sciences Centre, Hammersmith Hospital Du Cane Road London W12 0NN (UK) Tel. +44 208 383 8324, Fax +44 208 383 8306, E-Mail [email protected]

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Chapter 14 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 233–245

Skeletal Aspects of Non-Accidental Injury Karl Johnson Birmingham Children’s Hospital, Birmingham, UK

Abstract Inflicted non-accidental skeletal injuries form a small but important part of the spectrum of child abuse, with the majority of skeletal injuries occurring in children under 2 years of age. Radiology plays a vital role in the detection and evaluation of these skeletal injuries. A thorough detailed radiological evaluation should be undertaken in order to investigate appropriately a child for suspected inflicted non-accidental injury. This is to detect accurately, and possibly date, any injuries, exclude normal variants of growth which may mimic fractures and possibly to diagnose any underlying metabolic or genetic disorder of the bone which may predispose a child to fracturing. Any fracture may be the result of an inflicted injury or accidental event. It is therefore appropriate that any fracture that is identified is correlated with relevant appropriate clinical history. Certain injuries, such as rib or metaphyseal fractures, require a more specific method of causation and therefore have a higher degree of suspicion of being the result of an inflicted injury than other fracture types, such as skull and clavicular fractures, which are relatively non-specific in their mechanism of causation. In all cases, correlation with the clinical history is mandatory. While radiology does play an important role in the dating of injuries, the dating of fractures from the radiological findings is difCopyright © 2009 S. Karger AG, Basel ficult and imprecise.

One of the most challenging areas of paediatric medicine is that relating to child abuse. Child abuse covers a wide spectrum of injuries and assaults that includes social, sexual, emotional as well as physical events. Consequently, to diagnose a child as having suffered from an episode of abuse has significant social, criminal and civil implications [1]. Non-accidental injury is the term often used when a child has suffered an episode of physical abuse. The incidence of physical abuse of children is unfortunately not uncommon, with reports of between 4 and 16% of children being abused each year [2]. Child maltreatment and abuse significantly contributes to child mortality and morbidity [2]. Returning an abused child into the care of their abuser can result in up to 30% of them suffering further injury [3]. The investigation of children who have suffered potential abuse should be multidisciplinary and involve paediatricians, relevant medical subspecialties, and other

healthcare professionals. Radiology provides a relatively small, but important, contribution in the investigation of skeletal and visceral injuries. Visceral injuries are common in the older child whilst skeletal and cerebral trauma are typically a feature in the non-ambulant child. Each may co-exist or occur separately. Skeletal injuries commonly occur in the non-ambulant child, typically under two years of age but are more common under one year [4]. This chapter will deal with the radiological features of skeletal injuries associated with non-accidental injury and consequently almost exclusively refers to children under 2 years of age. The site and type of fracture or skeletal injury can never in itself be totally discriminatory of abuse or accident. The child’s age and development is vitally important, as an unexplained injury, such as a fractured femur in a non-ambulatory two month infant, should be treated completely differently from the same fracture in a 15-yearold adolescent [1]. For each injury, it is vitally important that a clear history of how the injury occurred is obtained. This history has to be carefully correlated with the clinical symptoms and radiological findings. From the radiological features alone, no fracture should be regarded as being pathognomonic of abuse [4].

Radiological Investigation of Suspected Abuse

A radiological skeletal survey is the standard initial imaging modality for assessing young children who are suspected of having been physically abused [5]. Typically, this should be performed on all children under 2 years of age in whom there is clinical suspicion. In the older child, a detailed discussion between the referring paediatrician and the radiologist about the potential yield of such an investigation and its value, measured against the radiation dose, should be discussed. The yield of imaging is higher in the one year old child [5]. The role of the skeletal survey is to detect any occult bony injury, obtain further information about any clinically suspected fracture, aid in the dating of injuries and also help provide any evidence of any possible underlying skeletal disorder or metabolic bone disease which may predispose the child to fracturing. The radiographic imaging should be of a high standard with good quality collimation and optimised exposure parameters. A variety of guidelines published by national radiological societies exists to help achieve the appropriate level of quality. Ideally, all radiology staff involved in carrying out the study should be appropriately paediatrically trained [5]. A skeletal survey consists of radiographs of each anatomical site and the standard projections required are shown in table 1. Supplementary views should include a lateral view of any fractures and coned down views of the metaphyses when the features suggest a fracture or are otherwise equivocal. Repeat radiographs of areas of uncertainty should be performed within 1–2 weeks. This will allow time for a

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Table 1. Projections required for a radiographic skeletal survey for the investigation of NAI Skull – anterior posterior (AP) and lateral; additional Townes view (if clinically indicated), skull films should be obtained even if a CT scan has been performed Chest – AP, to include the clavicles and oblique views of both sets of ribs Abdomen, including pelvis and hips Spine – lateral view of cervical, thoracic and lumbar ( if the whole of the spine is not seen on the chest and abdominal film then additional views may be required) Limbs AP views of both humeri, both forearms, both femora and both tibiae and fibulae Hands – posterior/anterior Feet – dorsi/palmar

radiographically detectable healing response to occur around potential injuries. This helps confirm or refute equivocal findings and also improves the detection of occult injuries. A repeat chest radiograph with oblique views of the ribs should be obtained routinely as the detection of rib fractures is increased by over 20% [6, 7]. The use of scintigraphy (nuclear medicine bone scan) is a complementary investigation to the skeletal survey. Used together, the two investigations will detect more fractures than either investigation alone. Bone scintigraphy gives a better detection rate for rib fractures. However, it is less sensitive in detecting skull, spine or metaphyseal fractures [8, 9]; the latter as a consequence of the normal high activity seen in the physes of the developing skeleton. As with the skeletal survey, it is important that the bone scan is done to a high technical standard and, in view of the relatively high radiation dose of the bone scan, the investigation should not be undertaken unless the quality of the images is satisfactory [5]. Computerised tomography (CT) may have a role in helping determine the nature of equivocal radiographic findings. It appears to be more sensitive than radiographs in detecting occult rib fractures [10]. Magnetic resonance imaging (MRI) and ultrasound are both useful in the evaluation of soft tissue lesions and high resolution ultrasound has been used for the identification of periosteal elevation in metaphyseal and rib fractures [4, 11, 12]. Ultrasound, CT and MRI are very useful, but complementary, investigations and it is important that the initial radiological investigation is a highquality skeletal survey.

Skeletal Features

No fracture can be regarded as diagnostic of non-accidental injury. Any fracture can be caused by an accident or can occur from an inflicted non-accidental injury [1, 4,

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Table 2. Specificity of fractures in NAI Reduced Skull Clavicle Long bone Periosteal reaction Epiphyseal separation

Increased Multiple fractures of different ages Metaphyseal Ribs (posterior) Acromion Digits Sternum Spine

13]. For any fracture, it is important that there should be an appropriate history of a mechanism of causation and clinical presentation. From a clinical view, it is important that any history takes account of the child’s development, such as its ability to crawl or walk. With the clinical presentation, different fracture types will manifest with different symptoms and this should also be considered. A non-accidental injury should be considered if no history to account for the injury is given or the account given differs on its telling. A review of the different fracture types and their relative specificity for nonaccidental injury is given in table 2. The list reflects the incidence of different fractures type within abused children. It also takes account of the fact that certain fracture types, such as rib fractures, require a more precise method of causation compared with other injuries, such as skull fractures. This classification system only provides a guideline, and each fracture and its clinical presentation should be fully assessed without prejudice.

Fracture Distribution

Fractures which are the result of abuse predominantly occur in the infant-toddler age groups. Studies have shown that 80% of all fractures from abuse are seen in children under 18 months of age and by contrast 85% of fractures not due to abuse occur in children over 5 years of age [4, 14]. Approximately 25–50% of fractures occurring in children under 1 year of age are due to abuse and there is an association between multiple fractures and abuse [4, 14–16].

Rib Fractures

Rib fractures are uncommon in children under 3 years of age and are the result of severe thoracic compression. It is thought that posterior fractures occur from levering the end of the rib over the transverse position of the vertebral body [17–19].

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An infant’s ribs are relatively elastic and allow a considerable degree of deformation prior to fracturing. Consequently, the force needed to produce rib fractures is significant and, in the absence of major trauma or an underlying bone abnormality, the presence of rib fractures in a child is suspicious for non-accidental injury. A child with rib fractures has a 70% chance of having been abused [4]. Rib fractures following cardiopulmonary resuscitation (CPR) are rare, with many large studies failing to detect fractures in large numbers of children who had undergone CPR [20–23]. Conditions that predispose children to bone fragility such as osteopaenia of prematurity or bone dysplasia such as osteogenesis imperfecta are a relatively common cause of rib fractures in the infant/toddler population [24–26]. Rib fractures following abuse can occur in any location, and may be unilateral or bilateral. Rib fractures are not often associated with chest wall bruising. However, fingertip bruising may be an associated clinical feature. In some cases, an acute rib fracture may not be easily detected on the initial radiograph because the position of the fracture is in the line of the X-ray beam. Repeat radiographs one to two weeks later will show evidence of callus formation around the fracture site, increasing the detection of fractures (fig. 1) [17].

Metaphyseal Fractures

Metaphyseal fractures are the result of micro-fractures across the metaphysis adjacent to the physis, with the fracture plane being parallel to the physis. The injury is believed to be the result of a severe shearing force, which is not typically replicated when a child falls or suffers from blunt trauma [27]. To cause a metaphyseal fracture therefore requires a relatively specific mechanism. It can be caused by pulling/ torsional forces applied to the limb, or allowing an unsupported limb to flail about violently. On radiographs classical metaphyseal fractures often appear as small thin slivers of bone separated from the shaft. Depending on how this bone is projected, it may create a ‘bucket handle’ appearance, or resemble a chip or corner type injury. The fracture fragment of the bone is thicker at the periphery, or it may appear as a thin disc or rim. Depending on the extent of the injury, there may not be any disruption or elevation of the adjacent periosteum and, as a consequence, the amount of subperiosteal haemorrhage and subsequent new bone and callus formation may be limited (fig. 2, 3) [27, 28]. Metaphyseal fracture can occasionally be confused with the normal beaking and irregularity seen around the metaphysis of the immature skeleton. Many of these normal variations in growth have been described and documented [29, 30]. If clinical doubt exists then either additional radiographs to view the findings in another imaging plane, or repeat radiographs to document possible bone healing can be helpful.

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a b

c Fig. 1. a AP chest radiograph taken on the day of admission shows irregularity around the lateral aspects of the rib suggestive of fracture. b Follow-up chest radiograph 8 days later shows more obvious early callus formation around the lateral ribs on the right side, indicative of healing fractures. c A post-mortem chest radiograph (5 days following the second radiograph) of the same child confirms the presence of fractures with callus formation of the anterior lateral right 4th to 8th ribs and also the left lateral 5th and 7th ribs. These fractures were confirmed at post-mortem. There are also healing fractures of the left posterior 2nd to 7th ribs. These fractures were more difficult to visualise on the initial chest radiograph and their detection has been aided by the healing response around them. Note that these fractures are in a line and show the same degree of callus formation, suggesting that they were all sustained during a single event.

Subperiosteal New Bone Formation

Subperiosteal new bone is seen as a hazy and indistinct margin separated from the cortex. It may become visible as part of a healing response to a fracture or as a result of subperiosteal haemorrhage caused by a gripping or twisting force applied to the limb that disrupts the periosteal attachment to the bone. It also occurs as a normal physiological response in the growing child, in response to fracture healing, due to elevation of the periosteum and subperiosteal haemorrhage. It can be seen in other pathological processes such as a response to infection or malignant infiltration of bone.

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Fig. 2. Metaphyseal fracture of the proximal left tibia shows a separated slither of bone from the proximal metaphysis which has a classic ‘bucket handle’ appearance.

Fig. 3. Metaphyseal fracture of the proximal left tibia showing a ‘bucket handle’ fracture.

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4 Fig. 4. Normal physiological subperiosteal new bone formation. This is a single lamina which does not extend as far as the metaphysis. Fig. 5. Pathological periosteal new bone formation along the shaft of the distal humerus which is thickened and extends into the metaphysis. Follow-up film showed remodelling and reabsorption.

5

Periosteal new bone formation due to growth typically occurs in children less than about four months of age. It can occur in multiple locations, with an asymmetrical distribution. The tibia is the commonest site, but it can occur in any of the long bones. It is typically smooth, not more than 1.8 mm in thickness on an AP radiograph, it is not layered and it does not extend beyond the metaphysis. Typically, it would not be expected to alter over a 2-week period (fig. 4, 5) [31]. Pathological, post traumatic subperiosteal new bone obviously occurs in the older child, but not all periosteal new bone formation in younger children is normal. On radiographs, it often appears thicker, is layered, can have a convex border, is greater than 2 mm in depth and can extend down into the distal metaphysis and the periphysis region. Serial radiographs over a 2-week period would be expected to show change.

Long Bone Fractures

Obtaining an appropriate history of a possible method of causation is very important in determining if a long bone fracture is the result of an inflicted injury. The

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likelihood of an unexplained fracture being the result of abuse increases in the nonambulant child, compared to the situation when a child is independently mobile. Multiple fractures are more common after physical abuse than after a non-abusive traumatic injury [4]. The morphology and appearance of a long bone fracture may suggest a possible mechanism of injury, which is of potential value when assessing the validity of any explanation given by the carers. For example, a spiral fracture will require twisting or torsional component to the force, while an oblique fracture typically is the result of a levering or bending type action, A transverse fracture is often due to a direct impact and a buckle or torus fracture is commonly the result of a compression injury. There have been a number of reports reviewing the incidence of long bone fractures in abused and non-abused children [4, 14, 16, 32–38]. While these statistics are important in assessing the likelihood of these injuries being abusive, it must be remembered that each individual fracture in each case should be assessed separately. Femoral fractures as the result of abuse are more commonly seen in children who are not yet walking and a child with a femoral fracture has a 25–33% chance of having been abused [4]. Spiral or oblique fractures of the femur are the commonest type of injury in the abused child who is under 15 months of age. The commonest location of fractures in both the abused and non-abused was the midshaft. For children under 18 months of age, 96% of all fibula/tibia fractures resulted from abuse [4]. A child under the age of 3 with a humeral fracture has a 50% chance of having been abused. Mid-shaft fractures of the humerus are more common in abuse, while supracondylar fractures are more likely to be associated with a non-abuse injury [4].

Skull Fractures

Skull fractures are more commonly seen after accidental trauma than following abuse. The commonest fracture site in both abused and accidental events is the parietal bone and the commonest fracture type is a linear pattern [4]. The significance of more complex fracture patterns has not been substantiated. An infant or toddler with a skull fracture has a 33% chance of having been abused [4]. Skull fracture can occur from falls and it is estimated that falls from about 3 feet in height result in a skull fracture in about 1–2% of cases. Falls from below 3 feet may still cause a skull fracture depending on the type of impact [39].

Unusual Fractures

Fractures at uncommon sites are usually the result of unusual mechanisms and injures. Those injuries that do not occur from routine paediatric trauma,

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Fig. 6. Healing fracture of the metacarpal of the thumb in a child which is most likely the result of a severe squeezing injury.

Fig. 7. Healing fracture of the right acromion.

particularly if there is no immediate explanation, should alert the clinician to assess the potential method for causation. Unusual injuries include fractures of the spine, scapula, sternum, pelvis and finger and toes in the non-ambulatory child (fig. 6, 7).

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Fig. 8. a Transverse fracture of the right femur with extensive soft tissue swelling. b Follow-up films 10 days later show very early subperiosteal new bone formation around the fracture site.

a

b

Fracture Dating

The dating of fractures is based upon the subjective evaluation of radiographic changes that occur around the fracture site. These changes include the presence and then resolution of any soft tissue swelling, the appearances of early subperiosteal new bone, followed by bridging callus across the fracture site and loss of fracture line differentiation. More organised callus formation and the appearance of lamellar bone are later features [40–42]. The dating of fractures is obviously open to subjective opinion as to the stage of fracture healing and also it must be recognised that there is a wide variation in the rate of healing, both between individuals and even within separate injuries within the same individual. The radiological features of bone healing are a continuum, with considerable overlap. Early radiographic features of callus can be first noted within 7 days after injury and are present in 50% by 4 weeks. More mature callus and early remodelling are seen at 8 weeks in most cases. Radiological dating can only suggest a relatively wide time frame and is at best an approximation [40–42] and estimates of the time of injury are made in terms of weeks rather than days. However, radiologists can clearly differentiate recent from old fractures (fig. 8) [41]. Importantly, any fracture must be correlated with the clinical history and the onset of appropriate clinical symptoms and signs. There is an assumption that when a fracture occurs that it will be painful. The nature and duration of this pain will vary between individuals and also possibly between different fracture types. Importantly,

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if there is a history of a child being asymptomatic followed by a description of a child showing signs of distress and loss of limb function, then this would be a more accurate method of dating an injury than the radiological features. The majority of childhood fractures are not associated with bruising [43].

References 1 Sugar NF: Diagnosing child abuse. BMJ 2008;337:a1398. 2 Gilbert R, Widom CS, Browne K, Fergusson D, Webb E, Janson S: Burden and consequences of child maltreatment in high-income countries. Lancet 2009;373:68–81. 3 Ellaway BA, Payne EH, Rolfe K, et al: Are abused babies protected from further abuse? Arch Dis Child 2004;89:845–846. 4 Kemp AM, Dunstan F, Harrison S, et al: Patterns of skeletal fractures in child abuse: systematic review. BMJ 2008;337:a1518. 5 Intercollegiate Report from the Royal College of Radiologists and the Royal College of Paediatrics and Child Health: Standards for radiological investigations of suspected non-accidental injury. Intercollegiate report from the Royal College of Radiologists and the Royal College of Paediatrics and Child Health. Standards for radiological investigations of suspected non-accidental injury. 6 Kleinman PK, Nimkin K, Spevak MR, et al: Follow-up skeletal surveys in suspected child abuse. AJR Am J Roentgenol 1996;167:893–896. 7 Zimmerman S, Makoroff K, Care M, Thomas A, Shapiro R: Utility of follow-up skeletal surveys in suspected child physical abuse evaluations. Child Abuse Negl 2005;29:1075–1083. 8 Haase GM, Ortiz VN, Sfakianakis GN, Morse TS: The value of radionuclide bone scanning in the early recognition of deliberate child abuse. J Trauma 1980;20:873–875. 9 Mandelstam SA, Cook D, Fitzgerald M, Ditchfield MR: Complementary use of radiological skeletal survey and bone scintigraphy in detection of bony injuries in suspected child abuse. Arch Dis Child 2003;88:387–390. 10 Wootton-Gorges SL, Stein-Wexler R, Walton JW, Rosas AJ, Coulter KP, Rogers KK: Comparison of computed tomography and chest radiography in the detection of rib fractures in abused infants. Child Abuse Negl 2008;32:659–663. 11 Markowitz RI, Hubbard AM, Harty MP, Bellah RD, Kessler A, Meyer JS: Sonography of the knee in normal and abused infants. Pediatr Radiol 1993;23:264– 267.

