Genetic Disorders of Endocrine Neoplasia (Frontiers of Hormone Research)

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Author: A. B. Grossman | P. L. M. Dahia | Charis Eng

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Genetic Disorders of Endocrine Neoplasia

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Frontiers of Hormone Research Vol. 28

Series Editor

Ashley B. Grossman

ABC

London

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Genetic Disorders of Endocrine Neoplasia Volume Editors

P.L.M. Dahia Boston, Mass. C. Eng Columbus, Ohio

17 figures, 5 in color, and 13 tables, 2001

ABC

Basel W Freiburg W Paris W London W New York W New Delhi W Bangkok W Singapore W Tokyo W Sydney

Patricia L.M. Dahia, MD, PhD

Charis Eng, MD, PhD

Department of Cancer Biology Dana-Faber Cancer Institute Harvard Medical School Boston, Mass., USA

Human Cancer Genetics Program Comprehensive Cancer Center Division of Human Genetics Department of Internal Medicine The Ohio State University Columbus, Ohio, USA

Library of Congress Cataloging-in-Publication Data Genetic disorders of endocrine neoplasia / volume editors, P.L.M. Dahia, C. Eng. p. ; cm. -- (Frontiers of hormone research ; vol. 28) Includes bibliographical references and index. ISBN 3 8055 7203 4 (hard cover : alk. paper) 1. Endocrine glands--Cancer--Genetic aspects. I. Dahia, (Patricia L.M.) II. Eng, Charis, 1962- III. Frontiers of hormone research ; v. 28 [DNLM: 1. Endocrine Gland Neoplasms--genetics. 2. Multiple Endocrine Neoplasia--genetics. WK 140 G328 2000] RC280.E55 G45 2000 616.99’44042--dc21 2001029067

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 2001 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISBN 3–8055–7203–4

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Contents

VII Preface Grossman, A.B. (London) 1 Introduction Dahia, P.L.M. (Boston, Mass.); Eng, C. (Columbus, Ohio)

8 Hereditary Endocrine Neoplasias: Fundamental Insights and the Practice

of Clinical Cancer Genetics Dahia, P.L.M. (Boston, Mass.); Eng, C. (Columbus, Ohio) 20 Identification and Characterization of Disease-Related Genes:

Focus on Endocrine Neoplasias Aguiar, R.C.T.; Dahia, P.L.M. (Boston, Mass.) 50 Clinical and Molecular Aspects of Multiple Endocrine Neoplasia Type 1 Chandrasekharappa, S.C. (Bethesda, Md.); Tean Teh, B. (Grand Rapids, Mich.) 81 Multiple Endocrine Neoplasia Type 2: Molecular Aspects Mulligan, L.M. (Kingston) 103 Multiple Endocrine Neoplasia Type 2: Clinical Aspects Gimm, O. (Halle-Wittenberg) 131 Von Hippel-Lindau Disease: Genetic and Clinical Observations Iliopoulos, O. (Boston, Mass.) 167 Hamartoma and Lentiginosis Syndromes: Clinical and Molecular Aspects Marsh, D.J. (Sydney); Stratakis, C.A. (Bethesda, Md.)

214 Subject Index

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Preface

Genetic syndromes have always fascinated endocrinologists, but in the face of their apparent rarity and obscure manifestations they remained somewhat of a minority interest. Then, in 1993, with the identification of the molecular defect in the Sipple syndrome, now referred to as multiple endocrine neoplasia type 2 (MEN 2), the whole field became massively transformed. In the first place, it became possible to screen directly for mutations for MEN 2 as well as familial medullary carcinoma of the thyroid and other related conditions, thus considerably simplifying the accurate prediction of risk and the follow-up of such patients. In particular, the presence of ‘hot-spots’ identifying with some precision genotype-phenotype correlations, for the first time gave the clinician the means to forecast and advise. Not only were patients given accurate predictive information, but many other unaffected relatives were spared the investigation and anxiety by knowing that they were mutation-free. This has also impacted directly on treatment, as certain knowledge of the affected relative has allowed for prophylactic thyroidectomy in early childhood in order to avoid later neoplasia. By contrast, the positional cloning of the MEN 1 gene took much longer than many expected, with many false starts, and then the eventual discovery of a gene which even now is only poorly characterised from a functional point of view. In addition, the huge number of scattered mutations, albeit with occasional ‘warm-spots’, and the lack of a close genotype-phenotype correlation, has meant that its clinical usefulness is presently limited. In spite of these caveats, there is no doubt that the recent molecular discovery of these and other genes such as those associated with von Hippel-Lindau syndrome, Cowden syndrome, and, very recently, one variant of the Carney complex, has led to an enormous increase in interest in these diseases. It is becoming increasingly clear that they are much more common than

VII

previously recognised, and that the genes involved may well be involved in the more frequent sporadic tumours. Most importantly, they are adding considerably to our understanding of cancer in general, similar to the hereditary disorders of the colon, and indubitably will add to more effective clinical management and eventually therapy. Patricia Dahia and Charis Eng have assembled an impressive group of authors in this volume, covering all of the major hereditary endocrine neoplasia syndromes: most, if not all, of these contributors were intimately involved in the initial discovery of the relevant genetic pathology. I am most grateful to the editors for putting together this review, which just a new years ago would have consisted of advisory clinical guidelines and genetic uncertainty. It is a tribute to all the workers in this field how far we have come in so short a time. Ashley Grossman, London

Preface

VIII

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Introduction Patricia L.M. Dahia, Charis Eng

This Volume at a Glance: An Overview of Hereditary Endocrine Neoplasias The genetics of endocrine cancers, which is the major focus of this volume, is clearly an area where new scientific discoveries that have occurred in the past few years have already been translated into clinical practice. This volume attempts to provide a state-of-the-art review of some of the most relevant inherited syndromes that share a higher susceptibility to the development of endocrine tumors. Endocrine neoplasia syndromes are commonly associated with hyperfunction features (hormone hypersecretion), such as the hyperparathyroidism seen in multiple endocrine neoplasia type 1 (MEN 1) and MEN type 2 (MEN 2) syndromes, pheochromocytoma in MEN 2 and hyperinsulinism in MEN 1, although not all endocrine hyperfunction syndromes are neoplastic in nature [see ref. 1 for a review]. Occasionally, mutations of genes usually associated with nonneoplastic hyperfunction syndromes [1–3] can be found in somatic tissue, but no germline mutations associated with neoplastic hyperfunction of these genes have been reported. These occasional mutations will not be addressed in this book. Instead, familial tumor syndromes for which the primary gene defect has been well characterized or, in one case (the Carney complex), recently identified will be presented. We start this volume by addressing a general aspect of today’s molecular medicine, namely, the strategies behind the characterization of novel genes, which, we hope, will help clinicians with little hands-on experience at the bench to

understand the technical elements involved in such studies. Following this introduction to cloning strategies and gene characterization in cancer, specific heritable endocrine neoplastic diseases will be discussed. Some of the disorders addressed have been described together before, such as the MENs. Others, such as the large group of hamartoma and lentigines syndromes, have not received much attention in the past, but in this volume, these syndromes have gained an entire chapter that deals with their endocrine, as well as nonendocrine, clinical and molecular aspects. The prototypic model of an inherited cancer syndrome where molecular medicine is practiced as the standard of care is MEN 2 (discussed in detail in the chapters by Mulligan and Gimm). In MEN 2, molecular analysis has allowed a full assessment of individuals at risk, the results of which alter therapeutic decisions. This syndrome comprises medullary thyroid carcinoma, pheochromocytoma and hyperparathyroidism. It has long been known that hyperplasia of thyroid C cells precedes the fully developed thyroid carcinoma in this syndrome, and biochemical stimulation tests with pentagastrin and/or calcium have been routinely used to characterize the presence of this preneoplastic defect, as extensively discussed in the two chapters on MEN 2 in this volume. Although better than having no means of screening, the serial measurement of stimulated calcitonin levels has its disadvantages. For example, it displays age-related sensitivity, which, of course, defeats the purpose, given that the disease can become manifest well before the age of 10 years. False positives and negatives have been amply documented (as discussed in the MEN 2 chapters). In addition, cumbersome and unpleasant annual biochemical tests are required for at-risk individuals, half of whom will eventually turn out not to have the syndrome, until the age of 35, when all individuals with MEN 2 would produce an abnormal test [4]. So, clinicians were left with a suboptimal strategy for ‘early diagnosis’ of the disease. Not until the gene defect responsible for the disease was characterized, in 1993 [5], was it possible to increase the accuracy and precision of the diagnostic measures for MEN 2. Molecular tests involving the screening of the receptor tyrosine kinase gene RET for mutations are now routinely employed in reference laboratories throughout the world. In 195% of MEN 2 families, a germline RET mutation can be identified [6]. As discussed in the chapter by Gimm, germline RET mutation testing should start with a known affected individual in a given MEN 2 family. Once the family-specific mutation is identified, other as yet unaffected at-risk family members may be subjected to gene testing only looking for the presence or absence of the family-specific mutation. This sort of strategy is 100% sensitive and 100% specific, and is not age related. Identification of the mutation in individuals of any age, including newborns, warrants prophylactic thyroidectomy. Absence of the family-specific mutation enables the physician to exclude these particular individuals from the annual screening and the need for further tests.

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Such clinical decisions based on molecular facts are the paradigm of molecular medicine in the 21st century. In parallel with the genetic advances in the MEN 2 and RET field, basic bench research on RET and its fundamental actions had been going on well before RET and MEN 2 were linked, and a vast body of knowledge has been amassed (see the chapter by Mulligan). The chapter by Chandrasekharappa and Teh in this volume addresses MEN 1. This syndrome comprises pituitary, parathyroid and pancreatic tumors inherited in an autosomal dominant manner. The putative gene for MEN 1 was mapped to 11q23 using linkage analysis [7]. Although the gene responsible for increased susceptibility to MEN 1 was mapped at almost the same time as that for MEN 2, the identification of the gene, MEN1, came 4 years after RET was associated with MEN 2 [8]. The MEN1 gene was found to encode a protein of unknown function and with no homology to other known proteins in the database. While there is still much to be discovered about the function of MENIN, it is already known that it is a nuclear protein with the ability to interact with the transcription factor JunD [9]. Several studies have already addressed the issue of genotypephenotype correlations in MEN 1 families. Unlike MEN 2, however, no clear association between mutation type and clinical feature(s) has been characterized in many families studied so far. Interestingly, related familial syndromes, such as isolated familial hyperparathyroidism, until recently not clearly established as an incomplete MEN 1 syndrome, have recently been shown to result from mutations clustered at a specific ‘warm-spot’ area of the gene. On the other hand, other syndromes also considered to potentially represent incomplete MEN 1, such as familial acromegaly, do not carry MEN1 mutations. These recent molecular data have already started to be taken into account in patient management. Although screening of the proband in a MEN 1 family requires the analysis of the entire gene, once the mutation is identified in a kindred, all individuals at risk can be assessed in a focused manner, much like the approach employed for MEN 2 genetic testing. Due to the high penetrance of the disease and the high morbidity, associated mainly with malignant pancreatic tumors, individuals with the mutation can be treated in an early phase of their disease. While no long-term study is available yet, it is expected that this knowledge will improve the prognosis of MEN1 patients. The intriguing data on the role of MEN1 in certain, but not all, associated inherited neoplasia syndromes, as well as other details on recent advances in the clinical and basic aspects of MEN 1, is discussed in detail in the chapter by Chandrasekharappa and Teh in this volume. von Hippel-Lindau (VHL) syndrome is an autosomal dominant disorder caused by disruption of the VHL gene located at 3p25. This syndrome is characterized clinically by vascular tumors, including retinal and central nervous system hemangioblastomas, clear cell renal cell carcinoma and cysts of the kidneys, liver and pancreas. Pheochromocytomas occur in association with specific alleles of

Introduction

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the VHL gene. Studies of the natural history of VHL showed a life expectancy of less than 50 years before surveillance protocols were developed. Mutation analysis of the VHL gene has allowed presymptomatic identification of affected family members and is also considered the standard of care in the United States and most, if not all, of Europe. Individuals who are found not to have inherited the mutant gene do not need to be monitored. Unlike most, if not all, inherited cancer syndromes, VHL is unique in that virtually 100% of VHL cases have been found to have mutations if Southern blot analysis is used together with PCR-based approaches [10]. Families may be characterized by the presence (type 2; 7–20% of families) or absence (type 1) of pheochromocytomas. The clearest genotype-phenotype correlation seen in VHL cases stems from the classification above. Most type 2 families have missense mutations, whereas most type 1 families are affected by deletions or truncating mutations. Prediction of the lifetime risk of pheochromocytoma can be aided by determination of the underlying mutation in patients without family histories of VHL. There has been great progress in studies involving the biological role of the VHL protein. It is now known that the VHL product is part of protein complexes that contain elongin B, elongin C and Cul-2 [for reviews see ref. 11, 12]. These proteins inhibit the transcription of hypoxiainducible mRNAs. VHL appears to play a role in ubiquitination. In addition, this protein can also interact with fibronectin and takes part in the assembly of a fibronectin matrix. It has been shown that VHL plays a role in the ability of cells to exit the cell cycle, through induction of the cell cycle inhibitor p27KIP1 [13]. Recent progress in these aspects of VHL research will be discussed in detail in the chapter by Iliopoulos. While several protein kinases have been implicated as oncogenes, and phosphatases have been known to frequently antagonize their function, there has been no direct demonstration of the role of phosphatases in tumor development [14]. The characterization of PTEN as a bona fide tumor suppressor gene has confirmed that a deficient phosphatase can lead to cancer, as detailed by the studies described in the chapter by Marsh and Stratakis in this volume. The chromosome 10q23 region has long been found to represent an important target area in several human tumors, such as glioblastomas, prostate and breast cancer, endometrial neoplasms and hematological malignancies [15–20]. In 1996, Cowden syndrome (CS), an inherited multiple hamartoma syndrome with a high risk of breast and thyroid tumors, was mapped to the region 10q22–23 [21]. Another hamartoma syndrome linked to the same chromosomal region is Bannayan-Riley-Ruvalcaba syndrome (BRR). BRR also has an autosomal dominant trait and is clinically characterized by macrocephaly, lipomatosis and pigmented macules of the glans penis, Hashimoto’s thyroiditis and mental and developmental delay [22]. PTEN mutations have now been identified in up to 80 and 60% of cases of CS and BRR, respectively [23–29]. Not only do the two syndromes share some clinical features,

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but the mutations in some families are also identical, suggesting that these two disorders are allelic. Recent studies have attempted to establish genotype-phenotype correlations in CS and BRR [26, 28] and the results are summarized in this volume in the chapter by Marsh and Stratakis. In brief, PTEN mutation-positive CS and BRR are possibly different presentations of a single syndrome and hence, both should receive equal attention with respect to cancer surveillance [28]. It is possible that other modulating factors might be responsible for the observed clinical variation between CS and BRR. Approximately 20% of CS and 40% of BRR families still remain without an identifiable germline PTEN mutation. Individuals from these PTEN mutation-negative families cannot presently rely on a molecular diagnostic test for the disease. Nonetheless, the rapid identification of PTEN as the CS-BRR susceptibility gene, SMAD4 as a juvenile polyposis gene and LKB1/STK11 as the major Peutz-Jeghers syndrome gene has allowed molecular differentiation of these three syndromes with inherited hamartomatous polyps. The chapter on hamartoses by Marsh and Stratakis finishes on a very exciting note: the recent identification and partial characterization of one of at least two genes for Carney complex. Carney complex is characterized by primary pigmented nodular adrenal disease causing pituitary-independent Cushing syndrome, myxomas affecting multiple organs (heart, skin and breast), spotty skin pigmentation (lentigines and blue nevi) and tumors of three endocrine organs (adrenal, pituitary and testis). At least two putative susceptibility loci exist, at 2p16 and 17q22–23 [30, 31]. As discussed in the chapter by Marsh and Stratakis, the gene on 17q22–23 was recently identified as the gene encoding the alpha regulatory subunit of protein kinase A, PRKARIA [32]. In general, the germline mutations in Carney complex have been truncating, loss-of-function mutations. The precise mechanism whereby loss-of-function mutations in PRKARIA determine the phenotype still needs to be determined, although several possibilities, based on the known function of protein kinase A, can be hypothesized, as outlined in the chapter by Marsh and Stratakis. Finding the other susceptibility genes for Carney complex might also prove helpful in this regard. These are exciting times for human cancer genetics, and particularly so in the field of endocrine cancer genetics. The translation of RET mutation testing for MEN 2 and all medullary thyroid carcinoma (MTC) presentations into the routine clinical setting was a landmark. Genetic testing for other endocrine neoplasias followed rapidly. However, the challenge for the 21st century is to accurately predict who among those with germline mutations will get which particular organ-specific manifestation and at what age. Pharmacological prevention and cure based on the knowledge of the fundamental function of these susceptibility genes and their downstream pathways should be our goal for the not-too-distant future.

Introduction

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Marx SJ: Clinical review 109: Contrasting paradigms for hereditary hyperfunction of endocrine cells. J Clin Endocrinol Metab 1999;84:3001–3009. Vassart G: New pathophysiological mechanisms for hyperthyroidism. Horm Res 1997;48(suppl 4):47–50. Liu G, Duranteau L, Carel JC, Monroe J, Doyle DA, Shenker A: Leydig-cell tumors caused by an activating mutation of the gene encoding the luteinizing hormone receptor. N Engl J Med 1999;341: 1731–1736. Ponder BAJ, Ponder MA, Coffey R, Pembrey M, Gagel RF, Telenius-Berg M, Semple P, Easton DF: Risk estimation and screening in families of patients with medullary thyroid carcinoma. Lancet 1988;i: 397–400. Mulligan LM, Kwok JBJ, Healey CS, Elsdon MJ, Eng C, Gardner E, Love DR, Mole SE, Moore JK, Papi L, Ponder MA, Telenius H, Tunnacliffe A, Ponder BAJ: Germline mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458–460. Eng C, Mulligan LM: Mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2, related sporadic tumours and Hirschsprung disease. Hum Mutat 1997;9:97–109. Larsson C, Skogseid B, Oberg K, Nakamura Y, Nordenskjold M: Multiple endocrine neoplasia type 1 gene maps to chromosome 11 and is lost in insulinoma. Nature 1988;332:85–87. Chandrasekharappa SC, Guru SC, Manickam P, Olufemi SE, Collins FS, Emmert-Buck MR, Debelenko LV, Zhuang Z, Lubensky IA, Liotta LA, Crabtree JS, Wang Y, Roe BA, Weisemann J, Boguski MS, Agarwal SK, Kester MB, Kim YS, Heppner C, Dong Q, Spiegel AM, Burns AL, Marx SJ: Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 1997;276:404– 407. Agarwal SK, Guru SC, Heppner C, Erdos MR, Collins RM, Park SY, Saggar S, Chandrasekharappa SC, Collins FS, Spiegel AM, Marx SJ, Burns AL: Menin interacts with the AP1 transcription factor JunD and represses JunD-activated transcription. Cell 1999;96:143–152. Stolle C, Glenn G, Zbar B, Humphrey JS, Choyke P, Walther M, Pack S, Hurley K, Andrey C, Klausner R, Linehan WM: Improved detection of germline mutations in the von Hippel-Lindau disease tumor suppressor gene. Hum Mutat 1998;12:417–423. Kaelin WG, Iliopoulos O, Lonergan KM, Ohh M: Functions of the von Hippel-Lindau tumour suppressor protein. J Intern Med 1998;243:535–539. Maher ER, Kaelin WG Jr: von Hippel-Lindau disease. Medicine (Baltimore) 1997;76:381–391. Pause A, Lee S, Worrell RA, Chen DY, Burgess WH, Linehan WM, Klausner RD: The von HippelLindau tumor-suppressor gene product forms a stable complex with human CUL-2, a member of the Cdc53 family of proteins. Proc Natl Acad Sci USA 1997;94:2156–2161. Myers MP, Tonks NK: PTEN: Sometimes taking it off can be better than putting it on. Am J Hum Genet 1997;61:1234–1238. Rasheed BKA, McLendon RE, Friedman HS, Friedman AH, Fuchs HE, Bigner DD, Bigner SH: Chromosome 10 deletion mapping in human gliomas: A common deletion region in 10q25. Oncogene 1995;10:2243–2246. Pfeifer SL, Herzog TJ, Tribune DJ, Mutch DG, Gersell DJ, Goodfellow PJ: Allelic loss of sequence from the long arm of chromosome 10 and replication errors in endometrial cancers. Cancer Res 1995;55:1922–1926. Bose S, Wang SI, Terry MB, Hibshoosh H, Parsons R: Allelic loss of chromosome 10q23 is associated with tumor progression in breast carcinomas. Oncogene 1998;17:123–127. Albarosa R, Colombo BM, Roz L, Magnani I, Pollo B, Cirenei N, Giani C, Conti AMF, DiDonato S, Finocchiaro G: Deletion mapping of gliomas suggests the presence of two small regions for candidate tumor-suppressor genes in a 17-cM interval on chromosome 10q. Am J Hum Genet 1996;58: 1260–1267. Trybus TM, Burgess AC, Wojno KJ, Glover TW, Macoska JA: Distinct areas of allelic loss on chromosomal regions 10p and 10q in human prostate cancer. Cancer Res 1996;56:2263–2267. Kobayashi H, Hosoda F, Maseki N, Sakurai M, Imashuku S, Ohki M, Kaneko Y: Hematologic malignancies with the t(10;11) (p13;q21) have the same molecular event and a variety of morphologic or immunologic phenotypes. Genes Chromosomes Cancer 1997;20:253–259.

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Nelen MR, Padberg GW, Peeters EAJ, Lin AY, van den Helm B, Frants RR, Coulon V, Goldstein AM, van Reen MMM, Easton DF, Eeles RA, Hodgson S, Mulvihill JJ, Murday VA, Tucker MA, Mariman ECM, Starink TM, Ponder BAJ, Ropers HH, Kremer H, Longy M, Eng C: Localization of the gene for Cowden disease to 10q22–23. Nat Genet 1996;13:114–116. Eng C: Cowden syndrome. J Genet Couns 1997;6:181–191. Liaw D, Marsh DJ, Li J, Dahia PL, Wang SI, Zheng Z, Bose S, Call KM, Tsou HC, Peacocke M, Eng C, Parsons R: Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nat Genet 1997;16:64–67. Lynch ED, Ostermeyer EA, Lee MK, Arena JF, Ji H, Dann J, Swisshelm K, Suchard D, MacLeod PM, Kvinnsland S, Gjertsen BT, Heimdal K, Lubs H, Moller P, King MC: Inherited mutations in PTEN that are associated with breast cancer, Cowden disease, and juvenile polyposis. Am J Hum Genet 1997;61:1254–1260. Nelen MR, van Staveren WC, Peeters EA, Hassel MB, Gorlin RJ, Hamm H, Lindboe CF, Fryns JP, Sijmons RH, Woods DG, Mariman EC, Padberg GW, Kremer H: Germline mutations in the PTEN/MMAC1 gene in patients with Cowden disease. Hum Mol Genet 1997;6:1383–1387. Marsh DJ, Coulon V, Lunetta KL, Rocca-Serra P, Dahia PL, Zheng Z, Liaw D, Caron S, Duboue B, Lin AY, Richardson AL, Bonnetblanc JM, Bressieux JM, Cabarrot-Moreau A, Chompret A, Demange L, Eeles RA, Yahanda AM, Fearon ER, Fricker JP, Gorlin RJ, Hodgson SV, Huson S, Lacombe D, Eng C, et al: Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation. Hum Mol Genet 1998;7:507–515. Longy M, Coulon V, Duboue B, David A, Larregue M, Eng C, Amati P, Kraimps JL, Bottani A, Lacombe D, Bonneau D: Mutations of PTEN in patients with Bannayan-Riley-Ruvalcaba phenotype. J Med Genet 1998;35:886–889. Marsh DJ, Kum JB, Lunetta KL, Bennett MJ, Gorlin RJ, Ahmed SF, Bodurtha J, Crowe C, Curtis MA, Dasouki M, Dunn T, Feit H, Geraghty MT, Graham JM Jr, Hodgson SV, Hunter A, Korf BR, Manchester D, Miesfeldt S, Murday VA, Nathanson KL, Parisi M, Pober B, Romano C, Tolmie JL, Trembath R, Winter RM, Zackai EH, Zori RT, Weng LP, Dahia PL, Eng C: PTEN mutation spectrum and genotype-phenotype correlations in Bannayan-Riley-Ruvalcaba syndrome suggest a single entity with Cowden syndrome. Hum Mol Genet 1999;8:1461–1472. Tsou HC, Ping XL, Xie XX, Gruener AC, Zhang H, Nini R, Swisshelm K, Sybert V, Diamond TM, Sutphen R, Peacocke M: The genetic basis of Cowden’s syndrome: Three novel mutations in PTEN/ MMAC1/TEP1. Hum Genet 1998;102:467–473. Irvine AD, Armstrong DK, Bingham EA, Hadden DR, Nevin NC, Hughes AE: Evidence for a second genetic locus in Carney complex. Br J Dermatol 1998;139:572–576. Stratakis CA, Carney JA, Lin JP, Papanicolaou DA, Karl M, Kastner DL, Pras E, Chrousos GP: Carney complex, a familial multiple neoplasia and lentiginosis syndrome. Analysis of 11 kindreds and linkage to the short arm of chromosome 2. J Clin Invest 1996;97:699–705. Kirschner LS, Carney JA, Pack SD, Taymans SE, Giatzakis C, Cho YS, Cho-Chung YS, Stratakis CA: Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit in patients with the carney complex. Nat Genet 2000;26:89–92.

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Dahia PLM, Eng C (eds): Genetic Disorders of Endocrine Neoplasia. Front Horm Res. Basel, Karger, 2001, vol 28, pp 8–19

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Hereditary Endocrine Neoplasias: Fundamental Insights and the Practice of Clinical Cancer Genetics Patricia L.M. Dahia a, Charis Eng b a

b

Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Mass., and Clinical Cancer Genetics and Human Cancer Genetics Programs, Comprehensive Cancer Center and Division of Human Genetics, Department of Internal Medicine, The Ohio State University, Columbus, Ohio, USA

Contents 8 10 11 13 15 16 16

Introduction Classifying Cancer-Related Genes Gene Methylation Assessing Human Sequence Variation: The Answer to Phenotypic Diversity? A Word on Genetic Counseling Ethical Issues under Debate References

Introduction The field of cancer genetics has grown remarkably in the past decades. This most likely reflects trends in cellular and molecular biology, where basic research and disease studies are presently converging towards a comprehensive and deeper understanding of the biological processes underlying common and rare disorders.

Both authors contributed equally to this work.

The discovery of genes responsible for inherited forms of cancer has already given us powerful tools for cancer risk assessment, premorbid prediction and genetic counseling. Indeed, the discovery of the susceptibility gene for multiple endocrine neoplasia type 2 (see the chapters by Mulligan and Gimm [this vol.]) was fundamental in catalyzing the inception of the new medical subspecialty of clinical cancer genetics. Despite these early triumphs, further genetic and functional studies of hereditary cancers, including inherited endocrine neoplasia syndromes, could help refine our powers of prediction, prevention and treatment. The study of cancer-prone families is a powerful approach to cancer control. Studying such families has allowed us to isolate genes which, when mutated, give a susceptibility to cancer. In this era of molecular medicine, the identification of a family-specific germline mutation in a given susceptibility gene means that presymptomatic predictions can be made for other at-risk and as yet unaffected family members, so long as genetic counseling and clinical cancer genetic consultation are provided in the setting of genetic testing. Identification of the germline mutation for cancer susceptibility provides a unique opportunity to predict patients’ lifetime risk for certain cancers. Surveillance and management protocols, when merged with the particular syndrome’s natural history, can be lifesaving. This past decade has also been distinguished by the great advances that have been made with respect to the molecular mechanisms responsible for many inherited endocrinopathies. This knowledge has had an enormous impact on the clinical management of both affected individuals as well as those at risk. While clinical cancer genetics is still in its infancy, the genetic understanding of the inherited endocrine cancers as they relate to clinical practice leads the way. Gene testing for multiple endocrine neoplasia types 1 and 2 and von Hippel-Lindau (VHL) syndrome is considered the standard of care in the United States [1–2] and most, if not all, of Europe. Clinical testing for PTEN mutations in Cowden syndrome and its allelic variants has recently become available [2; Eng, unpubl. results]. Although much progress has been made in the current knowledge of the molecular aspects leading to cancer, there is still much to be discovered. To understand completely the mechanisms involved in the genesis and progression of cancer, it is critical to have comprehensive knowledge at the DNA level, as well as the steps leading to the processing of genetic information in cellular function. While this knowledge has mostly translated into diagnosis, management and assessment of prognosis, it is hoped that the years to come will bring further insights and tools that will also enable full translation into therapeutic options that can be tailored to patients according to their most specific ‘molecular requirements’. Since ‘an ounce of prevention is better than a pound of cure’, we should work towards molecular knowledge that would lead to a comprehensive and effective prevention plan.

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Classifying Cancer-Related Genes It has long been known that many human cancers result from disruption in two major groups of genes, namely, proto-oncogenes and tumor suppressor genes, the latter of which also includes repair genes [3, 4]. Discussion about these groups of genes is not within the scope of this chapter and the reader is referred to several comprehensive reviews in the literature dealing with this topic [3–7]. While this classification may appear to be an oversimplification in light of the complexity of current knowledge of molecular oncology, it is still valid from a generic point of view. Despite its broad use and acceptance, however, some researchers have recently advocated a new classification/nomenclature involving cancer-related genes [8], as discussed below. As early as 1902, it was recognized that cancer results from an imbalance of positive and negative regulators [9]. It is well established now that cancer is a disorder which behaves in a stepwise fashion, and it is presumed that several genetic defects accumulate in a single cell to overcome its multiple repair mechanisms so that it is finally transformed into a cancerous cell [10]. Examples of genes belonging to these categories are mentioned in other chapters in this volume. While a single mutant copy of a proto-oncogene might be sufficient to disrupt the fine control under which a cell maintains its growth and proliferation, it is believed that the two alleles of a tumor suppressor and a repair gene must be disrupted for the resulting cancer-prone effects to ensue. The latter mechanism is usually associated with a single-allele defect that is inherited and present in the germline DNA, with the second allele disruption occurring somatically later in life. Alternatively, two somatic events affecting the two alleles would need to occur in nonfamilial cancer cases. This mechanism was first proposed by Knudson [11] and has been known since as Knudson’s two-hit theory. While a more complete definition of tumor suppressor genes would require the existence of lossof-function mutations, inactivation in familial as well as in sporadic tumors and reversion of the tumor phenotype by the wild-type allele, this very stringent concept of tumor suppressor genes has been challenged recently [8]. The DNA repair genes are a complex category of genes; their loss is required for a phenotype to emerge, similar to the classic tumor suppressor genes [12–14]. However, as these genes are associated with a mutator phenotype, which is ultimately responsible for the cancer phenotype, reintroduction of the repair gene wild-type allele is not able to suppress cell proliferation. Further, as a wider knowledge of the function of several of the tumor suppressor genes has emerged, it is clear that they are associated with a broad spectrum of categories, including transcription factors, cell cycle inhibitors and genes involved in mRNA stability, which precludes a single classification based on gene function. Haber and Harlow [8] have proposed that tumor suppressor genes should be defined as ‘genes that sustain loss-of-function

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mutations in the development of cancer’. This is a simple definition and yet it is broad enough to include the most important requirements of a tumor suppressor gene. It does not deal, however, with specific issues that might arise with certain genes, such as the effect of dominant negative mutations and epigenetic effects such as allele imprinting and haploinsufficiency. It remains to be seen whether this proposed simplified classification of cancer genes will stand the test of time in the rapidly changing field of human cancer genetics.

Gene Methylation To add yet another degree of complexity to understanding the pathogenesis of cancers, it has become increasingly evident that factors other than purely structural alterations of the DNA sequence or gene copy number play a role in cancer. Epigenetic factors, such as DNA methylation of CpG islands, mainly those located in the promoter region of genes, which may interfere with transcription of the target gene, appear to contribute to the development of a number of tumors [15–19]. The mechanisms of methylation-mediated gene silencing are not well understood at present. The enzymes involved in this process are DNA methyltransferases, which catalyze the transfer of a methyl group from S-adenosyl-methionine to cytosine residues to form 5-methylcytosine, a modified base that is found mostly at CpG sites in the genome [20–22]. The class of methyltransferases is a growing family of genes, with more subtypes being identified in recent years. Currently, it is believed that the process of gene inactivation involves the initial methylation of CpG islands located in regulatory regions of genes and the subsequent recruitment of binding proteins that preferentially recognize methylated DNA [23, 24]. These proteins in turn associate with histone deacetylase and chromatin remodeling complexes to cause the stabilization of condensed chromatin. Alternatively, the presence of 5-methylcytosine may interfere with the binding of transcription factors or other DNA-binding proteins to block transcription [21, 22]. It has recently been proposed that disrupted chromatin structure might, on the other hand, be able to interfere with genes targeted for methylation [25]. Several categories of genes have been associated with abnormal methylation at the somatic level, including cell cycle regulators, genes that suppress metastasis and angiogenesis and genes that repair DNA, suggesting that epigenetics plays an important role in tumorigenesis [21, 22]. There are several examples of the importance of methylation as a mechanism of inactivation of genes associated with endocrine tumors. In pituitary tumors, it has been demonstrated that the p16CDK4 cell cycle inhibitor is methylated in almost 80% of nonfunctioning tumors [26]. The p16 gene maps to a chromosomal region, 9p21, known to be deleted frequent-

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ly in pituitary tumors [27]. The putative first hit in these tumors has therefore been found to be methylation of one allele rather than mutation. Another disease model in which methylation plays a critical role in gene inactivation is the VHL gene. Hypermethylation of VHL has been found in about 20% of tumors examined, and results in the suppression of VHL transcription [28, 29]. This phenomenon has been observed as a somatic event in sporadic as well as in inherited tumors and represents an additional mechanism of VHL inactivation apart from mutations and deletions of the VHL gene. More recently, the protein and lipid phosphatase gene PTEN, which is the susceptibility gene for the inherited hamartoma syndromes Cowden/Bannayan-Riley-Ruvalcaba, has been suggested to be methylated in a number of sporadic prostate cancers [30]. While this finding has yet to be confirmed by independent researchers, it suggests that similar to VHL, PTEN may be inactivated through a variety of mechanisms in tumors. Another important mechanism of control of gene expression, which is also related to methylation, is the phenomenon of imprinting. Although we have not included in this volume any syndrome where this mechanism of gene regulation plays a primary role in the disease pathogenesis, the relevance of the concept of genomic imprinting deserves a brief mention. Of particular interest to the field of endocrine neoplasias, genomic imprinting has been shown to be involved in the pathogenesis of Beckwith-Wiedemann syndrome, which is associated with adrenocortical carcinomas. Genomic imprinting is established during gametogenesis, and causes differential expression of maternally and paternally inherited alleles [31]. Neither androgenetic nor parthenogenetic embryos are viable, underscoring the need for precise gene imprinting for normal embryonic and fetal development. This requirement has been proposed to have evolved because of an interparental genetic ‘battle’ for the utilization of maternal resources during gestation and postnatally [32, 33]. The requirement for monoallelic expression of a subset of genes has also resulted in the formation of susceptibility loci for several human disorders, including cancer. Since imprinting involves both cytosine methylation within CpG islands and changes in chromatin structure, imprinted genes are potential targets for dysregulation by epigenetic factors that modify DNA methylation and histone acetylation [34, 35]. Imprinted genes are often clustered, suggesting that imprinting may be under the control of long-range regulatory elements. The two larger known clusters of autosomal imprinted genes in the human genome are located at chromosomes 15q11–12 and 11p15. Alterations in these clusters are associated with the PraderWilli/Angelman syndromes and the Beckwith-Wiedemann syndrome, respectively [36]. The Beckwith-Wiedemann syndrome is a neonatal overgrowth syndrome that predisposes to cancer, and the importance of a maternally active allele located at 11p15 in tumorigenesis is supported by the finding that loss of constitutive heterozygosity at chromosome 11p15 in sporadic Wilms’ tumors specifically

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involves maternal alleles [36]. Evidence of a role for abnormal imprinting in human tumorigenesis is the detection of loss of imprinting leading to biallelic expression of IGF-2 in Wilms’ tumor [37]. However, loss of imprinting represents a gain of expression of an otherwise imprinted IGF-2 allele. This is in contrast with the notion that the loss of maternal alleles at chromosome region 11p15 is a crucial event in tumorigenesis and the Beckwith-Wiedemann syndrome. A novel epigenetic mechanism has been reported in human liver cancer whereby loss of expression is observed in maternally expressed genes [38]. This mechanism has consequences similar to those caused by physical loss of genetic material, hence it is in agreement with the loss of constitutive heterozygosity at 11p15 detected in various human neoplasms and may explain the low frequency of somatic mutations in specific 11p15 genes. This novel mechanism results in the loss of expression of various imprinted genes at 11p15 and has been designated gain of imprinting [39].