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12 Nimkin K, Kleinman PK, Teeger S, Spevak MR: Distal humeral physeal injuries in child abuse: MR imaging and ultrasonography findings. Pediatr Radiol 1995;25:562–565. 13 Taitz J, Moran K, O’Meara M: Long bone fractures in children under 3 years of age: is abuse being missed in Emergency Department presentations? J Paediatr Child Health 2004;40:170–174. 14 Carty H, Pierce A: Non-accidental injury: a retrospective analysis of a large cohort. Eur Radiol 2002; 12:2919–2925. 15 Warrington SA, Wright CM: Accidents and resulting injuries in premobile infants: data from the ALSPAC study. Arch Dis Child 2001;85:104–107. 16 Worlock P, Stower M, Barbor P: Patterns of fractures in accidental and non-accidental injury in children: a comparative study. Br Med J (Clin Res Ed) 1986; 293:100–102. 17 Kleinman PK, Marks SC, Adams VI, Blackbourne BD: Factors affecting visualization of posterior rib fractures in abused infants. AJR Am J Roentgenol 1988;150:635–638. 18 Kleinman PK, Marks SC Jr, Nimkin K, Rayder SM, Kessler SC: Rib fractures in 31 abused infants: postmortem radiologic-histopathologic study. Radiology 1996;200:807–810. 19 Kleinman PK, Marks SC Jr, Richmond JM, Blackbourne BD: Inflicted skeletal injury: a postmortem radiologic-histopathologic study in 31 infants. AJR Am J Roentgenol 1995;165:647–650. 20 Betz P, Liebhardt E: Rib fractures in children: resuscitation or child abuse? Int J Legal Med 1994; 106:215–218. 21 Bush CM, Jones JS, Cohle SD, Johnson H: Pediatric injuries from cardiopulmonary resuscitation. Ann Emerg Med 1996;28:40–44. 22 Feldman KW, Brewer DK: Child abuse, cardiopulmonary resuscitation, and rib fractures. Pediatrics 1984;73:339–342. 23 Spevak MR, Kleinman PK, Belanger PL, Primack C, Richmond JM: Cardiopulmonary resuscitation and rib fractures in infants: a postmortem radiologicpathologic study. JAMA 1994;272:617–618.

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24 Barsness KA, Cha ES, Bensard DD, et al: The positive predictive value of rib fractures as an indicator of nonaccidental trauma in children. J Trauma 2003; 54:1107–1110. 25 Schweich P, Fleisher G: Rib fractures in children. Pediatr Emerg Care 1985;1:187–189. 26 Thomas PS: Rib fractures in infancy. Ann Radiol (Paris) 1977;20:115–122. 27 Kleinman PK, Marks SC, Blackbourne B: The metaphyseal lesion in abused infants: a radiologic-histopathologic study. AJR Am J Roentgenol 1986;146: 895–905. 28 Kleinman PK, Marks SC Jr: A regional approach to the classic metaphyseal lesion in abused infants: the proximal tibia. AJR Am J Roentgenol 1996;166:421– 426. 29 Kleinman PK, Belanger PL, Karellas A, Spevak MR: Normal metaphyseal radiologic variants not to be confused with findings of infant abuse. AJR Am J Roentgenol 1991;156:781–783. 30 Kleinman PK, Kwon DS: Differential diagnosis IV: normal variants; in Kleinman PK (ed): Diagnostic Imaging of Child Abuse. St Louis, Mosby, 1998. 31 Kwon DS, Spevak MR, Fletcher K, Kleinman PK: Physiologic subperiosteal new bone formation: prevalence, distribution, and thickness in neonates and infants. AJR Am J Roentgenol 2002;179:985– 988. 32 Beals RK, Tufts E: Fractured femur in infancy: the role of child abuse. J Pediatr Orthop 1983;3:583– 586. 33 Coffey C, Haley K, Hayes J, Groner JI: The risk of child abuse in infants and toddlers with lower extremity injuries. J Pediatr Surg 2005;40:120–123. 34 Leventhal JM, Thomas SA, Rosenfield NS, Markowitz RI: Fractures in young children: distinguishing child abuse from unintentional injuries. Am J Dis Child 1993;147:87–92.

35 Merten DF, Radkowski MA, Leonidas JC: The abused child: a radiological reappraisal. Radiology 1983;146:377–381. 36 Shaw BA, Murphy KM, Shaw A, Oppenheim WL, Myracle MR: Humerus shaft fractures in young children: accident or abuse? J Pediatr Orthop 1997; 17:293–297. 37 Strait RT, Siegel RM, Shapiro RA: Humeral fractures without obvious etiologies in children less than 3 years of age: when is it abuse? Pediatrics 1995;96: 667–671. 38 Thomas SA, Rosenfield NS, Leventhal JM, Markowitz RI: Long-bone fractures in young children: distinguishing accidental injuries from child abuse. Pediatrics 1991;88:471–476. 39 Johnson K, Fischer T, Chapman S, Wilson B: Accidental head injuries in children under 5 years of age. Clin Radiol 2005;60:464–468. 40 Islam O, Soboleski D, Symons S, Davidson LK, Ashworth MA, Babyn P: Development and duration of radiographic signs of bone healing in children. AJR Am J Roentgenol 2000;175:75–78. 41 Prosser I, Maguire S, Harrison SK, Mann M, Sibert JR, Kemp AM: How old is this fracture? Radiologic dating of fractures in children: a systematic review. AJR Am J Roentgenol 2005;184:1282–1286. 42 Yeo LI, Reed MH: Staging of healing of femoral fractures in children. Can Assoc Radiol J 1994;45: 16–19. 43 Mathew MO, Ramamohan N, Bennet GC: Importance of bruising associated with paediatric fractures: prospective observational study. BMJ 1998; 317:1117–1118.

Karl Johnson Birmingham Children’s Hospital Steelhouse Lane Birmingham B4 6NH (UK) E-Mail [email protected]

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Chapter 15 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 246–280

Case Histories Sasha Howarda  Gillian Lyderb  Jeremy Allgrovea  Nick Shawb a

Barts and the London NHS Trust, London, and bBirmingham Children’s Hospital NHS Foundation Trust, Birmingham, UK

Abstract The conditions related to abnormalities of calcium and bone metabolism are large in number and cover problems of hypocalcaemia, hypercalcaemia, primary and secondary osteoporosis, rickets resulting from both vitamin D and phosphate metabolic disorders, and a series of miscellaneous conditions. Included in this chapter is a series of 36 cases drawn from our clinics and from colleagues who have presented these clinical problems at recent Advanced Courses in Paediatric Bone and Calcium Metabolism run by the British Paediatric and Adolescent Bone group. The series of cases is not fully comprehensive but is designed to cover the major aspects of bone and calcium related disorders and can be added to online in due course. Copyright © 2009 S. Karger AG, Basel

As we have indicated earlier, many aspects of this book are based on the Advanced Course in Paediatric Bone and Calcium Metabolism. A feature of this course is to ask delegates to bring a case to present which is relevant to one of the main themes of the course. These cases are an important aspect of the course as they illustrate many of the disorders presented in the lectures, the different modes of presentation and the issues involved in establishing the correct diagnosis and treatment. We therefore felt it was important to include a chapter of case histories in this book for similar reasons. Each case is a genuine case and is identified in the text of the individual chapters and some cases are referred to in more than one chapter. At the end of each case description, a series of ‘Key Learning Points’ is presented.

Although most of the cases have been obtained from our own clinics we are grateful to a number of additional individuals who have sent us their cases for inclusion. These include Dr. Caroline Brain, Dr. Wolfgang Högler, Professor Michael Patton, Dr. Fiona Ryan, and Dr. Birgit van Meijgaarden. In each case, the informed consent of the parents has been obtained to include details on their children to whom we are extremely grateful.

Cases Presented Hypocalcaemia Case 1: Autosomal-recessive isolated hypoparathyroidism Case 2: Familial autosomal-recessive hypoparathyroidism Case 3: Primary hypoparathyroidism secondary to 22q deletion (DiGeorge) syndrome Case 4: Hypoparathyroidism associated with KearnsSayre spectrum Case 5: Primary hypomagnesaemia Case 6: Autosomal-dominant hypocalcaemia Case 7: Autosomal-dominant hypocalcaemia Case 8. Pseudohypoparathyroidism type 1a Case 9: Pseudohypoparathyroidism type 1a presenting with soft tissue calcification (osteoma cutis and progressive osseous heteroplasia) Case 10: Pseudohypoparathyroidism type 1b (PHP1b) Hypercalcaemia Case 11 Primary hyperparathyroidism Case 12 Hyperparathyroidism jaw tumour syndrome Case 13 Tertiary hyperparathyroidism

Case 14 Neonatal severe hyperparathyroidism Case 15 Subcutaneous fat necrosis Rickets Case 16 Congenital rickets Case 17 Dilated cardiomyopathy secondary to vitamin D deficiency Case 18 Classical vitamin D deficiency rickets Case 19 Hypocalcaemic tetany secondary to vitamin D deficiency Case 20 Limb pain and weakness Case 21 Vitamin D-dependent rickets type I (1-alpha hydroxylase deficiency) Case 22 Vitamin D-dependent rickets type II (1,25(OH)2D end-organ resistance) Case 23 X-linked dominant hypophosphataemic rickets Case 24 Hypophosphataemic rickets secondary to an epidermal naevus Osteoporosis Case 25 Osteogenesis imperfecta type I Case 26 Osteogenesis Imperfecta type IV Case 27 Leukaemia presenting as osteoporosis Case 28 Osteoporosis pseudoglioma syndrome Case 29 Bruck syndrome (osteogenesis imperfecta with congenital joint contractures) Case 30 Idiopathic juvenile osteoporosis Miscellaneous Bone Disorders Case 31 Autosomal-dominant osteopetrosis Case 32 Infantile hypophosphatasia Case 33 Juvenile hypophosphatasia Case 34 Metaphyseal chondrodysplasia, Jansen type Case 35 Caffey’s disease Case 36 Cleidocranial dysplasia

Hypocalcaemia

Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.4–3.1) ALP, IU/l (<520) 25OHD, nmol/l (50–200) PTH, pmol/l (1.1–6.8) Mg, mmol/l (0.7–1.0)

At initial presentation

On representation

0.9 5.2 383 74

1.5 4.5 263

0.7

0.3 0.8

Her seizures resolved after treatment with alfacalcidol in a dose of 30–50 ng/kg/day and oral calcium supplementation of 2 mmol/kg/day to normalise plasma calcium levels. Twenty-one months later, mother gave birth to a second child, a male infant born at term in good condition. At 5 days of age, he presented with neonatal seizures and was also found to be hypocalcaemic (1.36 mmol/l). Genetic studies revealed homozygosity for the same mutation in both siblings, a mutation in exon 2 of the GCM2 gene on chromosome 6p24.2 (C140G>T), consistent with a diagnosis of autosomal-recessive isolated hypoparathyroidism. Family Tree

Key Learning Points 1 A high plasma phosphate in the presence of hypocalcaemia suggests a problem with PTH secretion or action. 2 Serum PTH should be checked whenever there is hypocalcaemia.

Case History 1 Case History 2 Autosomal-Recessive Isolated Hypoparathyroidism A female infant presented with neonatal seizures at 9 days of life secondary to hypocalcaemia. She had been born at 38 weeks after an uneventful pregnancy with a birth weight of 3.2 kg. Parents were not consanguineous but there was a history of mother having had transient hyperparathyroidism during pregnancy, and the patient’s low plasma calcium was initially felt to be secondary to this. However, despite being started on oral calcium treatment she represented at five weeks of age with further hypocalcaemic seizures.

Case Histories

Familial Autosomal-Recessive Hypoparathyroidism A 4-year-old boy presented to A&E with seizures after having relocated to a new area. His mother gave a history of seizures from the age of 1 year. He was reported to be clinically well between fits. Initial biochemistry revealed hypocalcaemia and he was treated with intravenous calcium which terminated the seizures. Further history from his previous hospital revealed multiple hypocalcaemic seizures with a diagnosis of hypoparathyroidism and non-compliance with treatment.

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On presentation after relocation Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.25–2.1) ALP, IU/l (<520) PTH, pmol/l (1.1–6.8) 25OHD, mmol/l (50–200) Mg, mmol/l (0.7–1.0) Urine Ca:Creat, mmol/mmol (0.06–0.74)

1.78 3.4 363 <0.32 31 0.8 0.1

He had been born at term via emergency Caesarian section with a birth weight of 3.3 kg. There were no developmental concerns and he was growing well. On examination, he had no dysmorphic features and systemic examination was entirely normal. Both parents were of Bangladeshi origin and were first cousins, with grandparents also first cousins. Family Tree

On regular Mother Maternal treatment (untreated) uncle (untreated) Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (0.8–1.5) ALP, IU/l (<520) PTH, pmol/l (1.1–6.8) 25OHD, nmol/l (30–164) Mg, mmol/l (0.7–1.0) Urine Ca:Creat, mmol/mmol (0.06–0.74)

2.51

1.36

1.22

1.44

2.7

3.00

177

212 –

0.6

155

39

59

0.79

0.7

1.4

same homozygous mutation of the GCM2 gene in the mother and maternal uncle. Key Learning Points 1 A detailed family history of other individuals with hypocalcaemia can lead to a rapid diagnosis. 2 Autosomal-recessive conditions are not necessarily confined to one generation within a family if consanguinity occurs in several generations. Case History 3

His mother was known to be hypocalcaemic but was on no regular treatment. The patient’s maternal uncle had presented aged 1 year with recurrent febrile seizures. Hypocalcaemia was discovered at the 7th seizure and a CT head showed scattered foci of calcification including the lentiform nuclei. His calcium normalised and seizures stopped after treatment with alfacalcidol was commenced. The patient was reviewed by the consultant who had previously looked after his maternal uncle, and a diagnosis of familial hypoparathyroidism was made. He was started on alfacalcidol at 50–100 ng/kg/day, Calcium Sandoz 1.5 mmol/kg/day and colecalciferol 6,000 IU/ day. Genetic studies showed the child to be homozygous for a nucleotide substitution (c.328C>T) in exon 2 of the GCM2 gene on chromosome 6p 24.2. Studies showed the

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Primary Hypoparathyroidism Secondary to 22q Deletion (DiGeorge) Syndrome (#188400) A teenage boy aged 13.6 years presented with sudden collapse and secondary respiratory arrest. He went on to have generalised seizures and hypocalcaemia was discovered on initial biochemistry.

On presentation Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (0.8–1.5) Mg, mmol/l (0.7–1.0) PTH, pmol/l (1.1–6.8) 25OHD, nmol/l (50–200) Urine Ca:Creat, mmol/mmol (0.06–0.74)

1.52 3.08 0.72 1.4 51 0.03

Howard  Lyder  Allgrove  Shaw

b

a

Fig. 1. Case 3. Lateral X-rays of spine in a boy of 13 who presented with hypocalcaemic seizures associated with 22q11 deletion syndrome. His mother observed that he lost height following the seizures and the X-ray shows vertebral collapse (a) that presumably occurred at the time. The subsequent X-ray (b) shows remodelling of the vertebrae, shown by the arrows.

He was noted to have swollen optic discs with retinal haemorrhages, although a CT head was normal. In addition, a lateral spine X-ray revealed collapse of several thoracic vertebrae (fig. 1) and a DEXA scan revealed low bone density. On further questioning, significant problems in his past medical history were discovered. These included: • Jitteriness at birth with a hoarse cry • Repeated episodes of croup-like illness after age 1 year • Cleft palate and hypernasal speech • Middle ear problems • Gastro-oesophageal reflux and constipation • One-year history of back pain • Acute loss of height at the time of his first convulsion (fig. 2) • Poor dentition, nail biting, trichotillomania and teeth grinding

Case Histories

•

Learning and behavioural difficulties – dyslexia and dyspraxia (behaviour seemed to improve during the summer months) Examination revealed dysmorphic features including long, narrow palpebral fissures slanting medially upwards, and abnormally formed low set ears. His height was <25th centile with weight >97th centile. In view of the clinical picture of hypoparathyroidism in combination with dysmorphism, genetic testing was done which showed a heterozygous 22q11 deletion. Because of the vertebral collapse, treatment with rPTH 1–34 was initially considered but rejected because of concerns about possible osteosarcoma formation. He responded well to oral treatment with alfacalcidol 12–18 ng/kg/day and calcium supplements (Calcium Sandoz 600 mg q.d.s.), with subsequent resolution of his optic abnormalities. He also required palatal surgery and speech and language therapy.

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200 * 180

160

140 Loss of height documented by mother 120

100

80

Fig. 2. Case 3. Growth chart of 13-year-old boy who presented with hypocalcaemic convulsions associated with 22q11 deletion syndrome. Following the seizures, his mother reported that he had lost height, which had occurred as a result of vertebral collapse.