Assessing Human Sequence Variation: The Answer to Phenotypic Diversity? All the diseases described in this volume are monogenic disorders, i.e. a single gene is responsible for the disease susceptibility. However, despite sharing the same genetic defect, it is clear that differences in the phenotype amongst distinct members of a family occur [40–42], as exemplified by some of the syndromes discussed in this volume (Cowden/Bannayan-Riley-Ruvalcaba syndrome, multiple endocrine neoplasia type 2 syndrome, VHL syndrome). The factors responsible for this phenotypic variation are not known. It is suspected that they might result from the effect of yet unidentified modulating factors. Some of these factors may represent interacting molecules present in the same signaling pathway of the target gene (exogenous modulating factors) or can represent small sequence variations within the gene itself (endogenous modulating factors) due, for example, to the existence of gene polymorphisms. There has recently been an increasing interest in the potential role of single-nucleotide polymorphisms (SNPs) as a component factor in the individual variation observed in certain disease phenotypes. This would be in agreement with the hypothesis that common genetic variants may contribute significantly to genetic risk for human diseases [43–45]. While this hypothesis still remains to be confirmed in large population-based studies and across a wide spectrum of gene-disease combinations, there are examples of disease associations with common alleles, such as the APOE allele in Alzheimer disease [46], the CCR5del32 in resistance to HIV infection [47] and the p53 codon 72ARG with certain subtypes of human papillomavirus-related cervical cancer [48], although these latter findings are somewhat controversial [49]. In addi-

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tion, it has been noted that there is an association between sequence variants in RET and sporadic medullary thyroid carcinoma [50] and Hirschsprung disease [51, 52], and between CUL2 and pheochromocytoma [53], although the functional significance of these associations still remains to be demonstrated. It is believed that these common variations within genes are present at high frequencies. It is estimated that there are close to 1 million SNPs in the human genome, with approximately 500,000 being non-coding SNPs, 200,000 being silent coding SNPs and 200,000 being replacement-coding SNPs. There are currently several studies under way interested in defining the nature of variation in human genes. Some of this work has been reported, and intends to provide a catalog of gene polymorphisms for association studies [54, 55]. These analyses use high-density variant detection arrays, which are probe arrays where oligonucleotides harboring specific variations have been immobilized and are hybridized with sequences from different individuals for genotyping. Instant analysis of multiple SNPs is likely to become an important tool to define associations previously overlooked when analyzing genes in relation to certain phenotypes. Sequence variation is almost certainly not the only underlying factor in phenotypical variation. Study of gene expression patterns may also disclose clues that may contribute to the understanding of the basis for differences observed in phenotypes, for example, in a given phase of disease development. Thus, while variant detection arrays are based on genetic variation of individual genes, there are also arrays composed of multiple genes that can be analyzed at once to determine the gene expression profile of a certain selected target template. These are the cDNA microarrays, which will, in the future, likely impact on cancer diagnosis, prognostication and therapy. Technical details and the applicability of these arrays are discussed in the chapter by Aguiar and Dahia [this vol.]. Microarray assays based on cDNA can measure the transcriptional effects of changes in gene function under different conditions [56–60]. They can reveal genes that are tissue specific and development specific, and genes that are affected by certain agents or that are responsive to particular modifications. A broad view of gene expression in cancer can bring clarity to previously unclear diagnostic categories. It represents a new way of approaching cancer therapeutics in the future. Surveys of gene expression in cancer will identify marker genes that will be used to stratify patients into molecularly relevant categories. This will improve the precision and power of clinical decisions. It can also provide a unique perspective on the development of new cancer therapeutics that could be based on a molecular understanding of the cancer phenotype. Such information will provide an important tool for future therapeutic strategies that can be individualized and will therefore hold higher chances of improving prognosis. However, we are currently far from this ideal; the scientific community is still pondering the bioinformatics challenge of merely analyzing these gargantuan amounts of data presented by a single array.

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Beyond genomics and transcript-level expression profiling comes the largescale analysis of proteins called proteomics [for a review, see ref. 61]. This topic is discussed in further detail in the chapter by Aguiar and Dahia [this vol.]. In brief, proteomics is a broad field and might be conveniently subdivided into three activities. The first involves protein microcharacterization for the large-scale identification of proteins and their role in posttranslational modifications. Secondly, examination of the proteomes of different cells (e.g. cancer versus normal) might be accomplished using a differential display approach for comparison of protein levels in health and disease. Thirdly, large-scale examination of protein-protein interactions and proteome-genomic interactions as they relate to health and disease would be the ultimate goal. Drug discovery will also be a major by-product of proteomic efforts. One of the main challenges of the future is to understand how genes and proteins interact to control the development or function of a system. It is to be hoped that by dissecting the critical components of such systems and understanding in depth the biology involved, we will eventually reach a level of knowledge that will allow the development of appropriate treatment for several human diseases.

A Word on Genetic Counseling Genetic counseling, in the broadest sense of the term, has been a very important integral part of these rapidly evolving times. The basic standards that have ruled traditional cancer genetics have been inundated by a mass of novel molecular data, with its consequences, limitations, drawbacks and, no doubt, improved power in terms of patient care. This is not to say that age-old dogmas such as careful evaluation of families for characteristic aggregation of tumor types among affected individuals, and the availability of affected persons for testing as being important issues in performing genetic testing and management are no longer in order. Indeed, in this era of molecular medicine, these rules hold more than ever before. Case reports illustrate the importance of genetic counseling as a component of cancer genetic risk assessment. Cancer risk assessment as part of the novel subspecialty of clinical cancer genetics is developing into a distinct discipline in which established empiric risk models are remodeled along with rapidly evolving genetic technologies for estimation of individual cancer risk [2, 62]. The genetic counseling process, as part of clinical cancer genetic consultation, includes exploration of patient risk perception, sources of anxiety related to cancer risk, patient education (specific cancer-related issues, prevention/intervention options), discussion of possible gene test options, test limitations and consequences of various gene test outcomes [63]. It is, in fact, the association of traditional genetic surveil-

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lance methods with modern information brought about by state-of-the-art technology that can provide the highest quality care to those affected by and at risk of inherited disorders. Although the impressive amount of genetic information which will soon become available will be critical for the development of new methods of screening and treatment, we should not lose sight of the impact of environmental influences on disease phenotypes. The importance of traditional epidemiologic studies will still remain in place for many years to come. This is an exciting era, in which some molecular advances can already be used to interfere with disease progression and management. This is merely the tip of the iceberg that heralds the future way of practicing medicine. We hope that the huge enthusiasm of those who work in the area and the general optimism that patient care will be improved are well founded. One way or the other, advances at the most basic genetic level will almost certainly lead us to practice translational medicine, a new concept in patient care and disease prevention.

Ethical Issues under Debate The more that is known about the importance of genes in defining an individual’s susceptibility to certain diseases, including cancer, the clearer it becomes that issues other than simply medical/scientific aspects are also relevant. Molecular epidemiologic information and the different ways it can be applied are increasingly becoming the source of much debate. It is not known presently how an individual’s molecular profile will affect his/her ability to interact with society. It is still unclear, for example, how such knowledge will influence health care insurance decisions, job opportunities and family planning, should this information be made public [64]. This ethical debate has only just begun, but already passionate advocates in both camps are thundering their opinions, with many favoring full disclosure of whichever ‘genetic truth’ is available, while others defend the right to a ‘genetic privacy’. The more conservative among us believe that for this and other related issues, a middle ground ‘of reason’ will likely be the right answer. We will certainly be faced with novel facets to this ever-growing topic in the years to come, but as physicians who practice molecular medicine, let us always remember ‘primum non nocere’ (above all, do no harm).

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Duerr EM, Gimm O, Neuberg DS, Kum JB, Clifford SC, Toledo SP, Maher ER, Dahia PL, Eng C: Differences in allelic distribution of two polymorphisms in the VHL-associated gene CUL2 in pheochromocytoma patients without somatic CUL2 mutations. J Clin Endocrinol Metab 1999;84: 3207–3211. Cargill M, Altshuler D, Ireland J, Sklar P, Ardlie K, Patil N, Shaw N, Lane CR, Lim EP, Kalyanaraman N, Nemesh J, Ziaugra L, Friedland L, Rolfe A, Warrington J, Lipshutz R, Daley GQ, Lander ES: Characterization of single-nucleotide polymorphisms in coding regions of human genes (published erratum appears in Nat Genet 1999;23:373). Nat Genet 1999;22:231–238. Halushka MK, Fan JB, Bentley K, Hsie L, Shen N, Weder A, Cooper R, Lipshutz R, Chakravarti A: Patterns of single-nucleotide polymorphisms in candidate genes for blood-pressure homeostasis. Nat Genet 1999;22:239–247. Khan J, Bittner ML, Chen Y, Meltzer PS, Trent JM: DNA microarray technology: The anticipated impact on the study of human disease. Biochim Biophys Acta 1999;1423:M17-M28. Debouck C, Goodfellow PN: DNA microarrays in drug discovery and development. Nat Genet 1999;21:48–50. Bowtell DD: Options available – from start to finish – for obtaining expression data by microarray (published erratum appears in Nat Genet 1999;21:241). Nat Genet 1999;21:25–32. Cheung VG, Morley M, Aguilar F, Massimi A, Kucherlapati R, Childs G: Making and reading microarrays. Nat Genet 1999;21:15–19. Ramsay G: DNA chips: State-of-the art. Nat Biotechnol 1998;16:40–44. Pandey A, Mann M: Proteomics to study genes and genomes. Nature 2000;405:837–846. Weitzel JN: Genetic cancer risk assessment. Putting it all together. Cancer 1999;86:2483–2492. Kelly PT: Will cancer risk assessment and counseling services survive genetic testing? Acta Oncol 1999;38:743–746. Severin MJ: Genetic susceptibility for specific cancers: Medical liability of the clinician. Cancer 1999;86:2564–2569.

Patricia Dahia, MD, PhD, Department of Cancer Biology, Dana-Farber Cancer Institute Harvard Medical School, 44 Binney Street SM1010, Boston, MA 02115-6084 (USA) Tel. +1 617 632 4664, Fax +1 617 632 4663, E-Mail [email protected] Dr. Charis Eng, Human Cancer Genetics Program Ohio State University Comprehensive Cancer Center 620 Medical Research Facility, 420 W. 12 th Avenue, Columbus, OH 43210 (USA) Tel. +1 614 688 4508, Fax +1 614 688 4245, E-mail [email protected]

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Dahia PLM, Eng C (eds): Genetic Disorders of Endocrine Neoplasia. Front Horm Res. Basel, Karger, 2001, vol 28, pp 20–49

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Identification and Characterization of Disease-Related Genes: Focus on Endocrine Neoplasias Ricardo C.T. Aguiar a, Patricia L.M. Dahia a, b Departments of a Adult Oncology and b Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Mass., USA

Contents 21 22 23 23 26 27 28 28 29 34 35 35 36 36 38 39 42 43

Introduction Positional Cloning Identification of a Candidate Area Familial Cancer Syndromes Sporadic Cancer Cloning of the Disease-Associated Gene Exon Trapping In silico Gene Discovery Gross Chromosomal Abnormalities Expression-Based Studies Differential Display Representational Difference Analysis Serial Analysis of Gene Expression Microarray-Based Approaches Protein-Related Strategies for Gene Discovery Analysis of Known Disease-Related Genes Conclusions References

Both authors contributed equally to this work.

Introduction The complete understanding of the molecular basis for every human disease, which at a minimum entails both the identification and functional characterization of genes (disciplines known as genomics and functional genomics, respectively), is the ultimate goal of modern medicine. The remarkable progress of recent years in this arena, largely related to disease gene identification, has already permanently changed our understanding of physiology and pathophysiology. Currently, this knowledge and techniques developed from it allow more precise diagnosis (including large-scale screening for carriers of disease-causing mutations), refined risk profiling and treatment surveillance, and will certainly be the blueprint for more rational drug design/discovery and ultimately will impact on treatment strategies and outcome. Obviously, the first step in this endeavor involves gene identification. In this chapter, we intend to introduce the reader to the most commonly utilized gene cloning techniques. Although some of these strategies can be used in a wide variety of situations, i.e. for Mendelian or non-Mendelian traits and malignant or benign disorders, the general approach used for gene identification can be remarkably specialized depending on the features associated with a particular disease. Therefore, we will indicate, whenever appropriate, the value of a given cloning strategy in distinct settings such as hereditary or sporadic endocrine cancers. Although the genes responsible for the disorders described in the following chapters of this volume have been characterized, many endocrine neoplasias, both inherited and sporadic, still remain without an identifiable primary molecular defect. Amongst these are certain familial forms of pheochromocytoma, familial papillary thyroid carcinoma, familial follicular thyroid carcinoma (and their associated syndromes, such as breast and thyroid cancer syndromes, for example), some inherited forms of parathyroid tumors and their sporadic counterparts. Nonfamilial endocrine cancers have only rarely been associated with molecular defects in genes commonly disrupted in other human cancers, such as the p53 gene, RAS and p16, among others. This paucity of data on endocrine tumors indicates that the majority of the molecular defects carried by these tumors are yet to be characterized, and the genes involved in endocrine tumorigenesis remain largely unknown. In view of the vast number of techniques currently available for gene identification, it is the aim of this chapter to provide an overview to endocrinologists of the strategies available to increase the molecular knowledge in this still unclear field. Finally, we will briefly discuss the analyses of the susceptibility genes for many endocrine tumor syndromes that have already been characterized, which frequently involve an entire host of different techniques.

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Fig. 1. Positional cloning strategies for gene identification. The figure shows a schematic representation of the techniques available for gene cloning and a stepwise application of the methods. Single-headed arrows represent the order in which the relevant techniques are usually applied. Double-headed arrows represent techniques that can be interchangeably used in the same step of the gene identification process. The asterisk indicates that molecular cytogenetic strategies could be used to further characterize the area initially identified by linkage analysis. SKY = Spectral karyotyping.

Positional Cloning The basic concept of positional cloning involves the initial identification of a chromosomal location (see sections below for a discussion of techniques), narrowed down to the smallest size possible, suspected to include the disease-associated gene. This is followed by the use of several strategies to identify the targeted gene and responsible mutations (fig. 1). Several familial and sporadic endocrine neoplasms (some of which are described in other chapters of this volume) have had their molecular basis estab-

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lished by the use of positional cloning strategies. In fact, once the candidate target area has been identified, the techniques used to clone the disease-related gene are virtually the same for familial and sporadic cancers. It is in the initial characterization of the candidate area, however, that these two groups of disorders differ most. In familial diseases, linkage analysis is widely used and is discussed in detail below. However, linkage analysis cannot be applied for directly mapping sporadic cancer genes. Hence, an important clue to the molecular basis of these tumors is not immediately available and, instead, other strategies are utilized to identify candidate genes, including mapping information obtained from polymorphic markers [loss of heterozygosity (LOH) studies] and the identification of gross or minute chromosomal defects. It should be noted, however, that the information from linkage analysis for familial neoplasia syndromes can also be applied to the study of sporadic counterparts of the component tumors, with results that vary according to the disorder in question [1–8]. The unique approaches to familial or sporadic cancers as well as the common strategies used after mapping of the candidate area are discussed in detail in the next sections.

Identification of a Candidate Area Familial Cancer Syndromes Very often, the first step towards identification of a susceptibility gene for familial cancers utilizes linkage analysis. This has been the case in many endocrine cancer syndromes, including the diseases discussed in this volume. A broad overview of the basis of linkage studies and information obtained from them is presented below. Linkage analysis is a powerful tool that has been widely employed in the search for susceptibility genes to human diseases. Its use started when it was recognized that human DNA variations could be assessed as genetic markers for such association studies [9]. Linkage studies have been widely employed in a variety of conditions, from rare syndromes to more common ones, and rely on genetic recombination to estimate the distance between certain loci [9–11]. This strategy takes advantage of naturally-occurring genetic variation represented by polymorphic markers, which are distributed throughout the genome, to analyze human families [12, 13]. Due to the meiotic process of recombination that occurs during cell division, markers that are close to the disease gene show a strong association with disease patterns in families. A statistical model is then applied to interpret the results [13–16]. The aim of linkage analysis is to establish whether two given loci segregate independently in meiosis. The closer the distance between two loci on the same

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chromosome, the higher the chance that their alleles are linked, i.e. that they are coinherited more than 50% of the time [10, 11]. By tracking recombination events, the region harboring the target gene can be narrowed down to distances in the order of hundreds to several thousand kilobases [11, 17]. Such markers can be used as a diagnostic tool and can help to characterize the actual disease susceptibility gene [12]. Typically, highly polymorphic markers such as microsatellite repeats for which the mapping location is known are used to determine haplotypes from affected and unaffected individuals of one or more kindreds where the disease is transmitted by inheritance [12, 15, 16]. While in the past the identification of haplotypes relied on the use of Southern blot analysis of relevant polymorphic areas based on differential restriction digestion (restriction fragment length polymorphism), currently, polymerase chain reaction (PCR) is the method of choice for the amplification of highly polymorphic microsatellites [18]. The primers are designed to flank repetitive areas of the genome that have high rates of heterozygosity and therefore are more commonly informative [18]. Once haplotypes are generated with the use of marker-specific PCRs from each individual, samples are analyzed for the presence of a genotype that segregates with the disease and is not shared by known unaffected individuals [19]. A null hypothesis of lack of association between a given genotype and the phenotype is then tested. If the null hypothesis is rejected, it is assumed that there is an observed association (or linkage) between the genotype in a specific area and the phenotype being studied [20]. The power of linkage analysis is highest for Mendelian disorders, i.e. diseases resulting from a defect in a single gene, where a clear correspondence can be observed between genotypes at a given locus and the phenotype in question. Typically, a stringent lod score threshold of 3, as suggested by Morton [9], is applied as evidence in favor of linkage (this corresponds to a p value of 10 –3 for a sequential test or 10 –4 for a fixed-sample size test). Conversely, the lack of association between a chromosomal region is assumed if a threshold of –2 or greater is obtained. Exclusion of linkage to certain areas is also of great relevance, especially as it helps to distinguish seemingly identical/related disorders [10]. Susceptibility genes for several inherited disorders, including endocrine neoplasias, which are discussed in other chapters of this volume, have been characterized after initial linkage studies identified the target loci (table 1). As mentioned above, linkage studies have successfully permitted the identification of several genes responsible for Mendelian disorders [21]. However, these diseases are in general rare in frequency, in contrast to the more prevalent multigenic traits. The discovery of the molecular bases of these more common disorders, including essential hypertension, diabetes mellitus and arthritis, has not enjoyed the same rate of success and remains largely a challenge for studies based on linkage analysis [20, 22].

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Table 1. Endocrine neoplasias identified by linkage analysis and cloning strategies used thereafter for the identification of the respective susceptibility genes Gene

Cloning strategy

Disease

References

RET

positional cloning, candidate gene testing

MEN 2

138, 139

MEN1

positional cloning, cDNA library screening, candidate gene testing

MEN 1

140, 141

PTEN

positional cloning, RDA, exon trapping, screening of cDNA library for phosphatase domains

Cowden syndrome/ Bannayan-RileyRuvalcaba syndrome

29, 30, 137

VHL

positional cloning, cDNA library screening genes encoded by a cosmid spanning the VHL locus

VHL disease

142

PRKARIA

positional cloning, candidate gene testing

Carney complex syndrome

143

A potential limitation of the linkage analysis approach to identifying diseasecausing loci is the phenomenon of genetic heterogeneity [10, 20]. This term generally refers to both allelic heterogeneity (when the variation occurs within the same locus) or nonallelic heterogeneity (variation in different loci). The latter expression is usually used interchangeably with the generic term genetic heterogeneity. While the first type of heterogeneity does not confound analysis of linkage amongst different families (in fact, it reinforces the causal relationship between the locus and the disease phenotype), the second type can be a source of problems with these studies. In this case, the study of large families is usually required to establish linkage to potential target loci [20]. When nonallelic heterogeneity occurs, it typically involves a limited number of distinct loci. Moreover, the relationship between genotype and phenotype within each family unit remains strong and each involved locus carries the potential to be characterized fully from affected families. Another issue relating to heterogeneity deals with the phenomenon of founder effect, which results from population isolates in which many affected individuals share the same disease mutation derived from a single ancestor [22–24]. Linkage studies, therefore, provide the preliminary information which serves as the basis for the employment of a variety of cloning strategies that are described in the sections below.

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Sporadic Cancer As mentioned above, in sporadic cancers, traditional linkage analysis is not a valid choice to help localize the gene in the first instance, and if positional cloning is to be pursued, other techniques have to be used to pinpoint an area harboring the gene of interest. All these strategies are aimed at the identification of a chromosome location containing an abnormality consistently found in association with a disease phenotype (fig. 1). These chromosomal abnormalities can be relatively small and appear most often as a second event in the development or progression of a malignancy. In these instances, LOH analysis (detailed below) is the technique of choice and has been successfully used to clone several disease-associated genes [25–30]. Conversely, the chromosomal abnormalities can be much more apparent and may involve structural (translocations, inversions, amplifications and deletions) as well as numerical abnormalities (loss or gain of entire chromosomes or changes in ploidy). These abnormalities are often identified by classical cytogenetics or related techniques such as fluorescence in situ hybridization (FISH), spectral karyotyping and comparative genomic hybridization (CGH). These techniques are usually employed in a setting that is related to, but distinct from, the molecular biology arena, namely molecular cytogenetics. Therefore, a detailed description of these techniques is beyond the scope of this chapter, and the interested reader is referred to classic textbooks and reviews for more information [31–36]. Nevertheless, standard definitions and technical assessment of these methods will be provided in the section detailing the cloning of the disease-associated gene from gross chromosomal abnormalities. It has long been known that two abnormal alleles need to be present for recessive traits to be observed. Initial observations were made in the study of retinoblastoma patients by Knudson [37]. According to his model, two mutations were required to transform a normal cell into a neoplastic cell. At the molecular level, mutations occurring in each allele of a gene would result in the disease phenotype. In familial cancers, mutation of one allele is inherited and therefore present in all cells, and the second mutation is acquired by the remaining allele later in life, and affects only the target tissue. In somatic cancers, two independent, acquired events occur. This model became widely known as the ‘two-hit theory’ and has been broadly confirmed in many settings. As the first ‘hit’ in the hereditary model is present in the germline, this allows carriers of the mutation to be tested using any cell type as a sample. Analysis of the affected neoplastic tissue reveals the second hit. From Knudson’s model, the concept of the tumor suppressor gene was created and several familial cancer genes were thus characterized [38–40]. An extension of this concept permits the characterization of tumor suppressor genes in sporadic cancers [41–43]. Common sequence variations distributed throughout the genome, known as polymorphisms (see the description of

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linkage analysis above), are used as the basis for characterizing deleted areas of the genome that might carry tumor suppressor genes [44]. By comparing polymorphic alleles from normal tissue and a cancerous sample in parallel, one can verify whether the heterozygosity present in the germline (which implies the existence of two alleles) has been lost by the tumor (meaning that one of the alleles has been deleted). The LOH of specific areas of the genome suggests the existence in these areas of tumor suppressor genes which contribute to the cancer phenotype [45–47]. Several tumor suppressor genes have been characterized in sporadic human tumors by using LOH analysis [48, 49]. With the identification of thousands of polymorphic markers throughout the genome, mapping areas of deletion has become increasingly precise and has facilitated the characterization of target genes [50]. One potential limitation of LOH analysis is the quality of the tumor sample being tested. Tumors are usually comprised of a mixture of cells, which includes normal contaminating cells admixed with the neoplastic tissue. This can give rise to equivocal results in LOH studies, as existing normal cells may mask the identification of deletions in certain areas. This limitation has been circumvented by the use of microdissection techniques that allow the separation of tumor cells from any contaminating cells and therefore yield clearer and more precise analysis of deletion areas in tumors [51–53]. In particular, laser capture microdissection is currently the most reliable technique to achieve the desired purity of studied samples [54].

Cloning of the Disease-Associated Gene Once the disease-associated candidate area is identified and narrowed down to a workable stretch of DNA (usually in the low megabases size), the challenge is to identify the genes residing inside this region. A significant problem, however, is that the expressed sequences, or cDNAs, may comprise only 1–5% of the total genomic DNA of interest [55]. For this purpose, several strategies have been routinely employed with variable degrees of success. These include: (1) screening of short genomic DNA segments for sequences which are conserved between species (zoo blotting) [28, 56]; (2) sequencing of large segments of genomic DNA in search of open reading frames [57]; (3) cloning of hypomethylated CpG islands, signaling the 5) end of some, but not all, transcription units [58]; (4) direct screening of cDNA libraries with large genomic clones [59, 60]; (5) somatic cell hybrid construction followed by subtractive cDNA hybridization [61, 62], and (6) a PCR-based cDNA selection approach. Although successful in many instances, some of these techniques could not be adapted to the analysis of large areas or were too complex and time-consuming to be routinely

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employed. However, one such approach, exon trapping [63–66], has endured the test of time and has been successfully used to clone many critically important genes [29, 67–69]. Exon Trapping The exon trapping strategy, also known as exon amplification, is based on the in vivo selection for splice sites flanking exon sequences in the genomic DNA [63, 66]. In brief, mammalian genomic DNA is shotgun cloned into a specifically designed vector with the multiple cloning site located in the middle of an intron (usually from the HIV-1 tat gene) flanked by splice sites and exons. The resulting constructs are transiently transfected into COS cells and the reporter gene is transcribed with the inserted genomic fragment. When a fragment containing an entire exon with flanking intron sequence is present, the exon is retained in the mature cytoplasmic RNA. Subsequently, a reverse transcriptase (RT)-PCR using plasmid-specific primers is used to detect the novel exon sequence. Since this approach does not depend upon the endogenous level of transcription, genes whose RNA expression patterns are tissue specific or developmentally regulated can be isolated with the same efficiency as ubiquitously expressed genes [63, 66]. Exon trapping is only limited by its requirement for functional 3) and 5) splice sites flanking a target exon. Therefore, intronless or single-intron genes will not be identified using this approach. In silico Gene Discovery When searching for novel genes, investigators must consider that, as a result of large-scale sequencing efforts, an extraordinary number of novel sequences are available daily at the public nucleotide sequence databases: GenBank (www. ncbi.nlm.nih.gov), EMBL (www.ebi.ac.uk) and DDBJ (www.ddbj.nig.ac.jp). Therefore, this wealth of data is clearly influencing the way genes are presently identified. For example, it is very likely that after only a single round of exon trapping (or any other gene-identifying technique), the cloned new sequence could be used to screen these databases and the entire effort be shifted to the application of computer-based methods, also known as in silico cloning. Moreover, with mapping of a large proportion of these novel sequences, traditional laboratory methods could in some instances be skipped altogether. For the identification of novel genes, the most widely used strategy is to search the Expressed Sequence Tag (EST) database (db EST) or, to limit the search to human sequences, the Human EST collection (both databases available at the BLAST search engine, www.ncbi.nlm.nih.gov/BLAST). ESTs are short single-pass DNA sequences obtained from either end of cDNA clones. These ESTs are derived from a vast number of cDNA libraries from both normal and malignant tissues. They are deposited into the public databases along with annotations indicating the tissue of

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origin as well as similarity to known genes or proteins. More importantly, because there is high redundancy in the EST collection, the UniGene database (www.ncbi.nlm.nih.gov/UNIGENE) has automatically partitioned GenBank sequences into a nonredundant set of gene-oriented clusters. Each UniGene cluster contains sequences that represent a unique gene derived from hundreds of thousands of novel EST sequences as well as previously characterized genes. It also contains related information such as the tissue types in which the gene has been expressed, map location, homologues, orthologues and putative function. Consequently, this collection clearly simplifies the task of gene discovery. In an illustrative example, one could use linkage analysis to map a candidate area and exon trapping to identify a partial cDNA, and then use this sequence to search the EST database, where overlapping clones could be identified and substantially increase the length of the target cDNA or even allow for the identification of the complete open reading frame. Alternatively, in perhaps a more contemporary example, after the linkage analysis or any other mapping strategy, one could search the databases for ESTs assigned to the chromosomal region of interest (www.ncbi.nlm.nih.gov/cgi-bin/Entrez/) and follow the steps described above to obtain a full-length open reading frame. Taking together the recent developments in the Human Genome Project and the arguments presented above, it is reasonable to speculate that once all the human genes have been identified and, importantly, mapped to a particular chromosomal location, the candidate gene approach will be the ‘last technique standing’ and will ultimately be used in every case of identification of a disease gene. Gross Chromosomal Abnormalities Translocations. The identification of genes disrupted by gross chromosomal abnormalities, chiefly translocations, has been one of the most potent approaches to cloning disease-associated genes. Most of the strategies employed in such enterprises were developed and refined during the cloning of genes involved in hematologic malignancies, mainly leukemias [70–73]. This bias reflects the ease with which specimens can be obtained in these diseases but possibly also a relatively higher frequency or propensity for chromosomal instability/abnormality in hematologic malignancies when compared to other human neoplasms. However, regardless of the disease in question, once a gross chromosomal abnormality such as a nonrandom balanced chromosomal translocation has been identified in association with a particular phenotype, the steps for cloning the genes potentially disrupted by such abnormalities are the same. In cases of chromosomal translocations, one must initially consider the candidate gene approach, a strategy likely to yield important results in the fastest fashion, particularly considering the daily updates of the human genome databases with regard to new genes/ESTs and their mapping. Frequently, especially if

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FISH was used to further define the features of the translocation, the areas encompassing the targeted genes are fairly small. In this case, if a gene with a function potentially relevant to the disease phenotype is found to map to that area, the first goal should be to assign the candidate gene to informative clones (by Southern blot or PCR). This approach would exclude irrelevant genes or confirm the potential involvement of important candidates and spare the usually limited source of patient material for more definitive experiments. Conversely, if the translocation breakpoint was identified by conventional cytogenetics and therefore could be located anywhere within a large area, the first attempt should be to identify genomic clones containing the candidate genes and use them as probes in FISH experiments. This could be accomplished by conventional screening of genomic libraries or by in silico cloning (see previous section). This initial mapping would allow both narrowing the area of study and identification of bona fide target genes. Even if the clone containing the candidate gene is found to be split by the translocation, proof that this gene is indeed the target for the disruption usually relies on Southern blot analysis of the patient’s DNA using probes derived from the candidate’s gene sequences. If abnormally migrating DNA bands are seen in multiple independent restriction endonuclease digestions, it is very likely that one side of the translocation has been found. The steps for cloning the second gene disrupted by the translocation will depend largely on the type and amount of patient material available. In this regard, since this is usually a major limitation, it should be considered from the very inception of the project to establish either a cell line or somatic cell hybrids harboring the abnormality of interest. Conversely, when there is sufficient good-quality RNA from the sample(s) of interest, probably the best and most elegant strategy is to construct a cDNA library and screen it with probes from the gene disrupted on the other side of the translocation. The next steps will involve full-length cloning of the partner gene (in the case of a novel transcript), as well as Southern blotting and chromosomal mapping to further confirm its involvement in the translocation. Finally, an RT-PCR spanning the breakpoint (with oligonucleotides from both genes) should be performed to demonstrate that the fusion is indeed transcribed and to clarify whether both or only one of the hybrid genes is transcribed (in the case of a reciprocal translocation). If construction of a cDNA library is not possible but DNA is available, a backup option is to establish a genomic DNA library and follow the same steps as outlined above. However, because most translocation breakpoints are intronic, one problem with this strategy is the size of the disrupted intron. If insufficient material is available to construct either a genomic or a cDNA library, one additional option is to take advantage of a PCR-based technique, namely rapid amplification of cDNA ends (RACE)-PCR, which does not require large amounts of starting material.

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In the RACE-PCR approach [74–76], one end of the eventual PCR amplicon is known, whereas the other side remains to be determined. Actually, this description resembles the situation encountered when only one side of a reciprocal translocation has been identified. Therefore, RACE-PCR could be used to identify partner genes located either 5) or 3) of the known end of the translocation. In brief, in a 5) RACE reaction, an anchor sequence is attached to a homopolymer, complementary to an artificial poly-A tail, and is used to synthesize the second cDNA strand (the reverse transcription should be performed with a gene-specific primer). In the 3) RACE approach, the homopolymer (plus the anchor sequence) is itself used to reverse-transcribe the RNA. Subsequently, nested PCR amplifications using oligonucleotides derived from the anchor and from the known end of the hybrid gene are performed and the PCR product is eventually sequenced. The RACE-PCR procedure is extremely robust and since its original description [74], many improvements have been reported [75, 77]. The problem with this strategy in the specific setting of cloning of translocations is that the precise location of the breakpoint is frequently unknown. Therefore, the exact position at which to design the gene-specific primers is difficult to predict and one might use oligonucleotides located either beyond the breakpoint region or too far away from it and hence, in both instances, amplify only or predominantly the untranslocated allele. If one end of the translocation is known and all the strategies described above to ‘cross the breakpoint’ fail, the candidate gene strategy, as described above, should be employed to identify the second target for disruption. If the search for candidate genes is not successful, the best approach is probably to identify the smallest possible clone which is split by the translocation in FISH experiments (and hence likely to contain the sequence of interest). Subsequently, techniques to isolate genes from complex genomic DNA, such as exon trapping (discussed in detail in the preceding sections), should be used, followed by the approaches used to identify the gene disrupted on the other side of the translocation, as mentioned above. A diagram summarizing the stepwise strategies employed to clone a chromosomal translocation is shown in figure 2. The single most common gross abnormality found in endocrine neoplasias is a rearrangement involving the RET tyrosine kinase receptor in sporadic papillary thyroid carcinoma, collectively known as RET/PTC. RET is not expressed in follicular thyroid cells under normal circumstances, but rather in C cells of neuroectodermal origin (see the chapters by Mulligan and Gimm [this vol.] on multiple endocrine neoplasia type 2 (MEN 2) for details). However, hybrid transcripts resulting from the fusion of the tyrosine kinase domain of RET to the 5) portions of other genes, which are normally expressed in follicular thyroid cells, have been found in a proportion of papillary thyroid cancers. In these instances, the promot-

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Fig. 2. Gene identification from chromosomal translocation. The figure shows the strategies employed when none, one or both genes disrupted by the translocation are known.

er of the fused gene drives the transcription of RET. At least eight different types of rearrangements involving RET have been reported [78–84]. RET/PTC fusions identified so far are ubiquitously expressed, not membrane bound, and contain coiled-coil domains required for constitutive activation of the RET tyrosine kinase domain. Attempts to correlate certain RET/PTC fusions with specific histological subtypes of papillary thyroid carcinoma and with epidemiologic factors, such as radiation exposure or subpopulation groups, have resulted in conflicting reports [85–88]. While the molecular basis for familial papillary carcinoma has not yet been elucidated [89], RET fusions do not appear to play a causal role in this setting and seem to represent a late event in the progression of certain inherited forms of papillary cancers [90]. Recently, a study has provided the first insight into the structural basis of one of the RET rearrangements, the RET-H4 fusion [91]. It has been found that despite the large linear distance between RET and H4 in chromosome 10, recombination results from their physical proximity in the nucleus. It has been shown that radiation is able to create a double-strand break in each gene at the same site in the nucleus. It is possible, therefore, that gene proximity is implicated in susceptibility to radiation-induced cancer. More recently, a balanced translocation in follicular thyroid carcinomas, t(2;3)(q13;p25), which results in fusion of the DNA binding domains of the thyroid transcription factor PAX8 to the peroxisome proliferator-activated receptor gamma (PPARÁ), was reported [92]. PAX8-PPARÁ1 mRNA and protein were

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detected in 5 of 8 thyroid follicular carcinomas but not in other thyroid disorders or cancers. Functional studies suggest that the fusion inhibits PPARÁ1 transactivation in a dominant negative manner. Further studies will be necessary to fully assess the prevalence of this fused transcript in follicular carcinomas, its specificity for this histological group and whether it is also present in rare familial forms of this tumor. Amplifications and Deletions. Other gross chromosomal abnormalities that could lend initial clues to the search for disease-associated genes are deletions and amplifications. CGH is the most frequently used molecular cytogenetic approach to genome-wide scanning of differences in DNA sequence copy number. In this assay, normal human metaphase chromosomes are competitively hybridized with two differentially labeled genomic DNAs (test and reference), which, upon fluorescence microscopy, reveal the chromosomal locations of copy number changes in DNA sequences between the two complements [93]. The application of CGH to DNAs extracted from fresh frozen specimens and cell lines of various tumor types has revealed a number of recurrent chromosomal gains and losses that were undetected by traditional cytogenetic analysis. CGH, however, has a limited (approximately 20 Mb) mapping resolution, making it difficult to detect events involving small regions or to clearly dissect closely spaced aberrations. Therefore, a microarray-based CGH, which would allow for higher-resolution studies, has been devised [94, 95]. An overview of microarrays is presented in a further section below. In the CGH version of microarrays, where over 30,000 radiation hybrid-mapped human genes are represented, fluorescence ratios of arrayed DNA elements provide a locus-by-locus measure of DNA copy number variation, and genomic resolution can be determined by the map distance between the targets. If CGH is used to map a candidate area, the steps for cloning the targeted gene include the use of techniques to narrow down the critical region, followed by a candidate gene approach or techniques aimed at isolating genes from complex genomic DNA (described in the preceding sections). Conversely, if array-based CGH is used, a positive result would immediately lead to identification of the targeted locus, for the gene itself is already characterized and immobilized to the chip. Gene dosage variations occur in many diseases. In cancer, decreases and increases in gene copy number contribute to alterations in the expression of tumor suppressor genes and oncogenes, respectively. Thus, the detection and mapping of copy number abnormalities provides an approach for linking molecular changes with disease phenotype and for localizing critical genes.

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Fig. 3. Nonpositional cloning techniques of gene identification. Expression-based strategies of gene identification (left side of figure) typically result in partial characterization of target genes. Full-length gene characterization requires the use of additional techniques, shown in the center of the figure. Simultaneous use of different approaches is commonly required in the process of gene cloning.

Expression-Based Studies Several expression-based approaches have proven useful for the identification of genetic lesions in cancer cells. Some of these techniques rely on the comparison of putative differences in mRNA abundance or gene expression between the cancer cell and its normal counterpart. In fact, when the target candidate gene is known, measuring gene expression is trivial and can be performed with many well-established techniques such as Northern blots, RT-PCR and RNase protection assay. However, if the goal is to clone novel genes potentially related to cancer development and/or progression, a different set of techniques should be employed. For this purpose, the most commonly used approaches are differential display (DD), subtractive hybridization, serial analysis of gene expression (SAGE) and, more recently, microarray-based strategies. The latter, however, has been more frequently used to characterize basic biologic processes, such as identification of the genetic basis for the observed clinical heterogeneity in human diseases and characterization of signaling and circuitry of multiple pathways [96– 103]. In this chapter, we will focus our discussion on the use of microarrays as a gene discovery tool. Figure 3 displays the techniques used for gene discovery using expression-based approaches in a simplified format.