60

40 0

Biochemistry following oral treatment:

Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (0.8–1.5) ALP, IU/l (<520) PTH, pmol/l (1.1–6.8) 25OHD, nmol/l (50–200)

250

On calcium supplements

On calcium and alfacalcidol

2.25

2.11

2.07

1.61 168 1.6 164

2

4

6

8

10

12

14

16

18

20

22

His vertebral collapse improved on treatment with alfacalcidol (fig. 1). Key Learning Points 1 If a child presents with hypocalcaemic convulsions, a detailed history may give a clue to the diagnosis. In retrospect, symptoms had been present for several years. 2 Prolonged hypoparathyroidism may result in low bone density because of the loss of the anabolic effect of physiological PTH levels. 3 Alfacalcidol is effective in reversing the hypocalcaemia and vertebral collapse. 4 Prolonged hypocalcaemia can result in the appearances of papilloedema without raised intracranial pressure.

Howard  Lyder  Allgrove  Shaw

Case History 4 Hypoparathyroidism Associated with Kearns-Sayre Spectrum (#530000) A 2-year-old girl presented with ptosis. She subsequently developed a right sided sixth nerve palsy which has left her with a right convergent squint. She had been born normally at term and was well otherwise but, at the age of 4.5 years, she complained of tingling in the hands and developed tetany. She was found to have hypocalcaemia. Vitamin D levels were normal, but PTH was low. A diagnosis of hypoparathyroidism was made and she commenced treatment with alfacalcidol and oral calcium supplements which have maintained her serum calcium at the lower end of the normal range with no evidence of hypercalciuria. There is no relevant family history. She has an older sister and older half sister both of whom are well, as is her mother. Her father died at the age of 41 of coronary heart disease associated with hypertension and hypercholesterolaemia. The parents are not consanguineous. On examination she had a right ptosis and right convergent squint. Her height was on the 25th centile together with some mild dysmorphic features. She had a history of learning difficulties and speech delay but no difficulties with swallowing or speech difficulties. A diagnosis of 22q11 deletion syndrome was considered but excluded by negative FISH analysis for 22q11. She was subsequently found to have a >5-kb deletion within her mitochondrial DNA that incorporates a number of respiratory chain enzyme subunits. She does not have either a DNA duplication, large scale rearrangement of DNA or the commonest point substitution of m.3243A>G. The finding is consistent with Kearns-Sayre syndrome. Her mother does not have the same mutation. Key Learning Points 1 Idiopathic hypoparathyroidism is not usually associated with focal neurological signs. 2 The presence of these should alert one to the possibility of a mitochondrial disorder. 3 Although mitochondrial disorders are inherited via the maternal line, many arise as de novo mutations. 4 Treatment of the hypoparathyroidism is undertaken as for any other form of hypoparathyroidism but the neurological and learning difficulties may require additional treatment. Case History 5 Primary Hypomagnesaemia (#602014) An Asian baby girl presented at 5 weeks of age with a history of three generalised shaking episodes. She had been previously well. She had been a normal term delivery

Case Histories

with a birth weight of 3.49 kg. She was bottle-fed and gaining weight well. She was the third child of her parents who were first cousins. Her initial plasma calcium was 1.67 mmol/l and she was felt to be having hypocalcaemic seizures and was started on calcium supplements. She subsequently developed diarrhoea and vomiting and repeat bloods were as follows:

Corr Ca, mmol/l (2.2–2.6) PO4, mmol/l (1.25–2.1) ALP, IU/l (250–1,000) Mg, mmol/l (0.7 – 1.0) Urine Mg:Creat, mmol/mmol (0.18–1.05)

1.72 2.46 518 <0.08 0.75

She had no features of rickets and her renal function tests, 25OHD and PTH levels were normal. A diagnosis of primary hypomagnesaemia was made. In view of the urine magnesium excretion being within the normal range it was likely she had the type that is due to a selective defect in intestinal absorption due to a mutation in the TRPM6 gene. She was treated with four doses of intramuscular magnesium sulphate and was subsequently discharged home on oral magnesium glycerophosphate taken three times daily. Although this has not normalised her plasma magnesium, which remains at 0.5 mmol/l, this is high enough to maintain her plasma calcium in the normal range. Key Learning Points 1 Although a rare condition, the possibility of hypomagnesaemia should be considered in all infants presenting with hypocalcaemia 2 The urine magnesium/creatinine ratio is a simple investigation to distinguish between an intestinal absorption defect and a renal tubular magnesium leak. 3 The hypocalcaemia is secondary to the hypomagnesaemia since the latter interferes with PTH secretion. 4 Systemic magnesium may be required to correct the hypomagnesaemia initially. 5 Magnesium glycerophosphate causes fewer gastrointestinal side effects than other magnesium salts.

251

Case History 6 Autosomal-Dominant Hypocalcaemia (#146200) A preterm baby born at 31 weeks’ gestation developed respiratory distress at birth and was ventilated for 24 h. She was noted to be hypocalcaemic on day 2 with the following results: On presentation Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.25–2.1) ALP, IU/l (<520) PTH, ng/l (1.1–6.8) Mg, mmol/l (0.7–1.0)

0.7 4.47 363 5 0.59

Chromosomes for 22q deletion were negative. Her mother had been diagnosed with primary hypoparathyroidism in childhood and had subsequently developed nephrocalcinosis and renal failure in pregnancy. The baby was treated with calcium supplements and alfacalcidol. She had multiple admissions over the next 2 years with episodes of hypocalcaemia and hypercalcaemia and was subsequently seen by a neurologist for developmental delay and seizures and was given the diagnosis of familial hypoparathyroidism. She developed nephrocalcinosis and biliary calculi at 2 years of age with subsequent referral to a nephrologist and hepatologist (fig. 3, 4). She was treated with bendrofluazide, potassium citrate and ursodeoxycholic acid to help reduce her renal and biliary calcification. At the age of 3 years she was noted to have a high urinary calcium/ creatinine ratio (1.1 mmol/mmol) and low plasma magnesium of 0.59 mmol/l. Her initial diagnosis was reviewed and it was felt that she could have autosomal-dominant hypocalcaemia due to an activating mutation in the calcium-sensing receptor gene. She had molecular genetic analysis and was found to have a C>A point mutation in exon 7 of the gene for the calcium-sensing receptor.

Fig. 3. Case 6. Ultrasound scan of liver showing a stone in the gall bladder.

Fig. 4. Ultrasound scan of kidney showing extensive nephrocalcinosis.

3 Treatment with vitamin D analogues such as alfacalcidol is difficult in this condition due to persistent hypercalciuria. 4 Asymptomatic affected individuals should not receive treatment to normalise the plasma calcium. 5 Theoretically rPTH1–34 might be of value in this condition. Case History 7

Key Learning Points 1 Hypocalcaemia with a high urine calcium excretion should suggest the diagnosis of autosomaldominant hypocalcaemia. 2 Plasma magnesium is often slightly low in this condition.

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Autosomal-Dominant Hypocalcaemia (#146200)[] A female infant presented with neonatal seizures at day 5 of life. She had been a normal term delivery with a birth weight of 3.2 kg. On examination she appeared well with no clinical evidence of rickets. Biochemistry showed hypocalcaemia:

Howard  Lyder  Allgrove  Shaw

At presentation Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.4 – 3.1) ALP, IU/l (<520) PTH, pg/ml (10–50) 25OHD, nmol/l (50–200) Mg, mmol/l (0.7–1.0) Urea and electrolytes Urine Ca:Creat ratio, mol/mmol (0.06–0.74)

1.66 2.91 376 35 15 0.88 normal 1.7

She was treated initially with intravenous calcium and commenced on colecalciferol 3,000 IU/day for 3 months.

Case History 8 Pseudohypoparathyroidism Type 1a (#103580) A 3.8-year-old girl presented with global developmental delay with normal vision and hearing. She was noted to have short stature and round facies with broad, short fingers. Baseline investigations revealed hypocalcaemia with a raised PTH level. She had been reviewed aged ten months due to maternal concerns that her development was poor and she looked like her two older brothers. However, no dysmorphic features were found and thyroid function was normal so she was not investigated further. On examination she had round facies, brachydactyly and shortened 4th metacarpals. Her weight was 15.4 kg (25th to 50th centile) and height 94 cm (10th centile). Her biochemistry is as shown in the table.

Family Tree

Parents were not consanguineous but the family reported that the paternal grandmother was being treated with oral calcium supplementation for hypocalcaemia of unknown cause. The child’s father was therefore examined, and found to have clinical features of hypocalcaemia including positive Chvostek’s and Trousseau’s sign, brisk reflexes and poor motor function. He was hypocalcaemic on investigation. Genetic studies showed her to be heterozygous for a novel mutation in the CaSR gene on chromosome 3q13.3-q21 (c2492A>C), consistent with a diagnosis of autosomal dominant hypocalcaemia. She was started on alfacalcidol in a dose of 25 ng/kg/day and oral calcium supplementation of 3 mmol/kg/day, which has normalised her biochemistry and she has had no further symptoms. Key Learning Points 1 In a neonate presenting with hypocalcaemia enquire about a family history and consider checking plasma calcium in both parents. 2 A serum PTH within the normal range in the presence of hypocalcaemia often suggests a problem of the calcium sensing receptor.

Case Histories

3.8 years 4.9 years 8.4 years (on (on presen- (on alfa- alfacalcidol tation) calcidol) and thyroxine) Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.25–2.1) ALP, IU/l (<520) PTH, pmol/l (1.1–6.8) TSH, IU/l (<4) FT4 (11–24) Urea and electrolytes Urine Ca:Creat, mol/mmol (0.06–0.74)

1.72

2.05

2.42

2.52

2.60

1.76

227 52.3 2.8 11.7 normal

191 19.6 6.8 9.4

0.9 19.1

<0.24

She was treated with alfacalcidol 25–40 ng/kg/day with normalisation of her plasma calcium. At 4.9 years she was noted to be hypothyroid and was started on thyroxine. She developed ongoing behavioural problems and has required extra help with schooling. At 12.1 years, she underwent a glucagon stimulation test which revealed growth hormone deficiency and she was commenced on growth hormone treatment for 5 years. She has two older brothers who are known to have PHP1a with hypothyroidism. Mother has phenotypic features of Albright’s hereditary osteodystrophy.

253

She was found to have a heterozygous missense mutation (F246S) in exon 10 of GNAS1 gene (Chrom 20q13.2). Key Learning Points 1 Pseudohypoparathyroidism type 1a is often associated with a history of previous concerns about developmental delay. 2 It is caused by mutations in the coding sequence of the Gsα gene, but only if inherited from the mother. 3 Other forms of hormone resistance, particularly to TSH, due to receptors that require the Gsα subunit, often occur in this type of pseudohypoparathyroidism. Case History 9 Pseudohypoparathyroidism Type 1a Presenting with Soft Tissue Calcification (Osteoma Cutis and Progressive Osseous Heteroplasia) (#103580) A female infant was referred with a soft tissue swelling over the ankle at 7 months of age. She had been a normal term delivery with a birth weight of 3.6 kg. She was under investigation for developmental delay and was being treated with thyroxine for acquired hypothyroidism. There was no family history of note and parents were not consanguineous. On examination she had soft tissue swelling over her ankles and knees, more on the right than left with restricted movements at those joints. No skeletal abnormalities were detected. She was noted to be hypocalcaemic on initial investigation. X-rays of the knees and ankles confirmed the presence of calcified tissue (fig. 5).

At presentation Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.25–2.1) ALP, IU/l (<520) PTH, pmol/l (1.1–6.8)

1.96 2.24 262 35.8

In view of the presenting features, a provisional diagnosis of pseudohypoparathyroidism type 1a was made. This was confirmed by genetic studies, which showed a heterozygous mutation in the GNAS1 gene on chromosome 20q13.2. Surgical resection of the bony lesions showed that the tissue was partially differentiated bone but no significant improvement in function was noted.

254

Fig. 5. Case 9. X-rays of limb in progressive osseous heteroplasia associated with GNAS mutation and mild pseudohypoparathyroidism type Ia. There is extensive soft tissue calcification of the lower leg particularly around the ankle and knee. Most of the calcification is on the medial aspect but there is also a small amount around the fibula.

She was commenced on alfacalcidol in a dose of 65 ng/kg/day and had surgical removal of the soft tissue lesions. A trial of calcium carbonate (as a phosphate binder) is in progress. Key Learning Points 1 Pseudohypoparathyroidism can present early in life with subcutaneous calcification (osteoma cutis) and hypothyroidism. 2 The soft tissue calcification is made up of differentiated bone. 3 Surgery does little to improve matters.

Howard  Lyder  Allgrove  Shaw

Case History 10 Pseudohypoparathyroidism Type 1b (PHP1b) (#603233) A 13.1-year-old male presented with his first tonic-clonic seizure. His past medical history included only mild asthma with a normal birth and developmental history. He has normal intelligence and is currently a medical student. Family history was also unremarkable and parents were not consanguineous. Family Tree

He had a normal systemic examination and no dysmorphic features. His weight was 40.8 kg (25th centile) and height 147 cm (9th to 25th centile). On initial investigations biochemistry revealed hypocalcaemia with hyperphosphataemia.

At presentation Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (0.8–1.5) ALP, IU/l (<520) PTH, pmol/l (1.1–6.8) 25OHD, nmol/l (30–164) Mg, mmol/l (0.7–1.0) Urea and electrolytes Urine Ca:Creat ratio, mmol/ mmol (0.06–0.74)

1.27 3.1 174 32 60 0.76 normal 0.01

A CT brain revealed bilateral basal ganglia calcification. He was treated with intravenous calcium to return his serum calcium to the normal range, and then commenced on alfacalcidol at 25 ng/kg/day with oral Calcium Sandoz at 100 mg/kg/day (2.7 mmol/kg/day) to maintain his normalised biochemistry. He went on to have genetic studies which revealed a GNAS1 epimutation at Chromosome 20q13 (mosaic methylation abnormality at NESP55, leading to disrupted normal methylation within the GNAS locus), consistent with a diagnosis of pseudohypoparathyroidism type 1b.

Case Histories

Key Learning Points 1 Symptomatic hypocalcaemia in the presence of raised PTH and normal 25OHD suggests a diagnosis of pseudohypoparathyroidism. 2 This condition can be erroneously diagnosed if vitamin D deficiency is present. 3 The presence of normal intelligence and lack of dysmorphic features suggests that this may be associated with type 1b rather than type 1a. 4 In type 1b, there is no mutation in the coding region of the Gsα gene, but variable epimutations are present. 5 Although in this case no features other than the hypocalcaemia were found, evidence is now accumulating that some patients with type 1b disease may show mild features of type 1a as well.

Hypercalcaemia Case History 11 Primary Hyperparathyroidism A 9-year-old girl presented with abdominal pain and vomiting. She was diagnosed with acute pancreatitis and during initial investigations she was found to be hypercalcaemic. Corr Ca mmol/l (2.15–2.65) PTH ng/l (11–35)

3.52 75

She was treated with intravenous fluids and a single dose of intravenous pamidronate 0.5 mg/kg which normalised her plasma calcium. At subsequent follow-up her plasma calcium ranged between 2.79 and 2.99 mmol/l and serum PTH between 111–148 pg/ml. In view of the diagnosis of primary hyperparathyroidism the following further investigations were performed: Mother’s plasma calcium and serum PTH Genetic analysis for MEN1 Renal ultrasound Sestamibi scan

Ultrasound of neck (fig. 6)

– normal – negative – normal – no parathyroid adenoma – welldefined mass lower left thyroid lobe suggestive of parathyroid adenoma.

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At presentation Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (0.8–1.5) ALP, IU/l (<520) PTH, pmol/l (1.1–6.8) Urea and electrolytes Urine Ca:Creat, mol/mmol (0.06–0.74)

Fig. 6. Ultrasound scan of neck with parathyroid adenoma in left lower thyroid lobe.

Subsequent Sestamibi scan

– confirmed an adenoma in this location

She subsequently had surgical removal of an adenoma of the lower left parathyroid gland weighing 0.321 g. Her plasma calcium and serum PTH then returned to normal. Key Learning Points 1 Hypercalcaemia needs to be considered as a cause for pancreatitis. 2 Primary hyperparathyroidism in a child may be the first manifestation of multiple endocrine neoplasia type 1. Case 12 Hyperparathyroidism Jaw Tumour Syndrome (#145001) A 15-year-old male presented with a first episode of renal colic. Imaging revealed a kidney stone and his serum calcium was grossly elevated on biochemistry: He was found to have a normal systemic examination with weight 52 kg (25–50th centile) and height 165 cm (10–25th centile).

256

3.73 0.81 548 7.8 normal 0.88

His mother was known to have primary hyperparathyroidism with renal stones, and she had undergone a total parathyroidectomy as treatment. Family history revealed familial hyperparathyroidism in several relatives in the maternal family (fig. 7). In view of his mother’s medical history the child’s bone profile had been checked aged seven years, and at that time he was found to be normocalcaemic. He had no past history of fractures and no other past medical history of note. Family Tree (see fig. 7 on next page.) In view of his presentation and a family history of hyperparathyroidism, he went on to have a radio-isotope scan of his parathyroids, which showed a tumour in the lower left gland. He therefore underwent a total parathyroidectomy and post-operatively required alfacalcidol, initially at 30 ng/kg/day and then weaned to 20 ng/kg/day. He also required oral calcium supplements for 1 year postoperatively. PrePostThree years parathyroi- parathyroi- post-paradectomy dectomy thyroidectomy Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (0.8–1.5) ALP, IU/l (<520) PTH, pmol/l (1.1–6.8) Urea and electrolytes Urine Ca:Creat (0.06–0.74)

4.29

2.72

2.43

1.08

1.36

1.83

102 <0/7

0.3

normal 0.24

Howard  Lyder  Allgrove  Shaw

Hyperparathyroid PT Carcinoma Jaw lesions Presumed affected Renal lesion

Fig. 7. Case 12. Family tree of HYP-JT syndrome. It can be seen that several generations of this family have been affected by a range of problems including parathyroid carcinoma, parathyroid adenoma, jaw tumours and renal cysts.