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Differential Display In the DD assay, cellular RNA is isolated and simplified pools of cDNA fragments are produced from the RNA samples being compared, their content being defined mostly by the sequence of the arbitrary set of primers used [104, 105]. The analogous pools, obtained from the compared RNA samples, are then resolved side by side on a polyacrylamide gel and fragments that are differentially expressed are excised from the gel, reamplified by PCR, cloned and sequenced. Once the differential expression is confirmed by Northern blot analysis, cloning of the full-length gene (in the case of novel cDNAs) should be pursued by classical methods, such as RACE-PCR and screening of relevant cDNA libraries as well as by searching the databases, as mentioned above. Classical DD offers several advantages over other methods aimed at cloning differentially expressed transcripts: it is the simplest technique for mRNA comparison for it does not require any special reagents or materials, several dozen randomly picked cDNA fragments can be checked simultaneously and more than two RNA samples can be compared at once. However, although this method has been successfully applied to the isolation of genes differentially expressed in cancers [76, 105–107] and nonmalignant conditions such as heart disease, diabetes and embryogenesis [105], the high false-positive rate and possible underrepresentation of minor mRNA fractions in the analysis have somewhat limited the applicability of DD. However, since its initial description [104], several modifications have been introduced that have improved the classical DD assay, such as the use of longer oligonucleotides (20 bases). Contemporaneous development in related areas, e.g. microarray-based techniques (see below), will likely make the use of DD variants rather sporadic. The pituitary tumor-transforming gene (PTTG), which has been associated with the development of endocrine tumors, was cloned by DD [106]. PTTG was originally cloned from a rat pituitary tumor cell line, where it was found to be expressed at higher levels than normal pituitary counterparts. Further studies identified the human orthologue (hPTTG) and verified that hPTTG is overexpressed in a variety of pituitary tumors of different lineages, particularly in more aggressive subtypes [108]. However, its expression is not restricted to pituitary tumors, suggesting that it may in fact be associated with general tumor invasiveness [109]. Representational Difference Analysis One alternative to DD is representational difference analysis (RDA), a PCRbased subtractive hybridization technique that identifies differences between two (usually normal and tumor) genomes. RDA was initially designed [110] for studies at the DNA level and was later adapted to mRNA analysis [111]. In the former, gene copy number, deletion mapping and LOH can be characterized and can-

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didate areas mapped. Obviously, when the analysis is performed directly between two mRNA samples, the candidate gene could be identified without laborious additional steps. Nonetheless, RDA is a relatively complex technique additionally limited by the number of templates (two) which can be compared with each other, and therefore has never quite enjoyed the popularity of other methods used for identifying differentially expressed transcripts, such as DD. However, one example of its use as one of the steps towards the characterization of a neoplasia-related transcript is the cloning of the PTEN gene [29]. Serial Analysis of Gene Expression SAGE is another technique that allows the identification of differentially expressed genes [112]. It is based on the identification of short nucleotide sequence tags (9–11 bp) located at the very 3) end of the cDNA (close to the polyadenylation tail), as a result of restriction digestion. These tags are sufficiently unique to identify a specific transcript [112, 113]. Anchoring primers are ligated to the sequences generated by the digestions and amplified. The resulting PCR products are concatenated, cloned and sequenced. Since the final product of the SAGE analysis is a list of tags, with their corresponding relative abundance (as determined by each tag’s count value), it portrays a digital representation of cellular gene expression. An advantage of SAGE, a fairly laborious technique, is that it appears to yield good-quality data about transcript abundance but, consequently, much of the sequencing effort generates predominantly high-abundance transcripts. Two major disadvantages of this technique stem from the ambiguity of tags (two distinct genes with the same tag sequence) and limited specificity (one gene with more than one assigned tag, due to alternative adenylation signals, polymorphisms etc.). In addition, due to the small length of the sequences involved, even low-rate sequencing errors can have important negative effects on SAGE tag results. Microarray-Based Approaches Advances in microarray technology, also known as ‘DNA chips’, have enabled massive parallel analysis of data. cDNA- or genomic DNA-based chips allow monitoring of gene expression, polymorphism detection and genotyping on a large scale. In microarray assays, oligonucleotide or larger DNA/cDNA fragments are immobilized onto glass slides and are hybridized with specific fluorescently labeled probes. Usually, two distinct fluorophores are used simultaneously and the intensity of the fluorescence emitted by each hybridized spot functions as a readout for the change in abundance of the gene. A complete analysis involves three basic steps: (1) sample preparation; (2) array generation and analysis of samples, and (3) interpretation of the data [114, 115]. The specificities of each study

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will determine the respective relevance of each of these components. Studies that rely heavily on profiles of individual samples, for example, will involve considerations of tissue acquisition and processing that would not be as critical in studies that aim to characterize specific pathways in cell lines. The selection of the array to be used will also vary depending on the study goals and may require generation of customized arrays or, alternatively, the use of commercially available chips. The nature of the array, in turn, may differ according to the needs of the study, with oligonucleotide-based chips or those based on larger gene fragments being the most common alternatives. Additional details related to array varieties are described in the chapter by Dahia and Eng [this vol.]. Microarrays containing sequences representative of all human genes are expected to soon permit the expression analysis of the entire human genome in a single reaction [116]. The latest available commercial chip set contains approximately 60,000 sequences, including 12,000 known genes, which by some estimates could, numerically at least, well encompass all existing human genes [117, 118]. However, despite the effort to use information from partially assembled contigs to avoid duplications, it is unlikely that this set contains a full representation of the human genome. Regardless of this potential limitation, ‘genome chips’ will provide unprecedented access to key areas of human health, including disease prognosis and diagnosis, drug discovery, toxicology, development and aging. While studies involving microarray analysis have thus far predominantly focused on gene expression profiles in distinct biological conditions [96, 97], another important application of this technology, and one that should not be underrated, is in the area of gene discovery. A recent example of the successful use of microarrays to characterize novel sequences is an elegant study in which a compendium of expression profiles corresponding to 300 mutations and chemical treatments in yeast was developed which creates unique expression ‘fingerprints’ [119]. These specific expression profiles define patterns that are unique for particular cellular pathways so that unknown mutations or uncharacterized genes can have their functional role revealed by the profile they create in comparison with the compendium. Hence, by using this approach, it was possible to identify genes required for sterol metabolism, cell wall function, mitochondrial respiration and protein synthesis. Similar approaches can be used for finding novel disease-associated genes in humans. It is expected that analysis of endocrine neoplasias by microarrays will shed light on the pathogenesis of these tumors and in particular may help identify genes responsible for the less common, familial forms of endocrine tumors. Microarray analysis may also aid in the characterization of modifier factors in inherited disorders for which the susceptibility genes have already been identified, such as the ones discussed in this volume. Such modifiers account for the variability in phenotype which so far could not be distinctively correlated with genotypic features.

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Protein-Related Strategies for Gene Discovery While nucleic acid-based techniques have been the mainstream method of gene identification, the natural step following the availability of a complete collection of genome sequences is assigning each gene a function. As proteins are the ultimate catalysts of biological functions, their study is an essential part of the integral understanding of whole organisms. Protein-based approaches can be individualized to identify binding partners of a protein of interest, lending some clues as to its functional pathway, or may be used for a large-scale search of relevant proteins in a given system. The latter techniques have a wider potential to characterize protein function, including a not yet fully developed ‘protein chip’, with which thousands of proteins can be analyzed simultaneously in a similar manner to that already discussed for DNA arrays. The identification of associated proteins by physical interaction is well illustrated by the yeast two-hybrid system. In this technique, yeast is transformed with a gene encoding a protein for which partners are being sought and is used as a ‘bait’ to identify interacting proteins [120, 121]. The two-hybrid method can be used not only to determine if two known proteins (i.e. proteins for which the corresponding genes have been previously cloned) interact, but also to identify previously unknown proteins that interact with a target protein. This approach has permitted the identification of several proteins that are part of complexes which delineate specific signaling pathways. An example of a protein identified by a yeast two-hybrid system in the context of endocrine-related genes is the recent characterization of PTTG-binding factor (PBF) [122]. PBF is a novel protein that is required for the transcriptional activation of basic fibroblast growth factor by PTTG. More recent strategies dealing with large-scale analysis of proteins are mass spectrometry and protein chips. This field of research, also known as proteomics, is founded on the critical relevance of proteins for biological functions as compared to the somewhat more restricted role of transcription profiles, since proteins are the true end product of a gene. The advent of mass spectrometry has revolutionized the field of proteomics. This powerful technique is based on the characterization of protein identity after gel separation and digestion into peptides. The mass spectrum of eluted peptides is obtained by a method known as matrix-assisted laser desorption ionization (MALDI), resulting in a ‘peptide mass fingerprint’ [123, 124]. A second step towards identification of the nature of the protein relies on the more complex process of tandem mass spectrometry based on ‘electrospray ionization’, which dissociates preresolved peptides into aminoterminal- or carboxyterminal-containing fragments, aiding in their identification. Analysis of posttranslational modifications of proteins is another important feature of proteomics which has the potential to provide important insights into how proteins function, informa-

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tion which cannot be inferred from the mere analysis of their sequence. While much progress has been made in this area (in particular with phosphorylation studies), there are no available high throughput methods for a global analysis of the modifications that proteins can undergo. Progress in the development of protein chips has lagged behind the DNA counterparts; they are more difficult to synthesize than nucleic acids, and also, immobilization on solid surfaces often results in the loss of their secondary or tertiary structure, which is usually associated with their inactivity. Parallel analysis of a large number of proteins will enable enormous progress in investigating disease associations, and will also be instrumental in the development and discovery of new drugs. Different versions of protein chips have been designed; proteins, peptides or antibodies can be immobilized onto arrays and screened with, for example, fluorescently labeled lysates, similar to DNA arrays [125]. Progress in the area of proteomics is certain to lend important information on biological processes and it is expected that the advances seen in the genomics field that have accumulated in the past years will be promptly transferred to the study of proteins in the short-term future.

Analysis of Known Disease-Related Genes In the previous sections, a comprehensive discussion of standard and novel techniques to clone disease-associated genes was presented. In this part of the chapter, strategies to identify abnormalities of known disease-causing genes will be briefly discussed. Much progress has been made in characterizing the molecular basis for several endocrine neoplasias, as specifically discussed in other chapters of this volume. Similarly, great advances have been brought about to allow for an increasingly easier assessment of gene defects that are associated with specific disease phenotypes. Techniques have been developed that attempt to reach the ideal goal for a routine molecular diagnostics analysis: to be highly sensitive, specific, easily reproducible, technically simple, amenable to adaptation for large-scale use and cost-effective. Several methods have achieved many of these goals satisfactorily, although not all of them at once, and have been used in practice to characterize gene defects as an essential tool for the diagnosis of endocrine neoplasias (examples of the applicability of some mutation screening techniques are shown in table 2). A detailed discussion of these techniques is not within the scope of this chapter. There are several reviews that deal comprehensively with each of these techniques available for such purposes and the reader is referred to some of these reviews for further information [126–129].

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Table 2. Techniques for mutation detection that have been used in association with some of the syndromes described in the text RET √ √ √

MEN1

VHL

PTEN

√ √ √

√

√ √

PRKARIA Mutation screening method(s)

√ √

√ √

√

√

√ √

√

DGGE SSCP dDF PCR + restriction digestion analysis heteroduplex analysis chemical cleavage detection protein truncation test direct sequencing

dDF = Dideoxy fingerprinting.

The choice of screening technique varies according to the peculiarities of the gene of interest, such as the length of the gene, distribution of mutations (existence of ‘hot-spot’ or ‘warm-spot’ regions) and the nature of the sequence (GC content, presence of nucleotide repeats, etc.). As a result, certain mutation detection techniques have come to be associated more often, although not exclusively, with the screening of specific disease genes (table 2). Mendelian disorders such as the diseases described in this chapter are the result of a single gene defect. Often this translates into small mutations, especially point mutations, which can be recognized by highly sensitive techniques, usually PCR-based. However, occasionally the gene abnormality is associated with gross defects, such as major deletions, translocations or rearrangements, which can be detected more easily by other techniques, such as Southern blot. This is the case with the von Hippel-Lindau (VHL) gene, for example, where up to 20% of kindreds are expected to have partial or complete deletion of the gene that is recognizable by Southern analysis, while the remainder have small mutations. A combination of PCR-based techniques (in particular, direct sequencing of PCR products) with Southern blot and FISH has been reported to enable the identification of VHL gene defects in virtually 100% of cases [130]. The detection of germline MEN1 gene mutations has been achieved in 80– 90% of studied families. Several methods have been employed in these studies, which may account for the variability in the detection rate, e.g. single-strand conformation polymorphism (SSCP), dideoxy fingerprinting and denaturing gradient gel electrophoresis (DGGE). The lack of ‘hot-spot’ areas within the gene has dic-

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tated the need to analyze all coding exons. Some MEN 1 cases have been associated with large deletions of the gene, which, similar to what was described with the VHL gene above, may be detected by Southern blot analysis [131]. In MEN 2 families, the frequency of RET mutations is higher and it varies according to the subtype of MEN 2. A variety of techniques have been described that have been employed in RET mutation analysis, including SSCP, DGGE, chemical cleavage analysis and direct sequencing. Due to the high GC content of RET, the success rates obtained with the use of screening techniques have not been optimal. Since MEN 2-associated mutations tend to cluster in certain regions of the gene, sequencing the entire coding region of RET is usually not required. Therefore, most researchers rely on direct sequencing for the identification of disease-causing mutations, although clinical laboratories employ a broad range of mutation screening technologies, with high accuracy. Once the mutation is identified and confirmed in one affected individual, the remaining family members can be subjected to a focused approach whereby the specific familyrelated mutation is investigated in the family, whenever possible by a combination of PCR followed by restriction analysis that differentially digests mutant and wild-type forms of RET. The detection of PTEN mutations has relied on several methods, including DGGE [132] (associated with possibly the highest detection rates amongst the screening techniques), SSCP [133] and the protein truncation test (most likely the method with the lowest levels of mutation detection [29]). In approximately 20– 40% of patients with Cowden and Bannayan-Riley-Ruvalcaba syndromes, respectively, PTEN mutations are not detected [134]. While large deletions affecting the PTEN gene have not been widely described, it is possible that a small fraction of cases carry grosser PTEN abnormalities that require the use of techniques such as Southern blot for their detection [135]. At an increasingly frequent rate, many researchers resort to direct sequencing of PCR products to analyze samples for mutations. The precise and definitive nature of this approach, allied with technical improvements which have greatly impacted on the quality of the results at reduced costs, as well as allowing its use on a large-scale basis, such as the use of capillary electrophoresis, are some of the factors in favor of using sequencing as the primary method of mutation detection in inherited cancer-related genes, instead of resorting to it as the confirmatory step in suspected samples from other screening techniques. However, DNA sequencing may fail to detect all mutations in a given sequence. A mutation that is present in only one allele of the gene, for example, can be masked and not promptly identified by sequence analysis. For identification of these refractory mutations, a novel technique was recently reported [136]. The method is based on the conversion of diploid human chromosomes into haploid, single chromosomes on a background of a rodent cell line, by fusing test cells with a rodent cell line.

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Hybrids containing a single copy of any human chromosome of interest permit the identification of individual alleles. The limitations of this method, named conversion, include the intensive labor and costs associated with the hybrid generation and screening process. The advances brought about by microarray technology may change the scenario of mutation detection in the not too distant future. There are already commercially available arrays with which samples can be screened for mutations in the whole coding region of the p53 tumor suppressor gene with a single hybridization reaction. The array is designed to identify single-base mutations and deletions of p53. It is expected that soon other commonly mutated genes will be available for chip-based analysis. While this new technology has great appeal, it is likely that it will not at first entirely replace other techniques, especially considering that less common mutations and those spanning larger deletions may not be predicted by the array and can therefore be missed. However, regardless of the obvious limitations of first-generation assays, mutation detection by microarray analysis may become one of the gold-standard methods in the years to come.

Conclusions There are many techniques available for gene identification, as broadly discussed above. There has been a recent trend towards the use of multidisciplinary bioscience initiatives to fully and better explore the vast amount of data available to researchers. As the methods vary in complexity so does the preference and choice of strategies for each particular condition. Several genes have been identified with the aid of multiple techniques and strategies. The use of combined approaches for cloning and the multiplicity of cloning methods is well illustrated, for example, by the PTEN gene. Besides being characterized by two independent groups using distinct positional cloning strategies [29, 30], PTEN was also identified by a third team using a targeted, ‘functional’ approach to cloning novel phosphatases [137]. The identification of all genes by the techniques described here is the starting point in the immense progress we are witnessing in biology. Characterization of the function of genes and their integration in the cell circuitry will enable the prediction of phenotypes with great precision. Critical pathways involved in the pathogenesis of several human diseases will be unveiled and this new knowledge will allow researchers to unravel protein structure and function throughout evolution. New paradigms in biology will need to be developed to incorporate the knowledge of novel and complex gene/protein networks. Finally, it is expected that this information will be translated into the development of more rationally targeted drugs which will ultimately advance the treatment of human diseases.

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136 Yan H, Papadopoulos N, Marra G, Perrera C, Jiricny J, Boland CR, Lynch HT, Chadwick RB, de la Chapelle A, Berg K, Eshleman JR, Yuan W, Markowitz S, Laken SJ, Lengauer C, Kinzler KW, Vogelstein B: Conversion of diploidy to haploidy. Nature 2000;403:723–724. 137 Li D-M, Sun H: TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor ß. Cancer Res 1997;57:2124–2129. 138 Mulligan LM, Kwok JBJ, Healey CS, Elsdon MJ, Eng C, Gardner E, Love DR, Mole SE, Moore JK, Papi L, Ponder MA, Telenius H, Tunnacliffe A, Ponder BAJ: Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458–460. 139 Donis-Keller H, Dou S, Chi D, Carlson KM, Toshima K, Lairmore TC, Howe JR, Moley JF, Goodfellow P, Wells SA: Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC. Hum Mol Genet 1993;2:851–856. 140 Chandrasekharappa SC, Guru SC, Manickam P, Olufemi SE, Collins FS, Emmert-Buck MR, Debelenko LV, Zhuang Z, Lubensky IA, Liotta LA, Crabtree JS, Wang Y, Roe BA, Weisemann J, Boguski MS, Agarwal SK, Kester MB, Kim YS, Heppner C, Dong Q, Spiegel AM, Burns AL, Marx SJ: Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 1997;276:404– 407. 141 Lemmens I, Van de Ven WJ, Kas K, Zhang CX, Giraud S, Wautot V, Buisson N, De Witte K, Salandre J, Lenoir G, Pugeat M, Calender A, Parente F, Quincey D, Gaudray P, De Wit MJ, Lips CJ, Hoppener JW, Khodaei S, Grant AL, Weber G, Kytola S, Teh BT, Farnebo F, Phelan C, Hayward W, Larsson C, Pannett AAJ, Forbes SA, Duncan Bassett JH, Thakker RV: Identification of the multiple endocrine neoplasia type 1 (MEN1) gene. The European Consortium on MEN1. Hum Mol Genet 1997;6:1177–1183. 142 Latif F, Tory K, Gnarra J, Yao M, Duh F-M, Orcutt M-L, Stackhouse T, Kuzmin I, Modi W, Geil L, Schmidt L, Zhou F, Weng Y, Duan DR, Dean M, Glavac D, Richards FM, Crossey PA, FergusonSmith MA, Le Paslier D, Chumakov I, Cohen D, Chinault CA, Maher ER, Linehan WM, Zbar B, Lerman MI: Identification of the von Hippel-Lindau disease tumor suppressor gene. Science 1993; 260:1317–1320. 143 Kirschner LS, Carney JA, Pack SD, Taymans SE, Giatzakis C, Cho YS, Cho-Chung YS, Stratakis CA: Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit in patients with the Carney complex. Nat Genet 2000;26:89–92.

Ricardo Aguiar, MD, PhD, Harvard Medical School, 44 Binney Street M513 Boston, MA 02115-6084 (USA) Tel. +1 617 632 3881, Fax +1 617 632 4734, E-Mail [email protected] Patricia Dahia, MD, PhD, Department of Cancer Biology, Dana-Farber Cancer Institute Harvard Medical School, 44 Binney Street SM 1010, Boston, MA 02115-6084 (USA) Tel. +1 617 632 4664, Fax +1 617 632 4663, E-Mail [email protected]

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Dahia PLM, Eng C (eds): Genetic Disorders of Endocrine Neoplasia. Front Horm Res. Basel, Karger, 2001, vol 28, pp 50–80

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Clinical and Molecular Aspects of Multiple Endocrine Neoplasia Type 1 Settara C. Chandrasekharappa a, Bin Tean Teh b a

b

Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institute of Health, Bethesda, Md., and Laboratory of Cancer Genetics, Van Andel Research Institute, Grand Rapids, Mich., USA

Contents 51 51 54 55 55 56 57 57 58 58 60 62 63 63 64 64 65 65

Introduction Clinical Manifestations of MEN 1 Phenotype and Genotype Correlation Phenocopy and Anticipation in MEN 1 Modifier Gene(s) in MEN 1 MEN 1 – Any Variants? Management of MEN 1 Patients Treatment Genetic Testing Illustrative Case Presentation Identification of the MEN 1 Gene and Mutations Germline Mutations Somatic Mutations in Sporadic Tumors Nature of Mutations The MEN 1 Gene: Organization and Expression Exon-Intron Organization MEN 1 Expression by Northern Analysis and in situ Hybridization MENIN Expression by Western Analysis and in Relation to the Cell Cycle

Both authors contributed equally to this work.

66 66 67 67 68 68 69 70 72 73 73 73

The MENIN Protein MENIN Is a Nuclear Protein MENIN Interacts with JunD, an AP1 Transcription Factor MENIN Represses JunD-Mediated Transcriptional Activation Suppression of Tumorigenicity in RAS-Transformed Cells MEN 1 Orthologs Mouse, Rat and Zebrafish Men1 Genes and Their Expression in Development Amino Acid Sequence Comparison of the MENIN Orthologs Chromosome Instability and the ‘Mitogenic Factor’ in MEN 1 The Future Acknowledgments References

Introduction Multiple endocrine neoplasia (MEN) is considered a pathological entity characterized by neoplasia of more than one endocrine organ. First described by Erdheim [1] in 1903 in a case with parathyroid enlargement and pituitary tumors, its hereditary nature was not recognized until a few decades later [2, 3]. In recent years, with advances in molecular biology, the underlying etiology of MEN syndromes is better understood, which has led to improvements in patient diagnosis and management. The syndrome is classically divided into types 1 and 2, but a spectrum of MEN variants have also been described. In this chapter, the clinical and molecular aspects of MEN type 1 (MEN 1) are described.

Clinical Manifestations of MEN 1 In 1954, Wermer [2] described ‘adenomatosis of endocrine glands’ affecting several members of a family which spanned two consecutive generations. This hereditary disease, also called Wermer’s syndrome and now MEN 1, is transmitted as an autosomal dominant trait with over 95% penetrance and an equal sex distribution [4]. Its prevalence is estimated to be 0.02–0.2/1,000. Whilst the majority of MEN 1 families are of Caucasian origin, MEN 1 has also been described in families from a variety of ethnic backgrounds [5, 6]. It is typically characterized by a triad of neoplasia affecting the parathyroid glands (90–97% of patients), enteropancreatic endocrine tissues (30–80%) and the anterior pituitary gland (15–50%) [4]. Not uncommonly, the patients may develop tumors in a range of other endocrine and nonendocrine tissues (table 1). The diagnosis of MEN 1 is usually based on a finding of tumors in two or more of the principal

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Table 1. Endocrine and nonendocrine tumors in MEN 1 Endocrine tumors Parathyroid hyperplasia/multiglandular disease Enteropancreatic endocrine tumors Anterior pituitary tumors Adrenocortical tumors Carcinoids (thymic, bronchus, gastrointestinal tract) Thyroid tumors Nonendocrine tumors Cutaneous and visceral lipoma Skin tumors: angiofibroma, collagenomas Ependymoma Leiomyoma of gastrointestinal tract Angiomyolipoma of kidney

MEN 1-related glands, i.e. parathyroid, enteropancreas and anterior pituitary. Familial MEN 1 refers to a kindred in which 1 member fulfills the criteria for MEN 1 and at least 1 other first-degree relative has one or more endocrine tumors of the three principal glands. However, there are also the so-called sporadic MEN 1 patients who have no family history and develop the disease as a result of de novo mutations. MEN 1-related parathyroid glands manifest multiglandular disease or hyperplasia rather than solitary adenoma, which is commonly found in sporadic primary hyperparathyroidism. Parathyroid carcinoma, a feature of familial hyperparathyroidism-jaw tumor syndrome [7, 8], however, is not known to be associated with MEN 1. Today, most MEN 1-related hyperparathyroidism cases are asymptomatic and commonly detected through routine biochemical investigations rather than presentation with ‘moans, groans and stones’, the hallmarks of hypercalcemia. In contrast with parathyroid hyperplasia, pancreatic tumors are a major cause of morbidity and mortality in MEN 1 [9–11]. The clinical presentation of endocrine pancreatic tumors is usually dependent on the type of polypeptide they secrete, whilst nonfunctioning lesions are frequently detected during routine radiological screening. Gastrinomas in the pancreas or duodenum usually give rise to persistent peptic ulcers (i.e. Zollinger-Ellison syndrome), whereas insulinomas usually cause symptoms of hypoglycemia and psychological disturbances. Other less common endocrine pancreatic tumors can secrete glucagons (glucagonoma), somatostatin (somatostatinoma) or vasointestinal polypeptide (VIPoma).

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In most cases, MEN 1-related pituitary tumors are either prolactin secreting or nonfunctioning, although growth hormone (GH)-secreting, adrenocorticotropic hormone-secreting and, rarely, thyroid-stimulating hormone-secreting tumors have been reported. Besides the hypersecretory effects of these hormones, such as galactorrhea, amenorrhea, impotence and gigantism, the lesions can be large enough to cause optic nerve impingement. Adrenocortical disease has been reported in up to one third of MEN 1 patients but, except for a few reported cases of malignancies, the majority of cases involve benign enlargement not associated with any disturbance in the hypothalamic-pituitary-adrenocortical axis [12]. These cases are usually detected through intraabdominal imaging investigations. Foregut carcinoids such as thymic and bronchial carcinoids are rare, but are known to be associated with MEN 1. In thymic carcinoids, there is a male predominance and the patients do not present with carcinoid or Cushing’s syndrome. Instead, they are usually detected through routine investigations, or, in some advanced cases, the patients present with local invasion causing dyspnea or chest pain [13]. To date, other lesions that have been associated with MEN 1 include lipomas, spinal ependymoma, renal angiomyolipoma and leiomyoma of the esophagus and a number of cutaneous lesions. With regard to the latter, facial angiofibroma and collagenoma have been identified in up to 75% of MEN 1 patients, who may also have confetti-like hypopigmented macules and multiple gingival papules [14]. Other common tumors, such as melanoma and thyroid tumors, have also been reported in MEN 1 patients, but whether they occur by coincidence or as part of the syndrome is yet to be established [15, 16]. The age of diagnosis for MEN 1 is dependent on two factors: (1) if the patient is symptomatic or asymptomatic and diagnosed by biochemical screening, and (2) the type of tumor that the patient develops [15, 17, 18]. Asymptomatic patients are diagnosed at an earlier age by biochemical screening than are those who are symptomatic. For example, Trump et al. [17] showed that in their symptomatic group, the cumulative percentages of patients who developed MEN 1 were 18, 52 and 78% at the ages of 20, 35 and 50 years, respectively. In the asymptomatic group who were diagnosed by biochemical screening, however, the cumulative percentages increased to 43, 85 and 94%, respectively, in the same age groups [17]. In a study of the largest MEN 1 kindred, it was shown that by the age of 20 years, two thirds of patients were found to have primary hyperparathyroidism, and by the age of 30 years, this figure increased to 95% [18]. In addition, both studies showed that endocrine pancreatic tumors reveal two patterns: gastrinoma occurs commonly in older groups of patients (e.g. above 30 or 40 years), whereas insulinoma tends to occur in young patients, some in their teens. For anterior pituitary tumors, most patients are diagnosed between their twenties and forties [15, 17, 18].

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Fig. 1. NLS-1 and NLS-2 and the JunD-binding domains in MENIN. The locations of NLS-1 and NLS-2 in the 610-amino acid MENIN are indicated. The amino acid sequences that could function as a single basic type or bipartite basic type nuclear localization signal in either NLS-1 or NLS-2 are underlined. The locations of three domains known to be required for JunD interaction are also indicated.

Phenotype and Genotype Correlation With the recent cloning of the MEN 1 gene (MEN 1) (see Identification of the MEN 1 Gene and Mutations below), several studies have tried to correlate the genotypes (i.e. mutations) with the clinical phenotypes, an exercise that has proven very fruitful in a number of familial cancer syndromes such as von HippelLindau disease and MEN 2 (see the chapters by Mulligan, Gimm and Iliopoulos [this vol.]). In MEN 1, however, these studies have been unsuccessful. Firstly, unlike MEN 2, MEN 1 has never been divided into any clinically distinct subtypes. The clinical presentation, age of onset and natural history of the disease are known to vary extensively even among members of the same family. Secondly, judging by the spectrum of mutations, which is broadly spread throughout the gene [19, 20], any correlation would be difficult. Still, the findings of predominantly missense mutations in familial isolated hyperparathyroidism (FIHP) are interesting and worth further studies. For example, in two large autosomal dominant FIHP families, which are characterized by parathyroid hyperplasia (one with 7 and the other with 14 affected individuals), two distinct missense mutations in close proximity in exon 4, E255K and Q260P, have been identified [21, 22]. Interestingly, these two mutations fall outside the sites for nuclear localization signals and JunD binding (fig. 1), suggesting a correlation of functionally ‘milder’ mutations with a milder form of disease. Our findings suggest that they form the MEN variant of FIHP (see below MEN 1 – Any Variants?).

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Attempts of genotype/phenotype correlation in sporadic tumors have not been fruitful either. In an analysis of MEN 1 somatic mutations in 51 sporadic gastrinomas, more than half of the mutations were found to be clustered in exon 2 [23]. However, there is no correlation with clinical characteristics, primary tumor sites and outcome of treatment between patients with or without a MEN 1 mutation. Phenocopy and Anticipation in MEN 1 Phenocopies refer to family members who have the phenotype but not the genotype of a disease. In a study of the largest MEN 1 family, 7 of 71 individuals satisfying the clinical diagnostic criteria for MEN 1 were found to be genetically negative (exclusion by mutation analysis and haplotyping) for MEN 1 [24]. These MEN 1 phenocopies comprise 4 cases of primary hyperparathyroidism, 2 of ‘nonsecretory’ pituitary adenomas and 1 case of coincident prolactinoma and hyperparathyroidism. The high incidence of phenocopies in this particular family can possibly be attributed to the coupling of two factors: (1) both mild hyperparathyroidism and asymptomatic pituitary neoplasia occur frequently in the general population [25–27], and (2) stringent biochemical and radiological screening tests were performed prior to genetic diagnosis in this well-known family. Anticipation, a phenomenon in which severity increases and the age of onset decreases in successive generations, has also been described in MEN 1 [28]. In a study of a large central European family, obligate gene carriers of earlier generations were either asymptomatic or died in old age of unrelated causes. In the subsequent generations, however, early onset of disease and malignant tumors with severe consequences were found. The family carries a nonsense mutation, but no other genetic abnormality, such as DNA repeat expansion, which is known to cause anticipation, could be detected to explain this phenomenon. Modifier Gene(s) in MEN 1 Modifier factors, both genetic and epigenetic, can influence the penetrance of certain disease phenotypes by interacting with the primary disease gene [29, 30]. In MEN 1, genetic and clinical studies in the largest MEN 1 family, Tasman 1, provided prima facie evidence supporting the existence of a disease-modifying gene capable of modulating the expression of the MEN 1 gene. Specifically, gene carriers from different branches of the whole Tasman 1 family have been found to develop endocrine neoplasia at differential rates. For example, more than 50% of gene carriers from two branches develop prolactinomas, whereas in the other branches, the prevalence of prolactinoma among gene carriers is less than 10% [31]. Secondly, studies of MEN 1-related thymic carcinoids also suggest the influence of a modifier gene that is sex related. Of the 42 reported MEN 1-related thymic carcinoids, only 2 are female, and loss of heterozygosity (LOH) of the

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MEN 1 region, a common finding in MEN 1-related tumors, has not been found in these tumors [32]. MEN 1 – Any Variants? In addition to classical MEN 1, a number of kindreds with component features of MEN 1, notably FIHP and familial pituitary tumors, have been considered as having variants of MEN 1. FIHP is characterized by hypercalcemia, elevated parathyroid hormone and hypercalciuria resulting from parathyroid tumors without evidence of other endocrinopathies. More than 100 kindreds, mostly small, have been reported. With recent advances in this area, the genetic bases for some of these families have been delineated. Genetically, they can be divided into four subgroups: (1) a true MEN 1 variant with demonstrated MEN 1 mutation (discussed above) [21, 22]; (2) a variant of hyperparathyroidism-jaw tumor syndrome, an autosomal dominant disorder characterized by parathyroid adenoma with increased risk of malignant transformation, fibro-osseous jaw tumor, Wilms’ tumor, kidney cystic disease and neoplasia [7, 33] – the susceptibility gene maps to 1q21-32, and a subset of FIHP families have been linked to this region [34]; (3) a variant of familial benign hypocalciuric hypercalcemia – one family which has been described has all the features of hyperparathyroidism including hypercalciuria, but like familial benign hypocalciuric hypercalcemia, the underlying etiology is associated with a mutated calcium sensing receptor [35], and (4) genetically undefined families. Most of these families are small, with two or three affected cases. They are neither associated with MEN 1 mutation nor linked to 1q. It is possible some of these families may represent chance clustering of sporadic hyperparathyroidism or may be linked to an additional, as yet unidentified chromosomal region. Familial pituitary tumors have been described, usually as familial acromegaly or familial prolactinoma [36, 37]. Although the mode of transmission is considered to be autosomal dominant, reduced penetrance is frequently found in these families. These families are usually small compared with those with classical MEN 1, and a number of them have additional clinical expression atypical of MEN 1. Although clinically, these syndromes are considered to be separate entities from classical MEN 1, whether these families are allelic to MEN 1 is not known. To date, linkage to MEN 1 was excluded in two kindreds with familial acromegaly, but no new locus has been assigned [38, 39]. On the other hand, in several families, supportive but not conclusive linkage to the MEN 1 region and LOH of the wild-type 11q13 alleles in the tumors has been found [40, 41]. However, to date, no MEN 1 mutation has been found [41–43], which may suggest that if this were caused by MEN 1 germline mutations, then they may be unique and lie in regulatory or intronic regions.

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Management of MEN 1 Patients Treatment MEN 1-related hyperparathyroidism is invariably a multiglandular parathyroid disease, and recurrence after subtotal parathyroidectomy is well known [44]. Hence, the surgery of choice is either total parathyroidectomy with autotransplantation or subtotal resection of three and a half parathyroid glands. The choice is dependent on the surgeon’s preference, since to date, there has been no conclusive long-term study comparing the two alternatives. With either approach, however, concurrent thymectomy is highly recommended for two reasons. Firstly, it will remove any supernumerary parathyroid gland in the thymus. Secondly, MEN 1 patients, particularly males, have a risk of developing thymic carcinoid, which has a poor prognosis with no known effective treatment [13, 32]. Pancreatic tumors are a major cause of morbidity and mortality in MEN 1 [9–11]. Gastrinomas are the most frequently encountered; these are symptomatic secretory enteropancreatic tumors giving rise to Zollinger-Ellison syndrome [45, 46]. These tumors are typically multicentric and include microgastrinomas (!6 mm) which are often not detectable by conventional preoperative imaging techniques. A number of studies have shown that control of hyperparathyroidism can ameliorate hypergastrinemia, and parathyroidectomy together with medical management of gastric hyperacidity produces a satisfactory symptomatic response [18, 47, 48]. However, as this strategy does not ‘cure’ the gastrinoma, which has a predilection for recurrence and metastasis, surgical approaches have been advocated [46, 49]. These include percutaneous transhepatic selective venous sampling for gastrin, distal pancreatectomy, duodenotomy, enucleation of pancreas tumors and peripancreatic lymph node dissection. Total pancreatectomy has also been proposed, but this approach is not widely practiced due to the high morbidity associated with it. The forms of medical treatment include symptomatic treatment such as omeprazole and H2 receptor blockers, but recently, the somatostatin analogue octreotide has been used to treat hypergastrinemic patients [50]. This agent suppresses gastrin secretion and may also have the added benefit of inhibiting gastrin-induced neuroendocrine cell hyperplasia and carcinoidosis in the gastric mucosa [51]. Surgical resection is generally recommended for all other types of symptomatic enteropancreatic tumors (e.g. insulinoma and glucoganoma) or those lesions greater than 3 cm in diameter [52]. Resection of the former group is generally curative and disease recurrence is uncommon [53]. For tumor localization, conventional radiological imaging can be augmented by somatostatin receptor scintigraphy and intraoperative ultrasonography [54]. The treatment for MEN 1-related pituitary tumors depends on the type and severity, but all patients should be closely followed up. Small and asymptomatic

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‘nonfunctioning’ tumors do not require specific therapy. For prolactinomas, a D2 dopamine agonist (bromocriptine) is the treatment of choice, but occasionally, surgery is indicated when intolerance is a problem. For nonfunctioning tumors and GH- and adrenocorticotropic hormone-secreting tumors, surgery and/or radiotherapy are usually indicated. Somatostatin has also been shown to be effective in treating GH-secreting tumors [55]. Genetic Testing With the identification of the MEN 1 gene, mutation analysis is now the standard tool for diagnosis. Various approaches, including single-strand conformation polymorphism, dideoxy fingerprinting and denaturing gradient gel electrophoresis, have been used with various degrees of success in mutation detection. However, due to the wide spectrum of mutations found throughout the gene, direct sequencing of all coding exons is still considered the gold standard. To date, in approximately 10–20% of MEN 1 families, mutations still cannot be found by sequencing. It is likely that some of these mutations may involve large deletions [56], which may be detected by Southern blot or may lie in regulatory or intronic regions. In these families, linkage analysis using chromosome 11q13 polymorphic markers is still useful (fig. 2). In this process, blood from at least two affected relatives and those seeking presymptomatic diagnosis is needed. There are two types of genetic markers available for this purpose, namely, restriction fragment length polymorphism markers and microsatellite markers. The method using the latter, which is polymerase chain reaction (PCR) based, is preferred due to its technical convenience, larger number of polymorphic alleles and the lower amounts of DNA required. Illustrative Case Presentation A healthy 36-year-old man (II-2) was found to have hypercalcemia on a routine medical checkup by his general practitioner. He was subsequently confirmed to have hyperparathyroidism. A detailed family history revealed that his brother (II-3) and a paternal cousin (II-5) had been operated for hyperparathyroidism. His father (I-2) had died from a perforated stomach ulcer and a paternal aunt (I-3) had been treated for prolactinoma. MEN 1 was suspected in this case and he was screened for pancreatic and anterior pituitary tumors. All biochemical tests (see discussion below) were normal but an abdominal computed tomography (CT) revealed a mass 8 mm in diameter in the tail of the pancreas. Magnetic resonance imaging (MRI) of his brain was normal. A subsequent neck exploration was performed which revealed four mildly enlarged parathyroid glands. The three most enlarged glands were removed. Half of the remaining left inferior gland was removed. Concurrent thymectomy was also performed. After a period of hypocalcemia, the calcium level was restored to normal. He has two teenage children

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Fig. 2. Pedigree of the case reported in this paper, showing genetic predictive testing for MEN 1 using linkage analysis, as no mutation was identified by mutation analysis. The filled symbols indicate affected family members and the arrow indicates the proband. Numbers alongside the two bars below each individual indicate the genotypes for the informative marker systems, listed next to the symbol for I-1. A filled bar with genotypes 4, 5 and 3 indicates the inferred mutant chromosome. Of the individuals in generation III, only III-1 carries the disease gene.