Genetic studies showed him to be heterozygous for 15 bp deletion from exon 3 to intron 3 of the HRPT2 gene on chromosome 1q21–q31 (germline mutation). The same mutation was subsequently found in his mother and a maternal cousin. This mutation is consistent with hyperparathyroidism jaw tumour syndrome and he has therefore been monitored for jaw and renal tumours with ongoing monitoring of his calcium levels. Key Learning Points 1 Hypercalcaemia associated with raised PTH and raised urine calcium excretion is diagnostic of primary or tertiary hyperparathyroidism. 2 In this case, the family history was suggestive of familial primary hyperparathyroidism. 3 Genetic analysis confirmed the diagnosis of HYP-JT syndrome. 4 These patients need continuous monitoring for the development of jaw and renal tumours.

Case Histories

Case History 13 Tertiary Hyperparathyroidism A teenage boy of African descent presented with ‘knock knees’ aged 13 years. His mother gave a history of significant bowing of his legs as a toddler, but an otherwise unremarkable past medical history. He was reported to be otherwise well in himself and on no medications. There was no family history of note and no consanguinity. Family Tree

On examination, he had genu valgum of his knees bilaterally with a rachitic rosary and bilateral wrist swelling. There were no dysmorphic features and weight was

257

52.7 kg (25th to 50th centile) with height at 155.8 cm (25th centile). Initial biochemistry showed that he was normocalcaemic although his vitamin D levels were low. The latter was treated with vitamin D 10,000 IU/day following which he became hypercalcaemic and features of hyperparathyroidism were revealed:

Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (0.8–1.5) Alkaline phosphatase, IU/l (<520) PTH, pmol/l (1.1–6.8) 25OHD, nmol/l (50–164) Urine Ca:Creat (0.06–0.74)

Initial investigations

After treatment with vitamin D

Before surgery

2.58

3.27

3.12

0.67

1.14

0.97

2260

1700

978

84

49.8

44.9

11

34 0.79

In view of this, a radioisotope scan was performed which localised a parathyroid adenoma which was thought to have developed secondarily to long-standing nutritional vitamin D deficiency. Being of West African origin, it is not known what part calcium deficiency may also have played. He underwent a partial parathyroidectomy to remove the parathyroid adenoma and was started on colecalciferol 3,000 IU/day for 3 months followed by vitamin D 400 IU/day. Following this, he remains normocalcaemic. Key Learning Points 1 In tertiary hyperparathyroidism caused by long standing vitamin D deficiency, hypercalcaemia may be masked by continuing low levels of vitamin D. Once these are corrected, hypercalcaemia may be revealed. 2 Hypercalcaemia with a non-suppressed serum PTH suggests primary or tertiary hyperparathyroidism. 3 Prolonged vitamin D (and/or calcium) deficiency can lead to the development of a parathyroid adenoma.

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Case History 14 Neonatal Severe Hyperparathyroidism (#239200) An Asian baby girl who was a normal term delivery presented at 9 days of age with a history of poor feeding and was noted to be hypotonic and oxygen-dependent. Her initial blood tests were as follows:

Corr Ca, mmol/l (2.2–2.6) PO4, mmol/l (1.5–2.5) PTH, ng/l (11–35) Urine Ca:Creat ratio, mmol/mmol (<1.5)

6.97 1.14 675 19.2

X-rays of wrist and knee showed extensive demineralisation and metaphyseal fractures (fig. 8). Her parents are first cousins. Her mother’s plasma calcium level was 2.54 mmol/l and her father’s plasma calcium 2.34 mmol/l. A diagnosis of severe neonatal hyperparathyroidism was made. Both parents were subsequently found to be heterozygous for a mutation of the calcium-sensing receptor and the girl is homozygous for the mutation. She was treated with intravenous hyperhydration (200 ml/kg/day) and two doses of intravenous pamidronate (0.5 mg/kg). Her calcium decreased over the next week and her oxygen requirement resolved. Her plasma calcium began to rise again and she was started on a low calcium formula feed. An ultrasound of her neck showed a right-sided parathyroid adenoma which was subsequently removed. However, her hypercalcaemia did not resolve and she remained on low calcium feeds to maintain normal plasma calcium levels. She subsequently required further parathyroid surgery with removal of all the remaining glands. She was started on alfacalcidol to maintain normal plasma calcium. Key Learning Points 1 The plasma calcium is sometimes grossly elevated. 2 Intravenous pamidronate can be used in this condition to lower life-threatening hypercalcaemia and provide some time prior to surgery. 3 Total parathyroidectomy is required for definitive treatment of this condition. 4 Following surgery, massive amounts of calcium may need to be infused to prevent symptomatic hypocalcaemia until the bones begin to heal.

Howard  Lyder  Allgrove  Shaw

Fig. 8. X-ray of wrist and knee at diagnosis showing demineralisation and metaphyseal fractures of the distal femur and proximal tibia.

Case History 15 Subcutaneous Fat Necrosis A male infant, born by emergency Caesarean section for poor CTG and reduced fetal movements, was referred during the 2nd week of life for hypercalcaemia. He had been born in poor condition with acidotic cord gases, and Apgars of 41/75/1010. Grade 1 hypoxic-ischaemic encephalopathy was diagnosed and he was treated with broadspectrum antibiotics for 1 week for presumed sepsis. A boggy, mobile swelling had been noted over his upper back from day 6 and several more swellings developed over his back, face and thighs (fig. 9). Biopsy confirmed a diagnosis of subcutaneous fat necrosis.

Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.4–3.1) ALP, IU/l (<520) PTH, pmol/l (1.1–6.8) Urine Ca:Creat ratio, mmol/mmol (0.06–0.74) U&E/FBC CRP (<5)

Case Histories

The infant was initially treated from day 11 with hyper-hydration. Despite this, the plasma calcium continued to rise and he received intravenous pamidronate at 0.5 mg/kg infused over 4 h on days 12, 13, 15, 26 and 27 of life. A renal ultrasound showed no evidence of nephrocalcinosis. Key Learning Points 1 A history of birth asphyxia often precedes the development of subcutaneous fat necrosis. 2 The mechanism for the hypercalcaemia in this condition is often due to unregulated synthesis of 1,25(OH)2D from macrophages that are present in the areas of fat necrosis. 3 Corticosteroids have also been used to control the hypercalcaemia in this condition

Day 6

Day 12 (on hyperhydration)

Day 13 (after 1st dose of pamidronate)

Day 25 (before 4th dose of pamidronate)

Day 28

2.62

3.15

2.78

3.07

2.73

2.08

2.16

1.61

2.71

1.92

133

97

95

106 2.5

109

N

N 15

N 10

1.11

N 130

N 34

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Fig. 9. Case 15. Photograph of thigh (a) and back (b) of child suffering from subcutaneous fat necrosis. Note the area of the thigh from which a biopsy had been taken.

a

b

Rickets and Vitamin D Deficiency

Initi- Day Day ally 20 21

Case History 16 Congenital Rickets An infant was born at 41 + 5 weeks’ gestation weighing 3.32 kg. The mother had suffered a fall onto her abdomen at 36 weeks and had some vaginal bleeding, abdominal discomfort and reduced fetal movements for 5 days. A fetal ultrasound was normal. She had been feeling more tired than in a previous pregnancy and there had been several stressful life events including the need to move into a hostel. The infant was born by face presentation, was floppy at birth and required some resuscitation. He was admitted to the local NICU and continued to require respiratory support in the form of CPAP. He was treated for sepsis despite negative markers. A chest X-ray revealed an abnormal ‘bell-shaped’ rib cage (fig. 11) and multiple fractures which were also present on X-ray of the humeri, tibiae and femora (fig. 10). The femora were bowed and the skull vault and vertebrae appeared undermineralised. Investigations showed (see following table): Further investigations including screening for GM1 gangliosidosis, Krabbe’s and metachromatic leukodystophies, infantile neuronal ceroid lipofuscinosis (INCL) and classical late INCL (INCL, CLN1, LINCL, CLN2), mucolipidosis I and II, mucoplysaccharidosis VII, α- and β-mannosidosis, Schindler’s disease, fucosidosis, GM2 gangliosidosis, aspartylglucosaminuria and Gaucher’s

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Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.4– 3.1) ALP, IU/l (<520) 25OHD, nmol/l (50–200) PTH, pmol/l (1.1– 6.8)

Day Day Day 27 35 43

2.07 2.26 2.33 2.17 2.52 2.38 1.27 1.28 1.34 1.45 1.79 1.92 324 384 833 <15 40 91.6

749 595 567 91

52.2 20.4 7.5

6.1

disease, were all negative. All of these conditions may give rise to skeletal abnormalities which can be confused with congenital rickets. Investigation of the mother showed: Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.4–3.1) 25OHD, nmol/l (50–200) PTH, pmol/l (1.1–6.8)

2.33 1.12 <15 6.0

Howard  Lyder  Allgrove  Shaw

4 The alkaline phosphatase level may not be particularly raised at the time of diagnosis, despite the grossly elevated PTH, but rises after the start of treatment as the bones begin to heal. 5 The PTH remained elevated, falling steadily, for 6 weeks after the start of treatment. Case History 17 Dilated Cardiomyopathy Secondary to Vitamin D Deficiency A 3-month-old male infant suffered a cardiorespiratory arrest following a 3-day viral illness. He had been previously well apart from mild prematurity (gestation 36 weeks) for which he had not required special care. He was fully breast-fed, with no vitamin supplementation for him or his mother. On presentation he had features of cardiac failure but no dysmorphic features. Biochemistry on presentation revealed severe hypocalcaemia: a

b Fig. 10. Case 16. X-rays of upper limbs before (a) and after 6 months of treatment (b) of a child with congenital rickets. There has been a marked improvement in mineralisation and clear healing of the rickets.

He was treated with oral vitamin D supplementation but continued to need respiratory support in the form of CPAP and remained in hospital for 4 months until respiratory function improved sufficiently to enable him to go home, still needing home oxygen which he continued to need until the age of 9 months. The mother was given stoss therapy in the form of 600,000 IU vitamin D intramuscularly. A chest X-ray taken 6 months after his initial admission showed considerable improvement in the shape of the chest and an increase in the lung volume consistent with the improvement in respiratory function (fig. 11). He continues to have some developmental delay although this is improving. Key Learning Points 1 Congenital rickets, like dilated cardiomyopathy (see below), is a potentially fatal consequence of severe vitamin D deficiency. 2 It requires a period of several months for the condition to recover. 3 It is a preventable disease.

Case Histories

Corr Ca, mmol/l (2.15– 2.65) PO4, mmol/l (1.2–2.1) ALP, IU/l (<520) PTH, pmol/l (1.1–6.8) 25OHD, nmol/l (50–200) Urea and electrolytes

On presentation

2 months after presentation (on treatment)

1.35

2.58

1.08 3391 unavailable 15 normal

1.48 939 12.7 148 normal

Echocardiogram studies showed dilated cardiomyopathy with a poorly functioning dilated left ventricle. He required ECMO and balloon atrial septostomy, ventilation and then CPAP over the following 2 months. He was treated with multiple diuretics, ACE inhibitor, digoxin and aspirin for cardiac management. He received colecalciferol for 10 months, and then changed to Dalivit (400 IU) daily for long-term vitamin D supplementation. Once his serum calcium normalised, cardiac function and growth improved significantly, although this required more than 1 year to do so. During much of this time he required continued treatment with diuretics.

261

Fig. 11. Case 16. Chest X-rays before (a) and after 6 months of treatment (b) of a child with congenital rickets. Note the ‘bell-shaped’ appearance of the rib cage and limited lung capacity at diagnosis. This has improved considerably by the end of six months. Note also the very ‘moth-eaten’ appearance of the upper humeral metaphysis on the first X-ray. This has improved a considerably in the second film.

b

Age

LVEDD, mm

LVESD, mm

FS (%) (NR >35%)

3 months 5 months 7 months 15 months

31 29 18 25

27 24 14 18

12 17–22 24 31

LVEDD = Left-ventricular end-diastolic diameter; LVESD = left-ventricular end-systolic diameter; FS = fractional shortening (LVEDD-LVESD expressed as a percentage of LVEDD).

Key Learning Points 1 Severe vitamin D deficiency can cause lifethreatening cardiomyopathy. 2 It is one of the few forms of dilated cardiomyopathy that is reversible. 3 Vitamin D levels should be routinely checked in infants with cardiomyopathy. 4 Breast-fed infants and their lactating mothers should receive vitamin D supplements. Case History 18 Classical Vitamin D Deficiency Rickets A 2.3-year-old African-Caribbean female presented to the Paediatric Department with bilateral wrist swelling. She had been a well child with a normal developmental history. She had been fully breast-fed until the age of 2 months, with solids introduced at 3 months. Neither child nor mother had taken supplemental vitamins. Her weight was 10.2 kg (2nd to 9th centile) and height 84 cm (9th to 25th centile). On examination she had bilateral swelling of wrists, knees and ankles. Her chest showed an obvious rachitic rosary (fig. 12).

262

The following biochemical results were found: PrePostMother Father treatment treatment Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.25–2.1) ALP, IU/l (<520) PTH, pmol/l (1.1–6.8) 25OHD, nmol/l (50–165) Urea and electrolytes Coeliac screen

1.9

2.34

1.05

2.08

2,315 79.4

600 5.0

4.3

5.7

<18

291

20

42

normal negative

X-rays showed the classical signs of rickets (fig. 13). She was treated with colecalciferol 3,000 IU daily for 3 months, followed by 400 IU daily with subsequent healing of her rickets. Key Learning Points 1 Breast-fed infants should receive vitamin D supplements. 2 Classical vitamin D deficiency rickets in the UK is usually seen in infants of African-Caribbean, South Asian or Middle Eastern ethnic origin.

Howard  Lyder  Allgrove  Shaw

Fig. 12. Case 18. Appearances of wrists (a) and chest (b) in classical rickets. In the latter note the presence of the ‘rickety rosary’ lateral and parallel to the costal margin.

a

b

Case History 19 Hypocalcaemic Tetany Secondary to Vitamin D Deficiency A 14-year-old male presented with a 6-month history of painful muscular spasms of his hands, feet, pectoral and abdominal muscles. He was noted to have a poor diet with no meat or vegetables and minimal dairy products. On examination he was noted to have positive Chvostek and Trousseau signs and pallor. His weight was 47.4 kg and height 165.9 cm (both 25th centile). Biochemistry showed hypocalcaemia with vitamin D deficiency: At presentation Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (0.8–1.5) a b ALP, IU/l (<520) Fig. 13. Case 18. X-ray appearances of wrist (a) and knee PTH, pmol/l (1.1–6.8) (b) in classical rickets. There is cupping and fraying of 25OHD, nmol/l (50–200) the metaphyseal margins of the radius and ulna, tibia, Mg, mmol/l (0.7–1.0) fibula and femur giving the so-called ‘champagne glass’ Urea and electrolytes appearance. FBC Thyroid function tests

Case Histories

1.46 1.67 420 25 <18 0.69 normal normal normal

263

His parents were non-consanguineous and there was no significant family history of note. Family Tree

He was treated with colecalciferol 6,000 IU daily for 3 months with oral calcium supplementation in a dose of 2 mmol/kg/day with resolution of his symptoms. At 3 months he was changed to vitamin D 400 IU daily. Key Learning Points 1 Vitamin D deficiency in adolescence often presents with hypocalcaemia rather than rickets. The alkaline phosphatase is not always raised. 2 A history of muscular spasms should prompt a check of the plasma calcium. 3 Treatment with intravenous calcium often gives instant relief of muscle spasm pain. Case History 20 Limb Pain and Weakness A 13-year-old Asian girl was originally seen by an orthopaedic surgeon with a 3-year history of pain in her feet and ankles with increasing difficulty in walking. This was labelled as ‘growing pains’. Her gait had become particularly abnormal over the previous year and she had not attended school for 6 months because of difficulty walking. She was a normal term delivery. She had beta-thalassaemia trait but had been well until 3 years ago. There was no history suggestive of an arthritic process. Her parents were second cousins and were fit and well, as were her 3 younger siblings. On examination she had an abnormal gait with trunk rotation. There was significant lower limb weakness and a positive Gower’s sign. An X-ray done of her pelvis showed evidence of osteomalacia with Looser’s zones (fig. 14). She had the following results of biochemical tests: Corr Ca, mmol/l (2.2–2.6) PO4, mmol/l (1.0–1.7) Alkaline phosphatase, IU/l (200–730) PTH, ng/l (13–29) 25OHD, nmol/l (50–165)

264

1.96 0.5 2,762 611 <17.5

Fig. 14. X-ray of Pelvis demonstrating Looser’s zones (pseudofractures) of the pubic rami.

She was started on ergocalciferol 10,000 units daily and calcium supplements 500 mg three times daily. She was referred to a neurologist because of her brisk lower limb reflexes and a positive Gower’s sign but it was felt her features were not consistent with a neurological condition. An MRI scan of her spine, EMG and nerve conduction studies were normal. Within 2 months of treatment, she came into the clinic walking stating ‘she feels much, much better’ and she had returned to school. Key Learning Points 1 ‘Growing pains’ do not cause difficulty in walking. 2 Vitamin D deficiency in an adolescent rarely presents as rickets. 3 Muscle weakness may be the presenting feature of severe vitamin D deficiency.

Vitamin D-Dependent Rickets Type I (1α-Hydroxylase Deficiency) (#264700) Case History 21 A female child was initially referred at 1.2 years with gross motor delay and symptoms consistent with tetany. Motor delay had been noted from the age of 1 year and she was not yet pulling to stand or walking. She had also been observed to have intermittent spasms of her hands. Her parents were first cousins, and gave a family history of severe rickets in a paternal aunt and paternal cousin.

Howard  Lyder  Allgrove  Shaw

On examination she had bilateral swollen wrists and a rachitic rosary. X-rays showed evidence of rickets and she was found to be hypocalcaemic on biochemistry. Initial presentation Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.25–2.1) Alkaline phosphatase, IU/l (<520) 25OHD, nmol/l (50–200)

1.27 1.29 1,018 51

She received intravenous calcium therapy and was commenced on alfacalcidol in a dose of 10 ng/kg/day and oral calcium supplements. However, despite this therapy over 3 months, she failed to maintain her plasma calcium within the normal range and required further intravenous calcium infusions. Alfacalcidol was stopped, and she was treated with 3 months of colecalciferol 3,000 IU/day. This failed to normalise her biochemistry and she subsequently developed seizures secondary to hypocalcaemia. She was then recommenced on alfacalcidol, 30 ng/kg/day, after which her calcium returned to the normal range and X-rays showed healing of her rickets.

Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.25–2.1) Alkaline phosphatase, IU/l (<520) 25OHD, nmol/l (50–165) 1,25(OH)2D, pmol/l (50–200) PTH, pmol/l (1.1–6.8)

After 3 months on colecalciferol without alfacalcidol

On treatment with highdose alfacalcidol

1.18

2.28

1.29

1.96

2,167

1,754

89

65

58 13.9

Genetic studies showed her to be homozygous for a mutation in exon 8 of the CYP27B1 gene on chromosome 12q13.1-q13.3 (Arg432>Cys), consistent with a diagnosis of 1α-hydroxylase deficiency. Her mother was found to

Case Histories

be a carrier for the same mutation (the parents were separated and father was not available for testing). Key Learning Points 1 Patients with this condition usually present by the age of 2 years with inability to walk, bony deformities or seizures. 2 1,25(OH)2D levels are not always low or undetectable despite an absence of enzyme activity. 3 The initial response to alfacalcidol may not be good if ‘physiological’ doses are used. 4 Vitamin D deficiency must be excluded first prior to treatment with alfacalcidol. Case History 22 Vitamin D-Dependent Rickets Type II (1,25(OH)2D End-Organ Resistance) (#277440) A 2.5-year-old boy was referred with failure to thrive and gross motor developmental delay. His height and weight were both well below the 0.4th centile. He had started sitting unsupported at 8 months and crawled at 13 months. He was unable to walk. He had previously been seen in the dermatology clinic with alopecia totalis. His parents were consanguineous. On examination, he had no body hair, including no eyebrows, an open anterior fontanelle and swollen wrists (fig. 15). He had been born at term of normal birth weight. His hair had been shaved for religious purposes and had never regrown. Initial investigations were as follows:

Corrected plasma calcium, mmol/l (2.12–2.62) Plasma phosphate, mmol/l (1.0–2.0) Alkaline phosphatase, IU/l (200–1,100) PTH, pmol/l (1.3–7.6) 25OHD, nmol/l (50–200)

1.34 1.32 2,829 56.1 14

A wrist X-ray showed irregular splayed metaphyses and osteopaenia. It was felt that he had nutritional vitamin D deficiency rickets and was treated with increasing doses of oral calcium and ergocalciferol with no improvement in his clinical or biochemical findings. Repeat assessment of his vitamin D metabolites showed the following: 25OHD, nmol/l (50–200) 1,25(OH)2D, pmol/l (43–144)

259 12,120

265

Following intravenous treatment, he started to grow and walk independently and a wrist X-ray showed healing rickets (fig. 16). At the start of treatment, his plasma magnesium and phosphate both fell, presumably because of increased bone mineralisation, and he required supplements with both intravenous magnesium and oral phosphate. Key Learning Points 1 Nutritional rickets that fails to respond to calciferol should prompt investigation for vitamin D-dependent rickets type I or II. 2 Alopecia occurs in two-thirds of cases and is due to a lack of vitamin D receptor action in the keratinocytes. It is a marker of more severe disease. 3 Intravenous calcium infusions are needed in the severe cases to heal the rickets. 4 Attention must be paid to magnesium and phosphate as well as calcium when treatment starts.

Phosphate Disorders Case History 23

Fig. 15. Case 22. Photograph of child with vitamin D-dependent rickets type II (by kind permission of the parents).

These showed adequate vitamin D status but a very high level of 1,25(OH)2D consistent with a vitamin D receptor defect. Subsequent mutation analysis confirmed a homozygous acceptor splice mutation in intron 2 of the vitamin D receptor confirming vitamin D-dependent rickets type II. An attempt was made to heal the rickets with increasing doses of alfacalcidol (up to 8 μg/day) and oral calcium supplements (75 mmol/day) with no obvious response. He was then commenced on 24-hour intravenous calcium and magnesium infusions along with oral phosphate supplementation which resulted in a good biochemical response. Results after 2 months of continuous treatment: Corr Ca, mmol/l (2.12–2.62) PO4, mmol/l (1.0–2.0) ALP, IU/l (200–1,100) PTH, pmol/l (1.3–7.6)

266

2.47 1.9 973 6.7

X-Linked-Dominant Hypophosphataemic Rickets (#307800) A 1.6-year-old girl presented with a broad-based gait. Her parents reported that she seemed to complain of pain in her legs at night, especially after walking. The pregnancy had been uneventful and she had a normal birth history with a birth weight of 3.8 kg. She had no previous medical history with no developmental concerns. Her father had been physically disabled for many years requiring a wheelchair for mobility. He had been given a diagnosis of osteogenesis imperfecta. Both parents were of Ecuadorian origin but non-consanguineous, and family history was otherwise unremarkable. Family Tree

On examination she had marked varus deformity at both knees and ankles bilaterally, with a 22° angulation at the tibial midshaft. There were no other clinical features of rickets.

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Fig. 16. Case 22. X-rays at diagnosis (a) and 3 months after the start of treatment (b) with intravenous calcium infusions. Note the initial frayed appearance to the radial metaphysis which has improved, though not yet returned to normal. a

She was found to be hypophosphataemic on biochemistry, with a raised serum PTH:

At presentation Corr Ca, mmol/l (2.15–2.65)

2.39

PO4, mmol/l (1.25–2.1) ALP, IU/l (<520) PTH, pmol/l (1.1–6.8) 25OHD, nmol/l (50–165) Urea and electrolytes Urine Ca:Creat, mmol/mmol (0.06–0.74) Fractional excretion of phosphate (<0.15%)

1.1 400 13.6 67 normal 0.16 0.73

Genetic studies showed her to be heterozygous for a novel splice site mutation (c.436 + 5G>A) in intron 4 of the PHEX gene on chromosome Xp22.2-p22, consistent with a diagnosis of X-linked hypophosphataemic rickets. Genetic studies on her father performed after his daughter’s diagnosis showed him to have the same mutation of the PHEX gene. She has been treated with oral phosphate supplements in a dose of 3 mmol/kg/day in divided doses and alfacalcidol, 30 ng/kg/day, which has helped to improve her skeletal deformity. However, she has now had bilateral osteotomies which have resulted in significant straightening of her legs and reduction in pain (fig. 17).

Case Histories

b

Key Learning Points 1 Don’t assume that the diagnosis of another bone disorder in a family member is always correct. 2 Hypophosphataemic rickets may be associated with a raised serum PTH at presentation although this is usually normal. 3 Treatment with oral vitamin D metabolites and phosphate will usually correct the biochemistry and heal the rickets but surgery is often required to correct the deformity. Case History 24 Hypophosphataemic Rickets Secondary to an Epidermal Naevus An 11-month-old Asian boy was referred to the Metabolic Bone Clinic with rickets. He had been a normal term delivery and was noted to have a giant congenital melanotic naevus covering 70% of his body (fig. 18). He had extensive plastic surgery for his naevus. He had a stormy postoperative course including a respiratory arrest and the development of renal failure. He also developed seizures and was found to have features of neurocutaneous melanosis on an MRI scan. Whilst in hospital he was noted to have a low plasma phosphate from 2 months of age with a low tubular reabsorption of phosphate for which he was subsequently started on phosphate supplements. At his local hospital he was noted to have rickets which was confirmed on X-ray for which he was started on ergocalciferol, assuming this was due to vitamin D deficiency. However, there was no improvement in biochemistry with this treatment (fig. 20).

267

Fig. 17. Case 23. X-rays of hypophosphataemic rickets before (a) and after (b) surgery for correction of bilateral varus deformity. Note that both tibiae and fibulae have undergone osteotomies and both lower femoral epiphyses have been pinned to allow differential growth of the medial growth plates. Note the coarse trabeculation of the femur shown in the first X-ray. This is characteristic of hypophosphataemic, in contrast to vitamin D-related, rickets. a

b

Investigations at age 11 months were as follows: Corrected calcium, mmol/l (2.2–2.6) Plasma phosphate, mmol/l (1.25–2.1) Alkaline phosphatase, IU/l (250–1,000) Serum PTH, ng/l (13–29) 25OHD, nmol/l (50–165) TmPO4/GFR, mmol/l (1.15–2.44)

2.46 0.56 4,897 96 89.5 0.52

X-ray confirmed marked changes of rickets (fig. 19). It was felt this could be related to his melanotic naevus and a subsequent dermatology review identified an epidermal naevus on his face. The following two additional investigations were as follows:

Fig. 18. Case 24. Photograph of baby showing melanocytic naevus on body and face and an epidermal naevus on the face.

1,25(OH)2D, pmol/l (43–144) FGF23, RU/l (<100)

21 232

He was started on phosphate Sandoz 125 mg q.d.s. and alfacalcidol 500 ng daily and subsequently there has been an improvement in his rickets.

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Fig. 19. Case 24. X-ray of knee (a) and wrist (b) showing marked splaying of the metaphyses in a child with hypophosphataemic rickets secondary to a melanotic naevus.

a

b

Key Learning Points 1 Asian infants can develop hypophosphataemic rickets. 2 An epidermal naevus can secrete FGF23 which inhibits renal tubular phosphate reabsorption and conversion of 25OHD to 1,25(OH)2D.

Osteoporosis Case History 25 Osteogenesis Imperfecta Type I (#166200) An 8-week-old baby boy presented to the casualty department. His 8-year-old sister had been putting him into the cot when she heard a crack. He was brought to hospital as he was not moving his left leg and was in pain. X-ray revealed a spiral fracture of the left tibia (fig. 20). He was born at 38 weeks’ gestation via an elective caesarean section with a birth weight of 3.2 kg. He had 3 healthy siblings and there was no history of fractures or other significant health problems in the family. Because of the unexplained fracture, a social services investigation was initiated because of the possibility of non accidental injury. Blood tests showed a normal bone profile. A skeletal survey did not identify any additional fractures. However, a skull X-ray identified multiple Wormian bones (fig. 21). The possibility of osteogenesis imperfecta prompted referral for a metabolic bone opinion. On examination, he was noted to have blue sclerae, plagiocephaly and a large anterior fontanelle. It was felt

Case Histories

Fig. 20. X-ray of spiral fracture of tibia.

that his clinical features were consistent with osteogenesis imperfecta type I. He has subsequently gone on to have further low trauma fractures.

269

On examination, he was noted to have lax ligaments and blue sclera (just detectable) but no evidence of dentinogenesis imperfecta. His weight was 9.6 kg (0.4th to 2nd centile), with height 80 cm (2nd to 9th centile). Initial biochemistry was unremarkable:

Age 2.0 years Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (0.8–1.5) ALP, IU/l (<520) PTH, pmol/l (1.1–6.8) Creatine kinase 25OHD, nmol/l (50–200) 1,25(OH)2D, pmol/l (43–144)

2.4 1.48 462 1.5 439 80 89

Fig. 21. Skull X-ray showing multiple Wormian bones.

Key Learning Points 1 Osteogenesis imperfecta may present as an unexplained low trauma fracture. 2 The possibility of osteogenesis imperfecta needs to be considered in all children presenting with unexplained fractures. Case History 26 Osteogenesis Imperfecta Type IV (#166220) A 2.0-year-old boy was referred for assessment after he presented to A&E having fallen at home and sustained a fracture of his radius and ulna. He was noted to have been a bottom shuffler until 23 months of age and had only taken his first steps a few days before he fell. The child had been previously assessed at the age of 10 months for possible failure to thrive and recurrent viral infections. He had been born at term via a forceps delivery in good condition with a birth weight of 2.9 kg. There was no family history of consanguinity and no family history of note. Family Tree

270

In view of the injury occurring after minimal trauma, a skeletal survey was performed. This revealed generalised osteoporosis, with multiple diaphyseal fractures involving several long bones, sustained over a long period of time, several posterior rib fractures, a fractured scapula, multiple Wormian bones in the lambdoid suture and bowing of the tibia. These finding were felt to be in keeping with osteogenesis imperfecta. He went on to have five further fractures over the following 4-month period. A hearing test showed a significant hearing impairment. Genetic analysis thus far has detected no mutation in the COL1A1/2 genes. He was commenced on intravenous pamidronate therapy aged 3.8 years in a dose of 1 mg/kg o.d. for 3 consecutive days every 3 months and an oral calcium and vitamin D supplement (Cacit D3) with a resulting decrease in frequency of fractures. Key Learning Points 1 Type IV OI is more severe than type I. 2 Although most forms of OI types I–IV are caused by mutations in either COL1A1 or COL1A2, a small proportion have no detectable mutation in either of these genes. 3 It is frequently associated with short stature. 4 It responds well to treatment with bisphosphonate therapy. 5 Any vitamin D deficiency should be corrected before treating with bisphosphonates.

Howard  Lyder  Allgrove  Shaw

Family Tree

On examination, he had an antalgic gait with pain on spinal and leg flexion, but otherwise normal systemic examination. Investigations revealed a high-normal corrected plasma calcium with a suppressed PTH.

Corr Ca, mmol/l (2.15–2.58) PO4, mmol/l (1.25–2.1) ALP, IU/l (<520) 25OHD, nmol/l (50–164) PTH, pmol/l (1.1–6.8) ESR FBC

Fig. 22. X-ray of thoracic and lumbar spine in a boy with leukaemia showing multiple vertebral compression fractures.

Case History 27 Leukaemia Presenting as Osteoporosis A 7.8-year-old boy presented with traumatic back pain after falling down the stairs. He was initially treated with simple analgesia, but attended A&E as his pain was not controlled. He was reviewed by orthopaedics and a spinal X-ray showed wedging of T8/10/11/12 (fig. 22). His GP was asked to arrange an MRI spine, which revealed partial collapse of T6–8. He was therefore referred to paediatrics who saw him 6 weeks after the original injury and admitted immediately for further investigation. Further questioning revealed a one year history of intermittent back pain, but nil else of note in his past medical or family history.

Case Histories

Blood film

Initial review in A&E

On admission under paediatrics

2.57 1.99 254

2.47 866 42 0.7

1 Hb 13.5 WBC 4.9 platelets 448 normal

A DEXA scan showed severe osteopaenia with a total body Z score of –4.1. He was initially managed with analgesia and spinal corset while under investigation, and commenced on colecalciferol 9,000 IU and a calcium and vitamin D supplement (Calcichew D3 forte) in view of the osteopaenia. In view of the normal blood film, a bone marrow biopsy was performed the week following admission which revealed acute lymphoblastic leukaemia. He was subsequently commenced on chemotherapy for the leukaemia and is now in remission with symptomatic improvement in his back pain. Key Learning Points 1 An acute presentation with ‘idiopathic osteoporosis’ may be the presenting feature of acute lymphoblastic leukaemia. 2 A bone marrow biopsy should be considered in such a case despite a normal blood film.

271

Fig. 23. Case 28. Appearances of the eyes in a child with osteoporosis pseudoglioma syndrome. Note the white ‘pseudogliomas’ in each eye.

Case 28 Osteoporosis Pseudoglioma Syndrome (#259770) A 1.3-year-old boy presented to the paediatric department with a fracture of his right femur. He had sustained multiple fractures with minimal trauma over the previous year, including fractures of his humerus and femur. He had been diagnosed with severe bilateral visual a b impairment after he was noted to have roving eye movements from 6 weeks of age (fig. 23). He had pale blue fundi Fig. 24. Case 28. X-ray appearances of femur of child with (secondary to retrolental masses) with variable horizon- osteoprosis pseudoglioma syndrome treated with suctal nystagmus and would habitually push his fingers into cessive courses of intravenous pamidronate. Although the overall bone density remains low, each dense line his eyes. He also had global developmental delay. X-rays revealed severe generalised osteoporosis with represents one treatment cycle. thoracic and lumbar kyphosis secondary to wedge compression fractures. Biochemical investigations were unremarkable. Genetic studies showed no mutation in the Norrie Although there was no family history of fractures gene. He had a homozygous mutation in the LRP5 gene on 11q13.4 consistent with a diagnosis of osteoporosis or visual impairment, after diagnosis both parents were found to be heterozygous for the same mutation with pseudoglioma syndrome. In view of the frequency of fractures he was com- low bone density (non-consanguineous). The patient’s menced on intravenous pamidronate, 1 mg/kg for 3 younger brother has also subsequently developed the consecutive days every 3 months as a 2-year course. same condition with a similar phenotype. Following this he was switched to oral risedronate, 2 mg/ kg once per week. This has resulted in a significant in- Family Tree crease in bone density. Repeat X-rays show the typical appearances of a growing child who has been treated with successive pamidronate infusions (fig. 24). Age, years

L1-L4 BMD % increase Total body % increase g/cm2 over over baseBMD baseline line g/cm2

10.3 12.0 13.1

0.237 0.235 0.489

–0.8 107

0.494 0.554 0.624

12 26.3

He has also required orthopaedic surgery including bilateral femoral osteotomies.

272

Key Learning Points 1 A combination of congenital blindness and recurrent fractures should suggest the diagnosis of OPG. 2 Norrie’s disease has a very similar ophthalmological appearance but is not associated with osteoporosis. 3 This is not classified amongst the osteogenesis imperfectas as the defect is not located in collagen but is within osteoblast signalling. 4 The bone abnormalities are clinically very similar to moderately severe osteogenesis imperfecta and

Howard  Lyder  Allgrove  Shaw

respond to treatment with bisphosphonates in the same way. Case History 29 Bruck Syndrome (Osteogenesis Imperfecta with Congenital Joint Contractures) (#609220) A 13-month-old female was referred from the Middle East with a history of multiple fractures. She was a normal term delivery with a birth weight of 2.7 kg. She had been noted at birth to have flexion deformities of both knees and elbows, and a programme of physiotherapy was undertaken to try to correct these. However, she sustained a fracture of her right radius and ulna and left femur whilst undergoing physiotherapy. Her parents were known to be first cousins, but there was no family history of note. Fig. 25. Case 30. X-ray of left ankle in a child with idiopathic juvenile osteoporsis. Note the very low bone density that is apparent even on plain X-ray.

Family Tree

On examination she had contractures at both knees and ankles with generalised weakness. Her teeth and sclerae appeared to be normal. Weight at referral was 7.14 kg (<3rd centile) and height 71 cm (10th centile). She was referred to orthopaedics in view of her fractures, and X-rays revealed multiple healing rib fractures and Wormian bones in the skull. Biochemistry showed a slightly raised serum phosphate but was otherwise unremarkable.

Genetic studies have shown no mutation of COL1A1 or COL1A2 genes but Bruck gene mutation studies (PLOD2) are pending. Key Learning Points 1 Bruck syndrome is often reported as a form of osteogenesis imperfecta but is not due to mutations in either of the two genes for type 1 collagen. 2 Some cases of Bruck syndrome are due to defects in the PLOD2 gene that codes for bone specific telopeptide lysyl hydroxylase.

At presentation

Case History 30 Corr Ca, mmol/l (2.15–2.58) PO4, mmol/l (1.25–2.1) ALP, IU/l (<520) 25OHD, nmol/l (50–200) PTH, pmol/l (1.1–6.8) Urine Ca:Creat, mmol/mmol (0.06–0.74) FBC/U&E

2.58 2.13 274 68 2.1 4.7 normal

Treatment was started with intravenous pamidronate 1 mg/kg for 3 consecutive days every 3 months, as well as oral calcium supplements with colecalciferol 400 IU/ day. A moderated programme of physiotherapy was also re-introduced.

Case Histories

Idiopathic Juvenile Osteoporosis (#259750) A 7-year-old girl presented with a 6-week history of limping with no apparent trauma. She had increasing difficulty running and walking down stairs. An X-ray of her left foot showed soft tissue swelling and osteopaenia (fig. 25). She had been a normal term delivery weighing 4.2 kg. She had no other symptoms and there was no significant family history. She was reviewed by a rheumatologist who noted slight swelling of her left ankle, hypermobile joints and flat feet. It was initially felt that her symptoms were due to joint hypermobility. Her gait continued to worsen and she underwent further investigations. She had a normal full blood count and film, CRP, bone profile, PTH and 25OHD levels and her anti-nuclear antigen was negative.

273

Fig. 27. Case 31. X-ray of pelvis demonstrating generalised sclerosis of bones with thick cortices and narrow medullary cavities in the femurs in a child with autosomal-dominant osteopetrosis.

Fig. 26. Case 30. Lateral X-ray of lumbar spine in a child with idiopathic juvenile osteoporosis. Note the biconcave appearance of the vertebrae.

2 It differs from osteogenesis imperfecta in that there is evidence of low bone turnover on bone biopsy.

Miscellaneous Bone Disorders A bone density scan using DXA showed a lumbar spine bone density Z score of –4.0. A pQCT scan of the left radius showed low trabecular and total density. A thoracic and lumbar spine X-ray showed multiple vertebral compression fractures (fig. 26). These investigations suggested idiopathic juvenile osteoporosis (IJO) and further tests were done to exclude other causes. A bone marrow biopsy was normal, excluding leukaemia, and an iliac crest bone biopsy showed active osteoblasts to be almost absent, consistent with IJO. A DNA sample showed no evidence of a heterozygous mutation in LRP5 which has been reported in some cases of IJO. She was treated with intravenous pamidronate 3 mg/kg every 3 months with her bone density returning to normal after 2 years. This has been associated with a functional improvement in her walking. Key Learning Points 1 Idiopathic juvenile osteoporosis will often present with back pain and difficulty walking rather than long bone fractures.

274

Case History 31 Autosomal-Dominant Osteopetrosis (#166600) A 9-year-old girl presented to her local hospital with a fractured neck of her right femur. She had been playing at school and ran into a wall. X-rays at time of the injury showed a generalised increase in bone density with thick cortices and narrow medullary cavities (fig. 27). Her full blood count and film was normal: Hb 10.4 g/dl; WBC 5.7 × 109/l; platelets 191 × 109/l. Her femur fracture required internal fixation and she was referred to the Metabolic Bone Service. Skull and lateral spine X-rays showed increased density of the skull, particularly the skull base. There was increased density of the spine with a ‘rugger jersey’ appearance to the vertebral bodies, consistent with osteopetrosis. She had assessments of her hearing and vision which were normal. The diagnosis was felt likely to be the autosomal-dominant type of osteopetrosis. Genetic analysis of the ClCN7 chloride channel gene on chromosome 16p13.3 demonstrated a missense

Howard  Lyder  Allgrove  Shaw

mutation in exon 19 (M584V/A1750G) confirming a diagnosis of autosomal-dominant osteopetrosis type II. Her parents subsequently had bone density scans of lumbar spine and hips. The father’s was normal, the mother’s scan showed extremely high bone density for age. Her younger sibling had a normal scan. Several years after presentation, her 78-year-old grandmother was also identified as having a very high bone density. Thus, it is apparent that her mother and grandmother are also affected although they have been asymptomatic. The girl has not sustained any further fractures after 5 years of follow-up and is otherwise well. Key Learning Points 1 Although the autosomal-dominant form of osteopetrosis is felt to be a condition affecting adults it may present in childhood. a b 2 Affected individuals may be asymptomatic for many Fig. 28. Case 32. X-rays of child with infantile hypoyears phosphatasia. X-rays shown are hand (a) and lower limb (b). Case History 32 Infantile Hypophosphatasia (#241500) A 7-month-old girl, presented with severe failure to thrive and vomiting, weight 4.15 kg (–4.33 SD), length 56 cm (–4.94 SD), head circumference 39 cm (–3.45 SD). She had evidence of muscular hypotonia as well as signs of rickets. She was a normal term delivery of white, non-consanguineous parents. She developed refractory neonatal seizures from day 6 of life, only responsive to vitamin B6 (pyridoxine-responsive seizures, PRS). She was noted to have a low alkaline phosphatase. She developed failure to thrive and vomiting from around 6 weeks of age.

Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.25–2.1) PTH, pg/ml (10–55) 25OHD, nmol/l (50–200) ALP, IU/l (133–347) Urine Ca:Creat ratio, mmol/mmol (<0.74)

4.06 1.44 0.7 277 4 4.98

Radiographic findings consistent with rickets, including delayed skeletal ossification and hypomineralisation (fig. 28). She had nephrocalcinosis on ultrasound. She was diagnosed with the infantile form of hypophosphatasia (HPP). Further investigations:

Case Histories

Bone ALP, IU/l (47–181) Leukocyte ALP, nmol/min/mg protein (2–8) Urine phosphoethanolamine (PEA), mmol/mmol Creat (9–25) Plasma pyridoxal phosphate (PLP), nmol/l (5–107) Urine inorganic pyrophosphate (PPi), μmol/g creat (<198)

<2 0.2 407 36,500 1,253

Genetic analysis confirmed that she had a compound heterozygous mutation in the TNSALP gene. Prednisolone (0.5–1.1 mg/kg/day) was the only effective drug for reducing plasma calcium to high-normal levels. Calcitonin injections and a thiazide diuretic were ineffective. Attempts to reduce the prednisolone dose resulted in recurrence of hypercalcaemia and vomiting. Oral pyridoxine was also continued (10–30 mg/kg/day), and seizures never recurred. Despite some improvements in growth and weight gain, the patient’s respiratory function worsened. She subsequently developed a flail chest with recurrent rib fractures and died from respiratory failure at age 9 months. Post-mortem analysis revealed severely impaired endochondral and intramembranous ossification with widened epiphyseal growth plates and hypertrophic chondrocytes in disarray.

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Key Learning Points 1 A low alkaline phosphatase is diagnostic for HPP. 2 Vitamin B6 (pyridoxine) provides a link between brain and bone metabolism: PLP is an essential coenzyme for synthesis of various neurotransmitters and biogenic amines. Despite high plasma PLP concentration, brain cell PLP levels are presumably low in HPP, causing seizures. 3 HPP and PRS are independent autosomal-recessive disorders, but HPP is also one of four metabolic defects causing PRS. 4 All reported cases of infantile HPP with a history of neonatal seizures were lethal. 5 Treatment with recombinant alkaline phosphatase has recently been used on an experimental basis. Case History 33 Juvenile Hypophosphatasia (#241510) A 6.9-year-old girl was referred for further information about her diagnosis of juvenile hypophosphatasia. She had initially had problems from the age of about 2 years when her primary dentition began to fall out despite having been somewhat delayed in erupting. Since then she had lost several teeth although her secondary dentition was appearing. She had had considerable problems with dental cysts over the previous few years. Her mother had also noticed that her eyes had become prominent, particularly on the left side. Her birth had been normal with a birth weight of 3.62 kg, 6 days after term. There were no neonatal problems and she had otherwise been well. She has three siblings who are all well. However, her maternal grandfather had lost all his teeth by the age of 21 and is now confined to a wheelchair with severe back problems. She has an aunt, uncle and three cousins all of whom are well. On examination she was well looking. Her height was on the 25th centile. General examination was normal. She had some exophthalmos, mainly on the left side with a suggestion of ridging over the left coronal suture. She had no incisors and the other teeth in her lower jaw looked abnormal. Initial investigations had shown:

ALP, IU/l (139–347) Urine phosphoethanolamine (PEA), mmol/mmol Creat (9–25) Plasma pyridoxal phosphate (PLP), nmol/l (5–107)

276

109 43

This corroborates the diagnosis of juvenile hypophosphatasia. A CT scan of the head confirmed the presence of some craniosynostosis of the left coronal suture. She has been referred to the neurosurgeons for an opinion on whether or not she requires surgery. Key Learning Points 1 Juvenile hypophosphatasia is thought to be the heterozygous form of the condition (c.f. infantile hypophosphatasia). 2 The diagnosis is based on the combination of clinical features, low alkaline phosphatase and raised plasma pyridoxal phosphate and urine phosphoethanolamine. 3 Premature tooth eruption is an important feature. They usually fall out with their roots intact (see fig. 12 in chapter 12). 4 There is currently no specific treatment but surgery may be required if raised intracranial pressure from craniosynostosis becomes a problem. 5 There is a tendency for the condition to improve with age although osteoporosis may supervene in later life and become disabling. Case History 34 Metaphyseal Chondrodysplasia, Jansen type (#156400) A male infant, noted to have an abnormal head shape and dysmorphic facies at birth, was referred to neurosurgery for investigation of possible craniosynostosis for which he required suture release at 10 months of age. On referral to paediatrics at 11 months, examination revealed turricephaly with a flat occiput, hypertelorism with proptosis and micrognathia. Skeletal abnormalities included broad thumbs and phalanges, and a rachiticlike appearance of his wrists and other long bones (fig. 29). His weight and head circumference were both <3rd centile with a length between the 10–50th centile. Parents reported a past history of recurrent coryzal symptoms with obstructive sleep apnoea. He had been a normal term delivery with a birth weight of 2.96 kg. There were several family members, including mother, grandmother and great-grandmother, with similar, although milder, facial features and a maternal uncle with skeletal dysplasia who had died aged 6 months. There was no consanguinity. Family Tree

>500

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Fig. 29. Case 34. X-rays of ankles (a) and femurs (b) in a child with Jansen’s metaphyseal chondrodysplasia. Note the foreshortened femurs characteristic of this condition and the appearances of severe hyperparathyroidism despite the una detectable levels of PTH.

b

On investigation he was found to have abnormal biochemistry including intermittent hypercalcaemia, with hypercalciuria.

At referral Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.25–2.1) ALP, IU/l (<520) PTH, pmol/l (1.1–6.8) PTHrP 25OHD, nmol/l (50–200) 1,25(OH)2D, pmol/l (43–144) Urea and electrolytes Urine Ca:Creat, mmol/mmol (0.06–0.74)

3.36 0.9 680 undetectable undetectable 45 163 normal 2.63

X-rays showed generalised osteoporotic changes with a ‘moth-eaten’ appearance consistent with hyperparathyroidism (fig. 22), and a renal US revealed nephrocalcinosis. Bone biopsy showed evidence of hyperparathyroidism. Genetic studies revealed a missense mutation in the PTH/PTHrP receptor on chromosome 3p21.1-p22 (I458R), consistent with metaphyseal chondrodysplasia, Jansen type. He was initially started on indometacin at a dose of 1 mg/kg/day with a low calcium diet. However, serum calcium remained high with a urine calcium/creatinine ratio at the upper limit of normal. An oral bisphosphonate (risedronate) was started at age fourteen years. This resulted in a slow but gradual decline in urinary NTX (as a marker of bone resorption) but, eighteen months later, this was converted to three monthly intravenous pamidronate, which effected a more rapid decline in NTX (fig. 30).

Case Histories

Corr Ca, mmol/l (2.15–2.65) PO4, mmol/l (1.25–2.1) Alp, IU/l (<520) Urine Ca:Creat, mmol/mmol (0.06–0.74) Urinary N-telopeptide crosslinks (NTX) mmol/l BCE/mmol Creat (<50)

On indomethacin

On risedronate

On pamidronate

2.88

2.63

2.53

1.36

1.39

0.99

779 0.73

477 1.0

230

1878

296

Key Learning Points 1 Jansen’s is a rare condition caused by activating mutations in the PTHrP/PTH1 receptor. 2 Hypercalcaemia and hypercalciuria are features that occur intermittently especially with intercurrent illness. 3 Growth, particularly of long bones, is impaired. 4 Bone turnover markers are grossly raised but may be used as an index of response to treatment. Case History 35 Caffey’s Disease (#114000) A 4-week-old boy was brought to hospital with generalised irritability. His mother reported that he had been noted to hold his legs in an abnormal position and was concerned that he was in pain. The pregnancy and birth history had been normal.

277

2,500

Start oral risedronate

Stop oral risedronate and Start intravenous pamidronate

2,000 1,500 1,000 500

Fig. 30. Case 34. Urinary excretion of NTX in response to oral risedronate and subsequently intravenous pamidronate treatment.

Fig. 31. Case 35. X-rays of a child with Caffey’s disease. The arrows show the grossly widened periosteum on the humeri (a) and tibia (b). Similar reactions are seen in other bones including the facial bones (not shown).

0 14.5

15.0

278

16.0

16.5

17.0

17.5

18.0

18.5

a

His father, a paternal uncle and a paternal great uncle were reported to have had similar bony abnormalities as neonates. Family Tree

15.5

b

On examination, he had bony lumps palpable over the jaw, his tibiae were abnormally shaped and he had soft tissue and bony swellings at distal ends of all long bones (legs > arms). There was skin tenderness on palpation and he was clearly in discomfort. His biochemistry was normal. Plain X-rays revealed widespread periosteal reaction throughout the skeleton (fig. 31). Genetic analysis showed a heterozygous mutation of exon 42 of COL1A1 gene on chromosome 17q21.31-q22 (c.3040C>T; Arg1014Cys). He commenced treatment with non-steroidal anti-inflammatory agents, initially ibuprofen but subsequently with naproxen, the latter resulting in a particularly good response.

Howard  Lyder  Allgrove  Shaw

4 The condition is usually self-limiting but may occasionally return in later life. 5 Despite being caused by a mutation in COL1A1, bone density is not reduced and there is no increased fracture risk. Case History 36

Fig. 32. Case 36. Photograph of child with cleidocranial dysplasia demonstrating the ability to appose the shoulders.

Key Learning Points 1 If the diagnosis of Caffey’s disease is suspected, it can be confirmed by rapid analysis of the appropriate exon of COL1A1 without having to sequence the whole gene. 2 Caffey’s disease can also be acquired by the prolonged use of prostaglandin E2 to maintain the patency of a ductus arteriosus in congenital heart disease. 3 The clinical signs and symptoms are thought to be related to high circulating levels of prostaglandins and respond well to non-steroidal antiinflammatory agents.

Case Histories

Cleidocranial Dysplasia (#119600) A female infant was referred for short stature at the age of 8 months. Birth weight was 2.98 kg at term. She was well but noted to have mild dysmorphic features including brachycephaly, upward slanting eyes and posteriorly rotated ears. Muscle tone in the legs was slightly increased but low around the shoulders. She was able to appose her shoulders (fig. 32). A clinical diagnosis of cleidocranial dysplasia was made. She had mildly delayed motor development but intellectual development has been normal apart from some speech delay. Short stature persisted and by the age of 6.2 years was –3.5 SD. Growth hormone response to glucagon stimulation was slightly subnormal (peak 15 mU/l) and she was given a trial of growth hormone. The parents are not consanguineous. Growth response to growth hormone has been poor even after the dose was increased to that used in skeletal dysplasia (e.g. Turner syndrome). Height SDS after three years was still only –3.44, so growth hormone has been stopped (fig. 33). Genetic studies have not been undertaken as they were not felt by the geneticists to be warranted as the clinical diagnosis was obvious. Key Learning Points 1 Cleidocranial dysplasia is usually caused by mutations in the Runx2 gene. 2 The diagnosis of skeletal dysplasia can sometimes be made on clinical and radiological grounds. 3 Short stature is often a feature of skeletal dysplasias 4 Response to growth hormone is often poor even when partial growth hormone deficiency can be demonstrated.

279

200

Height (cm)

180

160

*

140

120

100

80

60

Fig. 33. Case 36. Growth chart of child with cleidocranial dysplasia showing lack of response to growth hormone.

40 0

2

4

6

8

10

12

14

16

18

20

22

Conclusions The cases presented here go some way to demonstrating the spectrum of disorders that are encountered in clinics held especially for children with bone and calcium disorders. They represent a wide variety and, although some are related to problems that arise secondarily to environ-

mental factors such as vitamin D deficiency, most of them are genetic in origin. An understanding of the basic principles of genetics as well as the physiology of bone and calcium metabolism is therefore required to be able to make a correct diagnosis that will allow suitable treatment.