(III-1, III-2), a 30-year-old cousin (II-4) and a teenage nephew (III-3) who have all been healthy and asymptomatic. The following factors need to be considered in this case: (1) what is the appropriate management for the family?; (2) what is the optimum management for the patient?, and (3) what constitutes the necessary biochemical and radiological screening for MEN 1? (1) This case stresses the importance of a careful family history. It led to further investigations for MEN 1. All family members should be contacted for consent for genetic testing. If facilities for DNA analysis are not available, blood samples can be sent to centers that have a special interest in this area. Genetic testing will help establish the diagnosis of MEN 1 in this family, besides determining who the gene carriers are. In this case, no mutation could be identified by full sequencing of the MEN 1 gene, and linkage analysis using MEN 1-linked markers was performed (fig. 2). Those who tested negative could be exempted from further routine biochemical and radiological screening (II-4, III-2, III-3). Those who were

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positive (I-3, II-2, II-3, II-5, III-1) should be followed up carefully by biochemical and radiological investigations but, unlike in MEN 2, no surgical interventions should be performed until hyperfunction or neoplasia is seen. It is very important to explain the condition in detail to all family members. A simple information sheet or booklet is very valuable (one is available on the Internet at http:// www.niddk.nih.gov/health/endo/pubs/fMEN 1/fMEN 1.html), and psychological counseling should be made available. (2) Irrespective of the findings during neck exploration, removal of three and a half glands or total parathyroidectomy followed by autotransplantation should be performed together with thymectomy in MEN 1 patients, as in this case. The likelihood that this patient will have a recurrence is high, and there is also a risk that he will develop thymic carcinoid. His asymptomatic pancreatic tumor was treated conservatively and should be followed up carefully. (3) MEN 1 is a complex disease, and hence the patients and their families should be under the care of specialists. We recommend the following initial screening: (a) for parathyroid involvement: serum intact parathyroid hormone, total and ionized calcium, albumin and phosphate; (b) for endocrine pancreatic and adrenal involvement: chromogranin, fasting gastrin, glucose, C-peptide, abdominal ultrasound, CT scan or MRI; (c) for pituitary involvement: serum prolactin and MRI or CT of the pituitary; (d) for foregut carcinoid: CT of the chest, and (e) for gastroduodenal carcinoid: upper gastrointestinal endoscopy. All blood tests and an abdominal ultrasound should be performed yearly, and the other special tests should be considered according to the history, signs and symptoms and clinical examination of the individual.

Identification of the MEN 1 Gene and Mutations The MEN 1 gene was identified by positional cloning, a process whereby a disease gene is identified based mainly on its chromosomal location. LOH studies of tumors from MEN 1 patients followed by analysis of MEN 1 kindreds demonstrated that the MEN 1 gene is in the vicinity of a polymorphic marker, muscle glycogen phosphorylase gene (PYGM), in chromosomal region 11q13 [57]. Haplotype analysis confined the MEN 1 gene locus to a 2,000-kb interval flanked by the markers D11S1883 and D11S449 [58, 59]. Helped by powerful microdissection technology and using a large number of newly developed polymorphic markers from the MEN 1 region, careful and systematic LOH studies were also carried out to define reliable deletion profiles for tumors. These studies eventually narrowed this region to a smaller 300-kb interval between PYGM and the marker D11S4936 [60]. Genes from this interval were identified and tested for mutations in MEN 1 kindreds [61]. Deleterious germline mutations observed in one of these

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Fig. 3. Knudson’s two-hit mutation model. a In familial tumors, MEN 1 patients inherit one mutant gene copy and, at some stage later in life, acquire a second mutation which inactivates the remaining normal copy in the glands involved. The second hit is usually in the form of a chromosomal deletion. The two-hit process causes complete elimination of the tumor-suppressive activity of the responsible gene, thus setting off tumor development. b In sporadic tumors, inactivation of both copies of the MEN 1 gene occurs by two independent hits in the same somatic cell.

genes in 15 of the 16 probands confirmed the MEN 1 gene [62]. The MEN 1 gene encodes a 610-amino acid novel protein, termed MENIN. The cloning of the MEN 1 gene was also reported in a separate study a few months later [63]. To date, over 300 distinct mutations, both germline and somatic, have been reported [20, 64, 65]. The deleterious mutations observed in MEN 1 kindreds, and the loss of the wild-type allele observed in tumors from familial MEN 1 patients and sporadic tumors, support Knudson’s two-hit model of tumorigenesis for the MEN 1 gene (fig. 3). In addition, as predicted from this model, somatic mutations in sporadic endocrine tumors have also been observed.

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Fig. 4. Germline and somatic mutations of MEN 1. Truncation (nonsense, frameshift and splice) mutations are shown at the top of the MENIN diagram and the missense and in-frame deletions are shown at the bottom. The noncoding region is stripped, and the nuclear localization signals (NLS) are indicated.

Germline Mutations To date, over 250 independent germline mutations have been identified since the cloning of the MEN 1 gene in 1997 [20, 64, 65] (fig. 4). The extent of mutations detected in different studies varies from 60 to 95% of the MEN 1 kindreds analyzed. Genetic heterogeneity seems unlikely based on prior linkage analysis. The variation in the sensitivity of the method employed for mutation detection, larger deletions that escape PCR-based mutation detection and mutations that lie outside the coding region and splice junctions might contribute to the failure to identify MEN 1 mutations in a certain fraction of these kindreds. Diverse inactivating mutations spread throughout the gene once again suggest that inactivation of MENIN is responsible for tumorigenesis in this syndrome. Recurrence of a common mutation in multiple kindreds has been observed. Haplotype analysis of MEN 1 kindreds in certain geographical locations has allowed the identification of founder mutations and the time of their origin. Four kindreds from the Burin peninsula of Newfoundland have been shown to share a common haplotype and an ancestral R460X mutation [66], which appear to have originated in settlers who migrated from southwest England in the early 1800s. The 1466del12 mutation observed in eight Finnish families originated about 300 years ago in a small village in northern Finland, and the origin of the 1657insC mutation in four Finnish families can be traced to a couple who lived approxi-

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mately 150 years ago [67, 68]. Six kindreds sharing the 512delC mutation shared a common haplotype and originated from the Ohio/Pennsylvania region of North America, whereas 5 kindreds with 416delC who also shared a common haplotype could not be traced to a common geographical origin [19, 69, 70]. There are several mutations that recur in apparently unrelated families, including those with different ethnic origins and geographical locations [67]. The most common among them were found to be 359del4, K119del, 734del4 and 1650–1657delC, affecting amino acids 83–84, 119, 209–211 and 514–516, respectively. It turns out that these are either short deletions or insertions occurring at CpG/CpNpG-type direct repeats or single-nucleotide repeats (e.g. 1650– 1657delC). Such sequences are hot spots for mutation, as they represent sites of common mechanisms of mutation due to cytosine methylation or slippage of DNA polymerase [70, 71]. De novo mutations appear to account for about 10% of the total germline mutations [71]. Somatic Mutations in Sporadic Tumors LOH of the 11q13 region occurs in a fraction of certain sporadic counterparts of MEN 1-related tumors, suggesting the involvement of somatic MEN 1 mutations in these tumors. To date, somatic MEN 1 mutations have been reported in 13–21% of parathyroid adenomas [72, 73], 27–39% of pancreatic gastrinomas [74–77] and 36% of carcinoid tumors of the lung [78], pointing to MEN 1 mutations as the single major contributor to the tumorigenesis of these tumors. MEN 1 mutations have also been found in less than 5% of sporadic pituitary tumors [75, 79] and a limited number of lipomas [80] (fig. 4). However, no MEN 1 mutation has been found in sporadic adrenocortical neoplasms [81, 82]. MEN 1 mutations in a third of bronchial carcinoids have prompted mutation analysis of the other neuroendocrine tumors of the lung. Small-cell lung carcinoma and large-cell neuroendocrine carcinoma failed to show inactivation of MEN 1 [83]. Interstitial deletions of chromosome 11q13 have been reported in primary cervical tumors, cervical cancer cell lines, breast and head and neck cancers, hibernoma and neuroblastoma [84]. However, the MEN 1 gene has been excluded from cervical cancer cell lines, suggesting that a different tumor suppressor gene at 11q13 may be involved in these tumors [84]. Nature of Mutations More than two thirds of the MEN 1 germline or somatic mutations are inactivating mutations, nonsense and frameshift mutations, leading to truncation of the encoded protein, MENIN, if expressed. The remaining mutations are missense and in-frame deletions leading to specific alterations in otherwise full-length or nearly full-length protein (fig. 4). The frameshift mutations include deletions, insertions or splice sequence alterations. A large heterogeneous germline deletion

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has been reported in a Japanese kindred with 3 affected members. Lack of coding region mutations in any of the 3 affected cases and apparent hemizygosity at the polymorphic locus GCA→ACA (A541T) prompted quantitative PCR analysis of the MEN 1 region. The deletion spanned at least 7 kb, encompassing the entire coding region of the gene [85]. The retention of heterozygosity at the flanking markers suggests that the deletion may be ! 300 kb. It is likely that there may be more unrecognized instances of such large deletions in MEN 1 kindreds without a defined MEN 1 mutation.

The MEN 1 Gene: Organization and Expression Exon-Intron Organization The MEN 1 gene (GenBank accession No. U93237) consists of 10 exons extending across 7.2 kb, which is predicted to express a 2.8-kb transcript (GenBank accession No. U93236) [62]. The first exon is noncoding and constitutes most of the 111 nucleotides in the 5) UTR. The sequence around the start codon (gccATGg) of the 610-amino acid open reading frame is identical to the ‘Kozak consensus’. The 797 nucleotides in the 3) UTR have an unusual polyA signal (AATACA) located at –13 from the polyA tail. The exon sizes range from 41 to 1,296 nucleotides, and the introns range in size from 79 to 1,563 nucleotides. Two alternative forms of the MEN 1 transcript have been described. One has a 5) UTR that is 382 nucleotides longer, detected in human thymocyte cDNA [86]. The variant 5) UTR originates from an alternative splice signal sequence in the first intron, and does not alter the coding region of MEN 1. One other alternatively spliced transcript was identified among the sequences of 20 MEN 1 cDNA clones isolated from a human leukocyte cDNA library [61]. This rare message utilizes a splice donor located 15 nucleotides downstream in intron 2, thus extending exon 2 by 15 nucleotides. This transcript, if expressed, keeps the entire reading frame intact except that it has an additional 5 amino acids (U93236). However, scanning of cDNA libraries from nine different tissues failed to identify this alternative message and, therefore, it appears to represent a tiny fraction of the leukocyte library. The 610-amino acid MENIN sequence does not have homology to any other known protein sequence, nor does it reveal any recognizable functional or structural domains [62]. There are sequences that resemble leucine zippers but they lack the characteristic amphipathic structure. Although it has several phosphorylation sites, phosphorylation or any other posttranslational modification of MENIN has yet to be reported. From its sequence, the predicted size of MENIN is 67 kD, but the size estimated from its mobility on a denaturing gel varies in different reports, ranging from 67 to 76 kD.

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MEN 1 Expression by Northern Analysis and in situ Hybridization Northern analysis of polyA RNA from 20 different human tissues showed the expression of the 2.8-kb MEN 1 transcript in all of them [61]. The ubiquitous nature of MEN 1 expression is apparent in a variety of cell lines, including 10 cervical carcinomas [84], 13 small-cell lung carcinomas [83] and several human and rodent cell lines of endocrine and nonendocrine origin. In addition to the 2.8-kb transcript, a lower amount of an additional, larger transcript (approximately 4 kb) has also been observed in polyA RNA from thymus and pancreas. The origin and characteristics of this larger transcript are unknown at present [61]. Besides Northern analyses, the expression of MEN 1 transcripts has been studied by in situ hybridization in a variety of adult human tissues including liver, lung, kidney, spleen, esophageal mucosa, duodenal mucosa, thyroid, pancreas (acinar and islet), endometrium and placenta using a riboprobe containing the MEN 1 cDNA sequence [87]. The MEN 1 transcript was present in all the organ systems tested, but its level varied from a weak and homogeneous staining in lung, liver, kidney, spleen, mammary gland and thyroid, to a stronger one in placenta. The expression was also found to be much higher in the proliferative phase of endometrium than in the secretory phase. The increased proliferative activity, as measured by the expression of the proliferative marker Ki-67, was in parallel with prominent MEN 1 expression [87]. MENIN Expression by Western Analysis and in Relation to the Cell Cycle Several polyclonal antibodies raised against short peptide sequences of human MENIN have served well in the analysis of MENIN expression by Western blotting and immunofluorescence. The high sequence conservation allows some of these antibodies to cross-react with MENIN from other species, even as remote as zebrafish [88]. A wide variety of human, rodent and simian cell lines have been found to express MENIN. These include cells of endocrine (rat pituitary tumor GH3, insulinoma RIN-5F, glioma C6, pheochromocytoma PC12, mouse pituitary corticotroph ArT-20, human lung carcinoma, human medullary thyroid carcinoma) and nonendocrine origin (human embryonic kidney HEK293, human cervical carcinoma HeLa, Chinese hamster ovary cells, mouse NIH3T3, simian COS-1, human lymphoma and leukemia). MENIN expression is observed in all mouse tissues tested, including liver, brain, pancreas, spleen, heart, lung, testis and kidney [89–91]. Among the 12 fetal human tissue extracts analyzed, MENIN was readily detectable in brain cortex, kidney, pituitary, testis and thymus, but was weaker in thyroid and undetectable in liver, lung, pancreas and skin [92]. The latter may be due to low expression or degradation of the protein.

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MENIN levels have been found to be relatively constant in NIH3T3 cells throughout the G0, G1 and M phases of the cell cycle [89]. Similar findings are observed in HeLa cells, and by expression microarray studies [92, 93]. However, two additional studies, one measuring the expression at the RNA level and the other at the protein level, indicated transient changes in MEN 1 expression levels. Kaji et al. [94] measured MENIN by Western analysis at different time points upon serum stimulation of starved rat pituitary tumor (GH4C1) cells. Over a 24-hour time period, the MENIN levels decreased transiently by as much as half, and then increased as more cells entered the S phase. Cells arrested at the G1-S boundary showed 30–50% less MENIN than did cells at G0. Release of the blockage caused the reentry of cells into the S phase and increased the MENIN levels. At all these stages, MENIN remained predominantly in the nuclear fraction [94]. In an independent study, COS-1 cells (African green monkey kidney cells transformed with SV40T antigen) synchronized at the G1-S border were shown to contain about half as much (44%) MEN 1 RNA as in asynchronous cells [87]. Again, as the cells entered the S phase, the MEN 1 RNA levels increased back to the normal (asynchronous cells) level. The variation (or lack thereof) in MEN 1 expression observed at different stages of the cell cycle might reflect the use of different cell lines and different reagents to synchronize the cells. The functional significance associated with these subtle alterations in MEN 1 expression, if any, needs to be determined.

The MENIN Protein MENIN Is a Nuclear Protein MENIN resides primarily in the nucleus, as shown by immunofluorescence, Western blotting of subcellular fractions and epitope tagging with green fluorescent protein in a variety of cell lines [95]. This finding has been further confirmed by: (1) immunostaining of cultured cells from a human pituitary tumor cell line [96]; (2) immunofluorescence with MENIN antibodies of COS7 cells transfected to express rat MENIN [97], and (3) subcellular fractionation and Western analysis of rat pituitary GH4C1 cells [94], human lymphocytes [90] and HeLa cells [92]. Two nuclear localization signals are found in the C-terminal quarter of MENIN: NLS-1 (amino acids 479–497) and NLS-2 (amino acids 588–608). Scanning the nearly 300 independent MEN 1 mutations (see Identification of the MEN 1 Gene and Mutations above) reveals that none of the 69 missense or 23 in-frame deletions affects the NLS-1 or NLS-2 sequences. All the frameshift and nonsense mutations leading to a truncated protein lack either one or both signals; none retains NLS-2 and only about 10% retain NLS-1. Thus, most truncated MENINs, if expressed, are not likely to localize to the nucleus. Moreover, it has

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been reported that a substantial portion of MENIN in lymphocytes from a heterozygous MEN 1 gene mutation (1657insC), expressing a truncated MENIN (amino acids 1–516) retaining only NLS-1, is present in the cytoplasmic fraction [90]. The total amount of truncated MENIN is lower than that of wild-type MENIN. In addition, the truncated 516-amino acid MENIN from both nuclear and cytoplasmic fractions is degraded faster than wild-type MENIN, indicating that NLS-2 may be critical for the efficient transfer of MENIN to the nucleus, and might also contribute to the stability of the protein [90]. In contrast, studies of patient lymphoblastoid cell lines harboring germline nonsense mutations have failed to detect the truncated protein in the nucleus or cytoplasm [92]. This probably reflects the unstable nature of the mutant message or the protein. The nuclear localization of MENIN suggests a variety of possible functions, such as transcription regulation, DNA replication or cell cycle control. One of the ways to decipher the function of this nuclear protein is to identify its associated protein partners, as described below. MENIN Interacts with JunD, an AP1 Transcription Factor Application of the yeast two-hybrid system to screen an adult human brain cDNA library has resulted in the identification of the first recognized interacting protein for MENIN, the AP1 transcription factor JunD [98]. The MENIN-JunD interaction has been confirmed in vitro and in vivo by glutathione S-transferase fusion protein pull-down assays, coimmunoprecipitation analysis and the mammalian two-hybrid system [98]. The AP1 transcription factor family consists of Jun members (c-Jun, JunB and JunD) and Fos members (c-Fos, FosB, Fra-1 and Fra-2) [99, 100]. However, other members of the Jun/Fos family (JunB, c-Jun, c-Fos, Fra-1, Fra-2) show no direct interaction with MENIN [98]. Deletions of MENIN have been generated and tested for interaction with JunD in the yeast two-hybrid system and glutathione S-transferase fusion proteinJunD pull-down assays. Three regions, amino acids 1–40, 139–242 and 323–449, appear to be required for MENIN interaction with JunD (fig. 1) [98]. MENIN Represses JunD-Mediated Transcriptional Activation JunD belongs to the basic leucine zipper family of transcription factors that dimerize at their C-terminal leucine zipper domain. They form the AP1 complex when their basic domains bind to DNA. They regulate transcription from promoters that possess a specific DNA sequence called the consensus 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE) [101]. To measure the effect of MENIN on JunD-mediated transcription, a GAL4 and UAS system is used in which the fusion of GAL4 DNA-binding domain and JunD stimulates transcription of a reporter gene construct that possesses a GAL4-responsive DNA sequence (UAS). It has been found that the transactivation by a GAL4 DNA-

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binding domain-JunD fusion protein from a GAL4-responsive reporter is repressed by cotransfection of MENIN in 293wt cells. Similarly, the transactivation by JunD from a TRE-containing AP1 reporter was also repressed effectively by MENIN. However, the mechanisms by which MENIN represses JunD-driven transcription remain to be elucidated. In contrast to other members of the AP1 family of transcription factors, JunD has been shown to possess inhibitory effects on cell growth [102, 103], a tumor suppressor-like property, but so far has not been implicated in the pathogenesis of any tumors. This property and its suppression by MENIN, another tumor suppressor, appears paradoxical at the least. However, the tumor suppressor-like properties that have been observed for JunD may be due to the functional synergy between the possible suppressive effects of MENIN and JunD. It is also possible that JunD interaction with MENIN may provide MENIN with its tumor suppressor function. Elucidation of the pathways and identification of the genes regulated by the MENIN-JunD complex, and the subsequent effect of the gene products on cell growth and transformation, will be helpful in understanding the importance of MENIN-JunD interaction. Suppression of Tumorigenicity in RAS-Transformed Cells Demonstration of the tumor suppressor function of MENIN in vivo has been attempted by exploring the effect of MENIN expression on RAS-transformed NIH3T3 cells [104]. Several independent clones, stably expressing MENIN, were generated from transformed RAS-NIH3T3 cells. These clones overexpress MENIN by 7- to 27-fold, and are more ‘normal’ than the RAS-transformed cells in terms of morphology, growth rate and growth in soft agar and nude mice. These studies demonstrate that the stable overexpression of MENIN has a growth- and tumor-suppressive effect on RAS-transformed cells. These cells express MENIN in the nucleus, and thus the tumor/growth suppression may not be due to blocking of the transport of MENIN to the nucleus. Several other tumor suppressor genes have been evaluated for their tumor suppressor function utilizing the same system [105, 106]. Although this may not be an ideal system to understand the molecular mechanism by which MENIN exerts its tumor suppressor function, the tumor suppressor nature of MENIN protein can be demonstrated.