Dr. N.J. Shaw Department of Endocrinology, Birmingham Children’s Hospital Steelhouse Lane Birmingham B4 6NH (UK) Tel. +44 0121 333 8189, Fax +44 0121 333 8191, E-Mail [email protected]

280

Howard  Lyder  Allgrove  Shaw

Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 281–291

Appendix Appendix 1. Explanation of abbreviations appearing in the text Abbreviation

Definition

1,24,25(OH)3D

1,24,25-trihydroxy vitamin D

1,25(OH)2D

1,25-dihydroxy vitamin D

1,25(OH)2D2

1,25-dihydroxy ergocalciferol

1,25(OH)2D3

1,25-dihydroxy cholecalciferol (calcitriol)

24,25(OH)2D

24,25-dihydroxy vitamin D

25OHD

25-hydroxy vitamin D

aBMD

areal bone mineral density

A&E

accident and emergency

ACE

angiotensin converting enzyme

ACVR1/ALK2

activin receptor I/activin-like kinase II

AD

autosomal dominant

ADH

autosomal-dominant hypocalcaemia

ADHR

autosomal-dominant hypophosphataemic rickets

AED

anti-epileptic drug

AHO

albright’s hereditary osteodystrophy

AIRE

autoimmune regulator

ALL

acute lymphoblastic leukaemia

AMP

adenosine monophosphate

AN

anorexia nervosa

APECED

autoimmune polyndocrinopathy candidiasis ectodermal dystrophy

APS1

autoimmune polyendocrinopathy type I syndrome

ARHR

autosomal-recessive hypophosphataemic rickets

AVP

arginine vasopressin

Abbreviation

Definition

BMAD

bone mineral apparent density

BMC

bone mineral content

BMD

bone mineral density

BMDa

areal bone mineral density

BMDv

volumetric bone mineral density

BMP1

bone morphogenic protein I

BMP2

bone morphogenic protein II

BMPs

bone morphogenic proteins

BMT

bone marrow transplant

BMU

bone multicellular unit

BP

bisphosphonate

BPABG

British Paediatric and Adolescent Bone Group

BSP

bone sialoprotein

bTNAP

bone-specific tissue alkaline phosphatase

BUA

broadband ultrasound attenuation

BWt

body weight

Ca

calcium

CA2

carbonic anhydrase II

CALCA

alpha calcitonin/calcitonin gene-related peptide

cAMP

cyclic adenosine monophosphate

CaSR

calcium-sensing receptor

CBFA1 (Runx2)

runt domain, alpha subunit I

CCD

cleido cranial dysplasia

CD

coeliac disease

CD44

CD44 antigen

c-FOS

serum responsive factor

CGRP

calcitonin gene-related peptide

CLCN5

chloride channel 5

CLCN7

chloride channel 7

COL1A1

alpha 1 chain of type I collagen

COL1A2

alpha 2 chain of type I collagen

282

Appendix

Abbreviation

Definition

COX-2

cyclo-oxygenase 2

CP

cerebral palsy

CPAP

continuous positive airway pressure

CPR

cardio-pulmonary resuscitation

CRF

chronic renal failure

CRTAP

cartilage-associated protein

CT

calcitonin

CT

computerised tomography

CTSK

cathepsin K

CYP27B1

1-alpha hydroxylase

CYP2J2

cytochrome P450, subfamily IIJ, polypeptide II

CYP2R1

cytochrome P450, subfamily IIR, polypeptide I

D2

ergocalciferol

D3

cholecalciferol (colecalciferol when used therapeutically)

DBP

vitamin D-binding protein

DCT

distal convoluted tubule

DGS

DiGeorge syndrome

DMD

Duchenne muscular dystrophy

DMP1

dentine matrix protein I

DNA

deoxyribonucleic acid

DOTANOC

gallium 68-labeled 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid/1-Nal3-octreotide

DRTA

distal renal tubular acidosis

DSPP

dentin sialophosphoprotein

DTARD

distal renal tubular acidosis without progressive deafness

DXA

dual energy X-ray absorptiometry

ECMO

extra-corporeal membrane oxygenation

EDS

Ehlers-Danlos syndrome

EDTA

ethylene diamine tetracetic acid

EGF

epidermal growth factor

EGFR

epidermal growth factor receptor

Appendix

283

Abbreviation

Definition

ENPP1

ecto-nucleotide pyrophosphate/phosphodiesterase I

ENS

epidermal naevus syndrome

ESR

erythrocyte sedimentation rate

FACIT

fibril-associated collagen with interrupted triple helices

Fas (TNFRSF6)

tissue necrosis factor superfamily, member 6

FBH (FHH)

familial benign hypercalcaemia

FD

fibrous dysplasia

FDG

fluorodeoxyglucose

FGFs

fibroblast growth factors

FGF23

fibroblast growth factor 23

FGFR1

fibroblast growth factor receptor I

FHH (FBH)

familial hypocalciuric hypercalcaemia

FIH

familial isolated hypoparathyroidism

FIPH

familial isolated primary hyperparathyroidism

FISH

fluorescent in situ hybridisation

FLK-1 (KDR)

kinase insert domain receptor

FOP

fibrodysplasia ossificans progressiva

Fra-1 (FOSL-1)

FOS-like antigen I

FS

fractional shortening

FT4

free thyroxine

FXYD2 (ADP1G1)

FXTD domain-containing ion transporter regulator II (sodiumpotassium ATPase)

Fz

frizzled protein

GACI

generalised arterial calcification of infancy

GALNT3

UDP-nacetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 3

GATA3

GATA-binding protein III

GC

glucocorticoid

GCMB/A210GCM2

glial cells missing II

GDP

guanosine diphosphate

GH

growth hormone

GHD

growth hormone deficiency

284

Appendix

Abbreviation

Definition

GHRH

growth hormone-releasing hormone

GIO

glucocorticoid-induced osteoporosis

GLAST

glutamate/aspartate transporter

GNAS1

heterotrimeric G-protein α-subunit

GO

geroderma osteodysplasticum

Gsα

alpha subunit of G-stimulatory protein

GTP

guanosine triphosphate

HDR

hypoparathyroidism, deafness and renal anomalies

HFTC1

hyperphosphataemic familial tumoral calcinosis type I

HFTC2

hyperphosphataemic familial tumoral calcinosis type II

HFTC3

hyperphosphataemic familial tumoral calcinosis type III

HPP

hypophosphatasia

HHRH

hereditary hypophosphataemic rickets with hypercalciuria

HKAFO

hip-knee-ankle-foot orthoses

HMG

high mobility group

HOMG1

hypomagnesaemia with secondary hypocalciuria

HOMG2

renal hypomagnesaemia associated with hypocalciuria

HOMG3

hypomagnesaemia, hypercalciuria and nephrocalcinosis syndrome

HOMG4 (IRH)

isolated recessive renal hypomagnesaemia

Hox

homeobox

Hoxa3

homeobox A3

HR-CT

high-resolution computerised tomography

HRD

hypoparathyroidism, mental and growth retardation and dysmorphic features

HRPT2

hyperparathyroidism 2 gene

HSS

hyperostosis hyperphosphataemia syndrome

HVDRR (VDDR2)

hereditary 1,25(OH)2D-resistant rickets

HYP-JT

hyperparathyroid jaw tumour syndrome

IAP

intestinal alkaline phosphatase

IBD

inflammatory bowel disease

IGF-1

insulin-like growth factor I

Appendix

285

Abbreviation

Definition

IGF-2

insulin-like growth factor II

IGFBP

insulin-like growth factor binding protein

IGFBP-4

insulin-like growth factor binding protein IV

IGFBP-5

insulin-like growth factor binding protein V

IHH

Indian hedgehog

IJO

idiopathic juvenile osteoporosis

IL-1

interleukin I

IL-6

interleukin VI

IRH (HOMG4)

isolated recessive renal hypomagnesaemia

ISCD

International Society for Clinical Densitometry

JIA

juvenile idiopathic arthritis

JMC

Jansen’s metaphyseal chondropysplasia

JPD

juvenile Paget’s disease

KCS

Kenny-Caffey syndrome

KL

Klotho

KSS

Kearns-Sayre syndrome

LALP

liver alkaline phosphatase

LIMK

LIM domain kinase I

LNSS

linear naevus sebaceous syndrome

LRP5

low-density lipoprotein receptor-related protein V

LRP6

low-density lipoprotein receptor-related protein VI

LVEDD

left-ventricular end-diastolic diameter

LVESD

left-ventricular end-systolic diameter

MAS

McCune-Albright syndrome

M-CSF

macrophage colony-stimulating factor

MTC

medullary thyroid carcinoma

MELAS

mitochondrial encephalopathy, lactic acidosis and stroke-like episodes

MEN1

multiple endocrine neoplasia type I

MEN2

multiple endocrine neoplasia type II

MEN4

multiple endocrine neoplasia type IV

MENIN

multiple endocrine neoplasia type I tumour suppressor gene

286

Appendix

Abbreviation

Definition

MEPE

matrix extracellular phosphoglycoprotein

MRI

magnetic resonance imaging

mRNA

messenger ribonucleic acid

MTPDS

mitochondrial trifunctional protein deficiency syndrome

Na-K ATPase

sodium-potassium ATPase

NaPi-IIc/SLC34A3

solute carrier family 34 (sodium phosphate co-transporter), member 3

NCC

sodium chloride co-transporter

NCX1

solute carrier family 8 (sodium-calcium exchanger), member 1

NESP55

neuro-endocrine secretory protein 55

NESPAS

neuro-endocrine secretory protein anti-sense

NFTC

normophosphataemic tumoral calcinosis

NHERF/SLC34A1

solute carrier family 34 (sodium phosphate co-transporter), member 1

NMDA

N-methyl D aspartic acid

NO

nitric oxide

NSHPT

neonatal severe hyperparathyroidism

OGD

osteoglophonic dysplasia

OI

osteogenesis imperfecta

OMIM

online mendelian inheritance in man

OOM (TIO)

oncogenic osteomalacia (tumour-induced osteomalacia)

OPG

osteoprotegerin

OPN

osteopontin

OPPG

osteoporosis pseudoglioma syndrome

OPS

osteoporosis pseudoglioma syndrome

OPTA1

autosomal-dominant osteopetrosis type I

OPTA2

autosomal-dominant osteopetrosis type II

OPTB

autosomal-recessive osteopetrosis

OPTB1

autosomal-recessive osteopetrosis type I

OPTB2

autosomal-recessive osteopetrosis type II

OPTB3

autosomal-recessive osteopetrosis type III

OPTB4

autosomal-recessive osteopetrosis type IV

OPTB5

autosomal-recessive osteopetrosis type V

Appendix

287

Abbreviation

Definition

OPTB6

autosomal-recessive osteopetrosis type VI

OPTB7

autosomal-recessive osteopetrosis type VII

OSD

opsismodysplasia

OSTM1

osteopetrosis-associated transmembrane protein I

PAM

pulmonary alveolar microlithiasis

PDD

progressive diaphyseal dysplasia (Camurati-Engelmann disease)

PDD

pseudovitamin D deficiency rickets

PDGF

platelet-derived growth factor

PEA

phosphoethanolamine

PET-CT

positron emission tomography-computerised tomography

PGE2

prostaglandin E2

PHEX

phosphate-regulating gene with homologies to endopeptidases on the X chromosome

PHP1a

pseudohypoparathyroidism type Ia

PHP1b

pseudohypoparathyroidism type Ib

Pi

inorganic phosphate

PLAP

placental alkaline phosphatase

PLEKHM1

Plekstrin homology domain-containing protein, family M member I

PLOD1

procollagen-lysine, 2-oxoglutarate, 5-dioxygenase I

PLOD2

procollagen-lysine, 2-oxoglutarate, 5-dioxygenase II

PMCA1b

ATPase, Ca2+-transporting, plasma membrane, Ib

PO4

phosphate

POH

progressive osseous heteroplasia

PPHP

pseudopseudohypoparathyroidism

PPi

inorganic pyrophosphate

pQCT

peripheral quantitative computerised tomography

PRAD1

cyclin D1

PTG

parathyroid gland

PTH

parathyroid hormone

PTH1R

parathyroid hormone type I receptor

PTH2R

parathyroid hormone type II receptor

288

Appendix

Abbreviation

Definition

PTHrP

parathyroid hormone-related peptide

QCT

quantitative computerised tomography

QUS

quantitative ultrasound

RANK (TNFRSF11A)

receptor activator of nuclear factor kappa-β

RANKL

receptor activator of nuclear factor kappa-β ligand

rPTH 1-34

recombinant human PTH 1-34

RTA

renal tubular acidosis

Runx2 (CBFA1)

runt domain, alpha subunit I

SAMD9

sterile alpha motif domain-containing protein 9

sFRP-4

secreted frizzled-related protein 4

SI

système international

SIBLING

small integrin-binding ligand, N-linked glycoprotein

SLC12A3

sodium chloride co-transporter

SLC34A2

sodium-potassium co-transporter

SLC34A3

sodium-phosphate co-transporter

SLC4A1

solute carrier, family 4 (anion exchanger), member 1

SLE

systemic lupus erythematosus

SNP

single nucleotide polymorphism

SOS

speed of sound

SOST

sclerosteosis

Sox3

SRY-Box 3

Sox9

SRY-Box 9

SRY

sex-determining region of the Y chromosome

STX16

syntaxin 16

T3

tri-iodothyronine

TBX

T-Box

Tbx1

T-Box 1

TCBE

tubulin-specific chaperone E

TCIRG1

t cell immune regulator I

TGFB1

transforming growth factor beta I gene

TGF-β

transforming growth factor beta

Appendix

289

Abbreviation

Definition

TIO (OOM)

tumour-induced osteomalacia

TmP/GFR

threshold for tubular maximal reabsorption of phosphate

TNF-α

tissue necrosis factor-α

TNFRSF11A (RANK)

receptor activator of nuclear factor kappa-β

TNSAP

tissue non-specific alkaline phosphatase

TP/GFR

tubular phosphate reabsorption related to glomerular filtration rate

TRP

transient receptor potential

TRP (%TRP)

tubular reabsorption of phosphate

TRPM6

transient receptor potential cation channel, subfamily M, member 6

TRPM7

transient receptor potential cation channel, subfamily M, member 7

TRPV5

transient receptor potential cation channel, subfamily V, member 5

TRPV6

transient receptor potential cation channel, subfamily V, member 6

TSH

thyroid-stimulating hormone

UDF1L

ubiquitin fusion degradation gene

UV

ultraviolet

UVB

ultraviolet B

VBCH

van Buchem disease

vBMD

volumetric bone mineral density

VDDR1

vitamin D-dependent rickets type 1

VDDR2 (HVDRR)

vitamin D-dependent rickets type 2 (hereditary vitamin D resistant rickets)

VDR

vitamin D receptor

VEGF

vascular endothelial growth factor

VIP

vasoactive intestinal peptide

WBV

whole body vibration

Wnt

an elision of Wg (wingless, gene found in Drosophila) and Int (integration sites, found in mouse mammary tumour virus)

XLD

X-linked dominant

XLHR

X-linked hypophosphataemic rickets

XLR

X-linked recessive

μSv

microsieverts

290

Appendix

Appendix 2. Conversion factors SI to ‘conventional’ units Substance

SI

Conversion

Conventional Conversion

SI

Molecular weight

Ca2+

mmol/l

4

mg/dl

0.4

mmol/l

40

PO4

mmol/l

3.2

mg/dl

0.312

mmol/l

32

Mg2+

mmol/l

2.3

mg/dl

0.43

mmol/l

23

25OHD

nmol/l

0.4

ng/ml

2.5

nmol/l

400

1,25(OH)2D

pmol/l

0.416

pg/ml

2.4

pmol/l

416

24,25(OH)2D

nmol/l

0.416

nmol/l

2.4

nmol/l

416

PTH

pmol/l

10

pg/ml

0.1

pmol/l

~10,000

TmPO4/GFR

mmol/l

3.2

mg/dl

0.312

mmol/l

32

Creat (plasma) μmol/l

0.113

mg/dl

8.84

μmol/l

113

Ca/Creat

mmol/ mmol

3.07

mg/mg

0.325

mmol/ mmol

40/113

Urea (BUN)

mmol/l

1.4

mg/dl

0.71

mmol/l

60 (14)

Appendix

291

Author Index

Ahmed, S.F. 170 Allgrove, J. IX, 1, 8, 115, 246

Johnson, K. 233 Jüppner, H. 133

Bergwitz, C. 133 Bishop, N. 157

Lyder, G. 246 Mughal, M.Z. 191

Cheung, M. 218 Crabtree, N. 58

Rauch, F. 49

Davies, J. 93

Shaw, N. IX, 73, 246

Elmantaser, M. 170

Ward, K. 58

Glorieux, F.G. VII Grabowski, P. 32 Howard, S. 246

292

Subject Index

Abbreviations, table 281–290 Acquired hypoparathyroidism, hypocalcemia 80, 81 ADHR, see Autosomal dominant hypophosphatemic rickets AHO, see Albright’s hereditary osteodystrophy Albright’s hereditary osteodystrophy (AHO), GNAS mutation 85, 86 Alfacalcidol, dosing 224 Alkaline phosphatase isoforms 25 therapeutic applications 230 Anorexia nervosa, osteoporosis risks 181 Anticonvulsants, osteoporosis risks 181, 182 APECED hypoparathyroidism 81 secondary hypercalcemia 107 ARHP, see Autosomal recessive hypophosphatemia Autosomal dominant hypophosphatemic rickets (ADHR), features 147 Autosomal recessive hypophosphatemia (ARHP), features 147 Bisphosphonates adverse effects 220–222 benefits 220 drug selection and dosing 222, 223 hypercalcemia management 111, 112 hypercalcemia management 226 indications 219, 220 initiation and termination of therapy 222 osteogenesis imperfecta management 166, 167 osteoporosis management 185, 186, 219 overview 219 BMD, see Bone mineral density

BMP2, see Bone morphogenetic protein 2 Bone gene mutations and diseases table 36–39, 40 load bearing adaptation 44, 45 metabolism in children and adolescents 54, 55 nervous system interaction 44 nutrition and exercise impact 45, 46 skeletal development 33, 34 types 33 Bone biopsy histomorphometric parameters 52–54 labeling with tetracycline compounds 51, 52 pediatric indications 55, 56 sample processing 52 technique 50–52 Bone mineral density (BMD), see also Dualenergy X-ray absorptiometry; Magnetic resonance imaging; Quantitative computed tomography; Quantitative ultrasound definition of types 58, 59 measurement guidelines 69, 70 principles 60 osteoporosis, see Osteogenesis imperfecta; Osteoporosis Bone morphogenetic protein 2 (BMP2), therapeutic applications 229, 230 Bruck syndrome, case history 273 Caffey’s disease case history 277–279 features 206 gene mutations 196, 197 Calcilytic drugs, therapeutic applications 230

293

Calcitonin calcium homeostasis role 9 function 25 history of study 3, 4 hypercalcemia management 112, 226 Calcitriol dosing 225 hypophosphatemia management 228 Calcium body weight relationship 9 cascade 17 conversion from SI units 291 homeostasis 9, 93–95 transport 10, 11 Calcium-sensing receptor (CaSR) antibodies 84 calcium homeostasis role 94 mutations 18–20, 102–104 signaling 17, 18 structure 17 Camurati-Engelmann disease features 204, 205 gene mutations 196, 197 Cancer, secondary hypercalcemia 106, 107 CaSR, see Calcium-sensing receptor Cathepsin K, inhibitor therapy 227 Chemotherapy, osteoporosis risks 182 Child abuse, see Non-accidental injury Chronic renal failure, secondary hypercalcemia 108 Cinalacalcet, hypercalcemia management 226 Cleidocranial dysplasia case history 279, 280 features 215 gene mutations 198, 199 Collagens mutations and diseases 36, 37, 158, 161–163 types 40–42 Computed tomography, see Quantitative computed tomography Congenital hypoparathyroidism, hypocalcemia 78 Conversion factors, SI units 291 Diabetes, osteoporosis risks 181 Di George syndrome case history 248–250 hypocalcemia 79 Dual-energy X-ray absorptiometry (DXA) diagnostic potential 64, 65

294

limitations 61–63 principles 60, 61 size adjustment techniques allometric approach 63 bone mineral content for height 63 mechanostat functional model 64 regression models 63, 64 volumetric bone density for age 63 DXA, see Dual-energy X-ray absorptiometry Exercise, osteoporosis management 184, 185 Familial benign hypercalcemia (FBH), hypercalcemia and features 102, 103 Familial hypocalcemia, hypocalcemia 83, 84 Familial isolated primary hyperparathyroidism, hypercalcemia and features 99, 100 FBH, see Familial benign hypercalcemia FGF23, see Fibroblast growth factor-23 Fibroblast growth factor-23 (FGF23) assays 136, 137 glycosylation 15 history of study 5 hypophosphatemic disorders 143, 145–147, 149 Klotho complex 16, 141 mutations 140, 141 phosphate homeostasis regulation 133, 134 receptor 44 renal effects 15, 16 secretion regulation 16, 17 structure 14, 15 Fibrodysplasia ossificans progressiva (FOP) features 205, 206 gene mutations 196, 197 FOP, see Fibrodysplasia ossificans progressiva Fractures, see Non-accidental injury GACI, see Generalized arterial calcification of infancy Generalized arterial calcification of infancy (GACI) features 212, 213 gene mutations 198, 199 Geroderma osteodysplasticum (GO) features 208 gene mutations 196, 197 Gestational maternal hypocalcemia, neonatal hypercalcemia 110

Subject Index

Glucocorticoids hypercalcemia management 226 osteoporosis pathophysiology 174, 175 risks 178, 179 GNAS, mutation in Albright’s hereditary osteodystrophy 85, 86 GO, see Geroderma osteodysplasticum Growth hormone deficiency and osteoporosis risks 180 osteogenesis imperfecta management 167 HDR syndrome, hypocalcemia 79, 80 Hectoral, dosing 224 Hemodialysis, hypercalcemia management 112 Heparin, osteoporosis risks 182 Hereditary hypophosphatemic rickets with hypercalciuria (HHRH), features 147, 148 HHRH, see Hereditary hypophosphatemic rickets with hypercalciuria Hypercalcemia calcium-sensing receptor mutations 102–104 case histories 255–260 clinical features 95, 96 drug therapy bisphosphonates 226 calcitonin 226 cinalacalcet 226 glucocorticoids 226 loop diuretics 225, 226 phosphate supplementation 226 familial benign hypercalcemia 102, 103 familial isolated primary hyperparathyroidism 99, 100 genetic causes 98 gestational maternal hypocalcemia and neonatal hypercalcemia 110 hyperparathyroid jaw-tumor syndrome 101, 256, 257 hypophosphatasia 106 Jansen metaphyseal chondrodysplasia 105 laboratory findings 95, 97 Lightwood syndrome 105, 106 multiple endocrine neoplasia type I 100 type II 10 type IV 101 neonatal severe hyperparathyroidism 102, 104, 258

Subject Index

parathyroid gland abnormalities adenoma 101 carcinoma 101, 10 pathophysiology 95 primary hyperparathyroidism 96, 97, 255, 256 secondary hypercalcemia causes 99 chronic renal failure 108 drug induction 109 endocrine causes 107 immobilization 108 malignancy 106, 107 subcutaneous fat necrosis 109, 259, 260 tertiary hyperparathyroidism 102, 257, 258 treatment 110–112 Williams-Beuren syndrome 104, 105 Hyperparathyroid jaw-tumor syndrome case history 256, 257 hypercalcemia and features 101 Hyperphosphatemia clinical assessment 135–137 clinical signs 137–139 drug therapy loop diuretics 228 phosphate binders 227 gene mutations and disorders 144, 145 genetic testing 139, 140 hyperphosphatemic familial tumoral calcinosis clinical features 140, 141 gene mutations 141, 143 types 140, 141 Hypocalcemia acquired hypoparathyroidism 80, 81 autosomal-dominant hypocalcemia 252, 253 calcium-sensing receptor antibodies 84 case histories 247–255 classification and etiology 77, 78, 87, 88 clinical manifestations 75 congenital hypoparathyroidism 78 Di George syndrome 79, 248–250 drug therapy 226, 227 familial hypocalcemia 83, 84, 247, 248 HDR syndrome 79, 80 hypomagnesemia acquired hypomagnesemia 83 hypermagnesuria 82, 83 hypomagnesuria 82 overview 81, 251

295

Hypocalcemia (continued) isolated congenital hypoparathyroidism 78, 79, 247 Kearns-Sayre syndrome 80, 251 laboratory findings 75–77 osteopetrosis 89 physiological response 73, 74 pseudohypoparathyroidism overview 84, 85 type Ia 85, 253, 254 type Ib 85, 86, 255 type Ic 86 type II 86 treatment 89, 90 vitamin D deficiency 86, 88, 89 vitamin D deficiency and convulsions 120 Hypogonadism, osteoporosis risks 180 Hypomagnesemia case history 251 hypocalcemia acquired hypomagnesemia 83 hypomagnesuria 82, 83 treatment 90, 227 Hypophosphatasia case histories infantile hypophosphatasia 275, 276 juvenile hypophosphatasia 276 forms and features 210–212 gene mutations 196, 197 hypercalcemia and features 106 Hypophosphatemia autosomal dominant hypophosphatemic rickets 147 autosomal recessive hypophosphatemia 147 clinical assessment 135–137 clinical signs 139 epidemiology 135 gene mutations and disorders 144, 145 genetic testing 139, 140 hereditary hypophosphatemic rickets with hypercalciuria 147, 148 hypophosphatemia with osteoporosis and nephrolithiasis 148 Jansen’s metaphyseal chondrodysplasia 149 management calcitriol 228 phosphate supplements 228 McCune-Albright syndrome 149 opsismodysplasia 149

296

osteoglophonic dysplasia 148, 149 renal transplant hypophosphatemia 145, 146 Schimmelpenning-Fuerstein-Mims syndrome 149 tumor-induced osteomalacia 143, 145 X-linked dominant hypophosphatemia 146 Hypothyroidism, osteoporosis risks 181 IBD, see Inflammatory bowel disease Idiopathic hypercalcemia of infancy, see Lightwood syndrome Idiopathic juvenile osteoporosis (IJO) case history 273, 274 features 207 gene mutations 196, 197 IJO, see Idiopathic juvenile osteoporosis Immobilization osteoporosis risks 179, 180 secondary hypercalcemia 108 Inflammatory bowel disease (IBD), osteoporosis association 177, 178 Isolated congenital hypoparathyroidism, hypocalcemia 78, 79 Jansen metaphyseal chondrodysplasia case history 276, 277 features 213, 214 gene mutations 198, 199 hypercalcemia and features 105 hypophosphatemia 149 JPD, see Juvenile Paget’s disease Juvenile Paget’s disease (JPD) features 207, 208 gene mutations 196, 197 Kearns-Sayre syndrome case history 250 hypocalcemia 80 Klotho fibroblast growth factor-23 complex 16 mutations 141, 143 Leukemia, presenting as osteoporosis 271, 272 Lightwood syndrome, hypercalcemia and features 105, 106 Loop diuretics hypercalcemia management 225, 226 hyperphosphatemia management 228

Subject Index

LRP5 high bone mass disease from activating mutations 196, 197, 203 mutations and bone mass 40 Magnesium conversion from SI units 291 homeostasis 11, 12 metabolism 12 transport 12, 13 Magnetic resonance imaging (MRI), bone mineral density measurement 68 MAS/FD, see McCune-Albright syndrome and fibrous dysplasia McCune-Albright syndrome and fibrous dysplasia (MAS/FD) features 208–210 gene mutations 196, 197 hypophosphatemia 149 MEN, see Multiple endocrine neoplasia MRI, see Magnetic resonance imaging Multiple endocrine neoplasia (MEN), hypercalcemia type I 100 type II 10 type IV 101 NAI, see Non-accidental injury Neonatal severe hyperparathyroidism (NSHPT) case history 258 hypercalcemia and features 102, 104 NFTC, see Normophosphatemic tumoral calcinosis Non-accidental injury (NAI) fractures dating 243, 244 distribution 236 long bones 240, 241 metaphyseal fractures 237, 239 radiological investigation 234, 235 ribs 236, 237 skull 241 specificity 236 unusual fractures 241, 242 frequency 233 subperiosteal new bone formation 238, 240 Normophosphatemic tumoral calcinosis (NFTC), features 150 NSHPT, see Neonatal severe hyperparathyroidism

Subject Index

OI, see Osteogenesis imperfecta OPPG, see Osteoporosis-pseudoglioma syndrome Opsismodysplasia, hypophosphatemia 149 Osteoblast differentiation 35, 40 function 35 osteoporosis pathophysiology 173, 174 Osteoclast differentiation and function 42, 43 osteoporosis pathophysiology 173 Osteocyte functions 42 osteoporosis pathophysiology 174 Osteogenesis imperfecta (OI) case histories 269–274 classification and gene mutations 158, 161–163 epidemiology 157, 158 evaluation differential diagnosis 160 initial examination 159, 160 laboratory testing 160, 163 medical history 158, 159 management basilar invagination 166 bisphosphonates 166, 167 growth hormone therapy 167 intramedullary rodding 165, 166 overview 164, 165 spinal surgery 166 surgery and anesthesia 165 type I 269, 270 type IV 270, 271 Osteoglophonic dysplasia, hypophosphatemia 148, 149 Osteopetrosis distal renal tubular acidosis in type 3 disease 130 gene mutations 194, 195 hypocalcemia 89 management of autosomal-recessive disease 192, 193 radiological findings 200–202 types autosomal-dominant form 199, 274, 275 autosomal-recessive form 191, 192 iatrogenic osteopetrosis 200 intermediate autosomal-recessive form 198, 199

297

Osteopetrosis (continued) osteoclast-poor autosomal-recessive infantile form 193, 198 osteocyte-rich autosomal-recessive infantile form 192 osteopetrosis with renal tubular acidosis and cerebral calcification 200 Osteoporosis, see also Osteogenesis imperfecta case histories 269–274 definition for children 157, 171 diagnostic approaches 182–184 pathophysiology glucocorticoids 174, 175 osteoblasts 173, 174 osteoclasts 173 osteocytes 174 overview 171 prevention and treatment bisphosphonates 185, 186 general approach 184 physical activity 184, 185 secondary osteoporosis etiologies anorexia nervosa 181 anticonvulsants 181, 182 chemotherapy 182 diabetes 181 glucocorticoids 178, 179 growth hormone deficiency 180 heparin 182 hypogonadism 180 hypothyroidism 181 immobilization 179, 180 inflammation 177, 178 overview 175–177 vitamin D deficiency 181 Osteoporosis-pseudoglioma syndrome (OPPG) case history 272, 273 features 207 gene mutations 196, 197 Osteoprotegerin, therapeutic applications 229 Parathyroid glands defects 20 development 20 hypercalcemia adenoma 101 carcinoma 101, 10 Parathyroid hormone (PTH) calcium homeostasis role 9, 43, 44, 94 conversion from SI units 291

298

familial isolated primary hyperparathyroidism mutations 99 history of study 3 hypocalcemia response 73, 74 processing 21 receptors mutations 21, 22 signaling 21, 22 types 21 structure 20 synthesis 20, 21 target organs 22–24 therapeutic applications 230, 231 Parathyroid hormone-related peptide (PTHrP) calcium homeostasis role 9 function 24, 94, 95 history of study 4, 24 secondary hypercalcemia in malignancy 106, 107 structure 24 Paricalcitol, dosing 225 Phosphate binders for hyperphosphatemia management 227 body weight relationship 13 clinical assessment 135–137 conversion from SI units 291 homeostasis regulation 133, 134 metabolism 14–16 supplementation for hypercalcemia management 226 supplements for hypophosphatemia management 228 transport 14 POH, see Progressive osseous heteroplasia PRAD1, familial isolated primary hyperparathyroidism mutations 99, 100 Primary hyperparathyroidism case history 255, 256 hypercalcemia and features 96, 97 Progressive diaphyseal dysplasia, see CamuratiEngelmann disease Progressive osseous heteroplasia (POH) features 206 gene mutations 196, 197 Pseudohypoparathyroidism case histories 253–255 overview 84, 85 treatment 90 type Ia 85, 253, 254 type Ib 85, 86, 255

Subject Index

type Ic 86 type II 86 PTH, see Parathyroid hormone PTHrP, see Parathyroid hormone-related peptide Pulmonary alveolar microlithiasis, features 151 Pycnodysostosis clinical features 202 gene mutations 194, 195, 202 management 203 QCT, see Quantitative computed tomography Quantitative computed tomography (QCT) diagnostic potential 68 limitations axial quantitative computed tomography 66 peripheral quantitative computed tomography 67 principles 65 techniques 65–68 Quantitative ultrasound (QUS), bone mineral density measurement 69 QUS, see Quantitative ultrasound RANK ligand, inhibitor therapy 227 Renal transplant hypophosphatemia, features 145, 146 Renal tubular acidosis (RTA) distal renal tubular acidosis 129 distal renal tubular acidosis and elliptocytosis 129, 130 distal renal tubular acidosis with progressive deafness 129 osteopetrosis type 3 130 osteopetrosis with renal tubular acidosis and cerebral calcification 200 overview 128 treatment 130 Rickets case histories 260–269 history of study 2 hypophosphatemia autosomal dominant hypophosphatemic rickets 147 hereditary hypophosphatemic rickets with hypercalciuria 147, 148 laboratory findings 117 renal tubular acidosis distal renal tubular acidosis 129

Subject Index

distal renal tubular acidosis and elliptocytosis 129, 130 distal renal tubular acidosis with progressive deafness 129 osteopetrosis type 3 130 overview 128 treatment 130 vitamin D deficiency rickets calcium deficiency rickets 120, 121 classical rickets 119 congenital rickets 118, 260, 261 metabolic gene defects 25-hydroxylase 122 vitamin D-dependent rickets type 1 122, 123, 264, 265 vitamin D-dependent rickets type 2 123, 128, 265, 266 vitamin D-dependent rickets with normal receptor 128 treatment 121, 122 RTA, see Renal tubular acidosis SCFN, see Subcutaneous fat necrosis Schimmelpenning-Fuerstein-Mims syndrome, hypophosphatemia 149 Sclerosteosis features 203 gene mutations 194, 195 Sclerostin, inhibitor therapy 227 SI units, conversions 291 Subcutaneous fat necrosis (SCFN) case history 259, 260 hypercalcemia and features 109 Tertiary hyperparathyroidism case history 257, 258 hypercalcemia and features 102 Thyrotoxicosis, secondary hypercalcemia 107 TIO, see Tumor-induced osteomalacia TRP5, calcium transport 11, 44 TRP6 calcium transport 11, 44 magnesium transport 12, 13 Tumor-induced osteomalacia (TIO), hypophosphatemia 143, 145 Van Buchem disease features 203 gene mutations 194, 195 Vitamin A toxicity, secondary hypercalcemia 109

299

Vitamin D calcium homeostasis role 9, 94 conversion from SI units 291 daily recommended intake for children 225 history of study 2 supplementation alfacalcidol 224 calcitriol 225 toxicity 224 synthesis 25, 26, 28, 223 Vitamin D deficiency, see also Rickets calcium deficiency rickets 120, 121 case histories 260–266 classical rickets 119, 262, 263 congenital rickets 118 dilated cardiomyopathy 118, 119, 261, 262 gene defects in etiology 124–127 generalized aches and pains 120 hypocalcemia 86, 88, 89 hypocalcemic convulsions 120 metabolic gene defects 25-hydroxylase 122 vitamin D-dependent rickets type 1 122, 123

300

vitamin D-dependent rickets type 2 123, 128 vitamin D-dependent rickets with normal receptor 128 osteoporosis risks 181 overview 116 stages 117 treatment 90 Vitamin D receptor ligands 28 mutations 28, 29, 123, 128 Williams-Beuren syndrome, hypercalcemia and features 104, 105 X-linked dominant hypophosphatemia (XLH), features 146, 266, 267 XLH, see X-linked dominant hypophosphatemia

Subject Index