MEN 1 Orthologs The isolation of MEN 1 orthologs from different species offers advantages for the study of the biology of MENIN. For example, one could inactivate the MENIN function by homologous recombination in the mouse, visualize the role of MEN 1 in early development in the zebrafish and evaluate the effect of (trans-

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genic) MENIN overexpression in desired tissues in Drosophila. In addition, interspecies sequence comparisons of MENINs may also identify functionally significant domains that should be highly conserved among species. To date, the full yeast (Saccharomyces cerevisiae) sequence [107] and the subsequent decoding of the complete sequence of the roundworm Caenorhabditis elegans [108] have not revealed any MEN 1-orthologous sequences. However, the MEN 1 orthologs from the mouse, rat and zebrafish have been characterized in detail. Mouse, Rat and Zebrafish Men 1 Genes and Their Expression in Development The MEN 1 orthologs from the mouse [86, 89, 91, 97, 109], rat [86, 97] and zebrafish [88, 110] have been identified and characterized. The MEN 1 chromosomal region in the mouse (19 B-C2) and zebrafish (LG7) share genes from human 11q13, and therefore the MEN 1 loci among humans, mice and zebrafish appear to be from shared, evolutionarily conserved syntenic regions. In the rat, Northern analysis has shown high expression of gene transcripts at early embryonic stages on days 14, 16 and 18 of gestation [97]. Mouse Men 1 expression has been observed as early as in 7-day-old embryos, and in all subsequent embryonic stages and adult tissues analyzed. Both mouse and rat Men 1 express two transcripts of different sizes (2.7 and 3.1 kb), depending on whether intron 1 is spliced or not. Both messages are expressed in similar amounts in all tissues. A 2.7-kb message has been observed in RNA isolated from different stages of zebrafish development, from 1-cell embryos to adult fish. The presence of the Men 1 message in embryos at the 1-cell stage suggests that it is expressed maternally. Although Northern analysis suggests a single message in zebrafish, sequence analyses of multiple transcripts indicate the utilization of two transcription start sites 14 nucleotides apart. The variant transcripts in the mouse, rat and zebrafish do not alter the open reading frame, but whether they differ in their translational efficiency remains to be discovered. The expression of Men 1 in the mouse, analyzed by whole-mount in situ hybridization, appears to be ubiquitous in early development (8.5- to 17-day embryos), although slightly higher levels of expression are observed in the cranial ganglia, sensory ganglia and neural tube in 10.5-day embryos, and in the thymus, skeletal muscle, brain and spinal cord in 17-day embryos. Expression in adult tissues is also observed in almost all tissues analyzed; however, increased expression has been observed in the brain (Purkinje cells and hippocampus) and testis (Sertoli cells) [89, 91, 109]. In various adult (8–12 weeks old) and fetal (18 days p.c.) murine tissues, quantitative PCR of reverse-transcribed mRNA (RT-PCR) suggests higher expression of men1 in hematopoietic and neuroendocrine tissues compared to nonendocrine tissues [86].

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Ubiquitous expression is observed by in situ hybridization in zebrafish embryos, from cleavage through to somitogenesis stages [88]. At the end of somitogenesis (24 h after fertilization), prominent staining is detected in the trunk in the intermediate cell mass, the site of primary hematopoiesis in fish. Strong expression in maturing blood cells has been confirmed from histological sections and by lack of staining in the intermediate cell mass in two bloodless mutants, vampirem262 and vlad tepesm651 [88, 111]. The strong expression in maturing blood cells resembles the increased expression in mouse hematopoietic tissues as measured by quantitative RT-PCR [86]. Expression is also detected in the central nervous system, including the spinal cord, and in head and fin mesenchyme. Forty-eight hours after fertilization, expression is not apparent in the blood, but is detected in the endoderm, including the liver primordium, as well as in the retina, tectum, otic vesicle, fin bud mesenchyme and branchial arches. Expression in mesenchymal tissues may have some relevance to the phenotypic expression of human MEN 1, as tumors of mesenchymal origin such as lipoma, angiofibroma and ependymoma are known to be components of the syndrome. Embryonic development expression patterns in the zebrafish appear to be similar to those in the mouse [88]. Amino Acid Sequence Comparison of the MENIN Orthologs The amino acid sequences of rat (AB023400), mouse (AF109389) and zebrafish (AF212919) MENIN were compared to the sequences from human MENIN (U93236) (fig. 5). MENIN in zebrafish (617 amino acids) and mice (611 amino acids) is longer than in humans and rats (610 amino acids). The protein sequences are highly conserved through evolution. Zebrafish, mouse and rat MENIN share 67, 96.7 and 97.2% identity, respectively, with human MENIN. Among the 105 amino acids that are altered due to disease-causing missense mutations and inframe deletions in humans, 90% are identical in zebrafish, and all but two are identical in rats and mice. This observation confirms the importance of these evolutionarily conserved residues (fig. 5). Homology to NLS-1 and NLS-2 suggests that similar to humans, two nuclear localization signals may function in rats,

Fig. 5. Alignment of amino acid sequences of MENIN orthologs. Human (Hu), rat (Ra), mouse (Mo) and zebrafish (Zf) MENIN sequences (in single-letter code) are compared. The amino acid residues identical with human MENIN are indicated by a dot. The filled circles on top of the human sequence indicate the 105 amino acids that are altered due to missense mutations and in-frame deletions in MEN 1 patients. The three JunD-binding domains [98] are boxed, and the two nuclear localization signals (NLS-1 and NLS-2) [95] are underlined.

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mice and zebrafish as well. All three regions in MENIN required for JunD binding are nearly identical in the mouse and rat; however, the central 139- to 242amino acid region appears to be most conserved in the zebrafish.

Chromosome Instability and the ‘Mitogenic Factor’ in MEN 1 Reports of chromosomal instability in either lymphoblastoid cells or fibroblasts from MEN 1 patients have indicated that both types of cells increase chromosomal breakage, rearrangements and polyploidy (trisomy) [112–114]. The rearrangements are most evident in short-term lymphocyte cultures, and include tricentric chromosomes, dicentric and ring chromosomes, double minutes, single minutes and acentric fragments. Tomassetti et al. [115] reported the enhancement of instability by treatment of phytohemagglutinin-stimulated lymphocyte cultures from MEN 1 patients with the alkylating agent diepoxybutane (DEB). The latter increases chromatid breakages, gaps and exchange figures. In another study, the effect of DEB on premature centromere division in lymphocyte cultures from 15 MEN 1 patients and 11 age-matched control subjects was investigated [116]. Nine of the 15 patients with MEN 1 had germline mutations, while no MEN 1 mutations were identified in the remaining subjects. It was found that the addition of DEB increased the frequency of premature centromere division in patients with MEN 1 mutation compared to that in normal individuals and those patients without an identifiable MEN 1 mutation. These results suggest that MENIN may play a role in the repair of damaged DNA and/or in the maintenance of the integrity of DNA. Plasma of MEN 1 patients has previously been shown to have mitogenic activity on cultured bovine parathyroid cells [117]. This ‘mitogenic factor’, estimated to have an apparent molecular weight of 50,000–55,000 kD, has subsequently been found to be a fibroblast growth factor-like factor that may be secreted by the pituitary tumors [118, 119]. However, its role in MEN 1 has not been established. Tumor suppressors are known to regulate growth factor gene expression. For example, the VHL gene has been shown to suppress the expression of vascular endothelial growth factor, which is highly expressed in renal cell carcinoma, a feature of von Hippel-Lindau disease [120]. However, in addition to further characterization of this mitogenic factor, its relationship to MENIN and its role in MEN 1, if any, needs to be established.

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The Future Undoubtedly, the cloning of the MEN 1 gene is one of the cornerstones in MEN 1 research. It has made possible the early diagnosis and detection of mutation carriers who can be clinically evaluated more frequently. The biochemistry, molecular biology and cell biology of MENIN is being evaluated in order to understand its biological role. The identification of its first recognized interacting protein, JunD, is a good start. Isolation of other MENIN-interacting proteins, and the development of animal models, particularly a mouse MEN 1 knockout model, should help understand additional functions of MENIN and the role of MENIN in tumorigenesis. It will be important also to look for genetic abnormalities, other than the MEN 1 gene, that might be involved either as modifier genes or as aids in the progression of tumor development. In keeping with the concept of the clonal evolution or multistep process of tumorigenesis, the mutations of the MEN 1 gene are expected to serve as the initial genetic triggers. A mouse model should also help understand the process that leads to the development of endocrine tumors and their progression to malignancy and metastases. The elucidation of this pathway, which is less understood in MEN 1, will have great clinical implications.

Acknowledgments S.C.C. would like to thank the members of the NIH-MEN 1 research group, collaborators in the studies on MEN 1, for insightful discussions. We would like to thank Steve J. Marx for critical reading of the manuscript and for an updated MEN 1 mutation diagram. We would also like to thank Lynn Ritsema for preparation of the manuscript.

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94 Kaji H, Canaff L, Goltzman D, Hendy GN: Cell cycle regulation of menin expression. Cancer Res 1999;59:5097–5101. 95 Guru S, Goldsmith PK, Burns AL, Marx SJ, Spiegel AM, Collins FS, Chandrasekharappa SC: Menin, the product of the MEN 1 gene, is a nuclear protein. Proc Natl Acad Sci USA 1998;95: 1630–1634. 96 Huang S, Zhuang Z, Weil RJ, Pack S, Wang C, Krutzsch HC, Pham TA, Lubensky IA: Nuclear/ cytoplasmic localization of the multiple endocrine neoplasia type 1 gene product, menin. Lab Invest 1999;79:301–310. 97 Maruyama K, Tsukada T, Hosono T, Ohkura N, Kishi M, Honda M, Nara-Ashizawa N, Nagasaki K, Yamaguchi K: Structure and distribution of rat menin mRNA. Mol Cell Endocrinol 1999;156: 25–33. 98 Agarwal S, Guru SC, Heppner C, Erdos MR, Collins RM, Park SY, Saggar S, Chandrasekharappa SC, Collins FS, Spiegel AM, Marx SJ, Burns AL: Menin interacts with the AP1 transcription factor JunD and represses JunD-activated transcription. Cell 1999;96:143–152. 99 Rahmsdorf H: Jun: Transcription factor and oncoprotein. J Mol Med 1996;74:725–747. 100 Karin M, Liu Z, Zandi I: AP-1 function and regulation. Curr Opin Cell Biol 1997;9:240–246. 101 Angel P, Herrlich P: General structure of AP1 subunits and characteristics of the Jun proteins; in Angel P, Herrlich P (eds): Fos and Jun Families of Transcription Factors. Boca Raton, CRC Press, 1994, pp 3–14. 102 Pfarr C, Mechta F, Spyrou G, Lallemand D, Carillo S, Yaniv M: Mouse JunD negatively regulates fibroblast growth and antagonizes transformation by ras. Cell 1994;76:747–760. 103 Mechta F, Lallemand D, Pfarr CM, Yaniv M: Transformation by ras modifies AP1 composition and activity. Oncogene 1997;14:837–847. 104 Kim Y, Burns AL, Goldsmith PK, Heppner C, Park SY, Chandrasekharappa SC, Collins FS, Spiegel AM, Marx SJ: Stable overexpression of MEN 1 suppresses tumorigenicity of RAS. Oncogene 1999;18:5936–5942. 105 Luo X, Reddy JC, Yeyati PL, Idris AH, Hosono S, Haber DA, Licht JD, Atweh GF: The tumor suppressor gene WT1 inhibits ras-mediated transformation. Oncogene 1995;11:743–750. 106 Eliyahu D, Michalovitz D, Eliyahu S, Pinhasi-Kimhi O, Oren M: Wild-type p53 can inhibit oncogene-mediated focus formation. Proc Natl Acad Sci USA 1989;86:8763–8767. 107 The yeast genome directory. Nature 1997;387:5–105. 108 Genome sequence of the nematode C. elegans: A platform for investigating biology. The C. elegans Sequencing Consortium. Science 1998;282:2012–2018. 109 Bassett J, Rashbass P, Harding B, Forbes SA, Pannett AA, Thakker RV: Studies of the murine homolog of the multiple endocrine neoplasia type 1 (MEN 1) gene, MEN 1. J Bone Miner Res 1999; 14:3–10. 110 Khodaei S, O’Brien KP, Dumanski J, Wong FK, Weber G: Characterization of the MEN 1 ortholog in zebrafish. Biochem Biophys Res Commun 1999;264:404–408. 111 Weinstein B, Schier AF, Abdelilah S, Malicki J, Solnica-Krezel L, Stemple DL, Stainier DY, Zwartkruis F, Driever W, Fishman MC: Hematopoietic mutations in the zebrafish. Development 1996; 123:303–309. 112 Gustavson K, Jansson R, Oberg K: Chromosomal breakage in multiple endocrine adenomatosis (types I and II). Clin Genet 1983;23:143–149. 113 Benson L, Gustavson KH, Rastad J, Akerstrom G, Oberg K, Ljunghall S: Cytogenetical investigations in patients with primary hyperparathyroidism and multiple endocrine neoplasia type 1. Hereditas 1988;108:227–229. 114 Scappaticci S, Maraschio P, del Ciotto N, Fossati GS, Zonta A, Fraccaro M: Chromosome abnormalities in lymphocytes and fibroblasts of subjects with multiple endocrine neoplasia type 1. Cancer Genet Cytogenet 1991;52:85–92. 115 Tomassetti P, Cometa G, Del Vecchio E, Baserga M, Faccioli P, Bosoni D, Paolucci G, Barbara L: Chromosomal instability in multiple endocrine neoplasia type 1. Cytogenetic evaluation with DEB test. Cancer Genet Cytogenet 1995;79:123–126. 116 Sakurai A, Katai M, Itakura Y, Ikeo Y, Hashizume K: Premature centromere division in patients with multiple endocrine neoplasia type 1. Cancer Genet Cytogenet 1999;109:138–140.

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117 Brandi M, Aurbach GD, Fattorossi A, Quarto R, Marx SJ, Fitzpatrick LA: Antibodies cytotoxic to bovine parathyroid cells in autoimmune hypoparathyroidism. Proc Natl Acad Sci USA 1986;83: 8366–8369. 118 Zimering M, Brandi ML, deGrange DA, Marx SJ, Streeten E, Katsumata N, Murphy PR, Sato Y, Friesen HG, Aurbach GD: Circulating fibroblast growth factor-like substance in familiar multiple endocrine neoplasia type 1. J Clin Endocrinol Metab 1990;70:149–154. 119 Zimering M, Katsumata N, Sato Y, Brandi ML, Aurbach GD, Marx SJ, Friesen HG: Increased basic fibroblast growth factor in plasma from multiple endocrine neoplasia type 1: Relation to pituitary tumor. J Clin Endocrinol Metab 1993;76:1182–1187. 120 Gnarra J, Zhou S, Merrill MJ, Wagner JR, Krumm A, Papavassiliou E, Oldfield EH, Klausner RD, Linehan WM: Post-transcriptional regulation of vascular endothelial growth factor mRNA by the product of the VHL tumor suppressor gene. Proc Natl Acad Sci USA 1996;88:8405–8409.

Settara C. Chandrasekharappa, Genetics and Molecular Biology Branch National Human Genome Research Institute, National Institute of Health Bethesda, MD 20892-442 (USA) E-Mail [email protected] Bin Tean Teh, Laboratory of Cancer Genetics, Van Andel Research Institute Grand Rapids, MI 49503 (USA) E-Mail [email protected]

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Multiple Endocrine Neoplasia Type 2: Molecular Aspects Lois M. Mulligan Department of Paediatrics, Queen’s University, Kingston, Ont., Canada

Contents 81 83 84 86 87 88 91 91 92 93 93

Introduction RET Ligand/Co-Receptor Complexes RET Mutations and MEN 2 RET Mutation Genotype and Disease Phenotype RET Mutations in Sporadic Tumours RET Downstream Signalling RET-Associated Diseases Hirschsprung Disease Papillary Thyroid Carcinoma Other Pathologies and Beyond References

Introduction Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominantly inherited cancer syndrome characterized by medullary thyroid carcinoma (MTC), with or without pheochromocytoma (PC) and hyperparathyroidism (HPT) (see the chapter by Gimm [this vol.]). The clinical phenotype of this disease and its diagnosis and management are all discussed in detail in the chapter by Gimm [this vol.]. Here, we will examine the molecular causes and effects of MEN 2, and how the nature of these events sets this disease apart from other heritable human cancers.

Fig. 1. Diagram of the RET receptor showing the structural domains of the protein and the members of the RET signalling complex. Members of the GDNF family act as soluble RET ligands. The GFR· family of cell surface-bound proteins act as co-receptors for presentation of the GDNF ligands to RET. All three components of this multimeric complex must be present to trigger activation of the RET kinase and downstream signalling.

MEN 2 is unusual amongst inherited cancer syndromes, which are generally caused by inactivation of a tumour suppressor, in that it is caused by germline activation of an oncogene, RET (REarranged in Transfection) [1, 2]. The RET proto-oncogene encodes a receptor tyrosine kinase which is required for the normal growth and maturation of cells derived from the neural crest [3–5]. RET expression is developmentally regulated, with the highest levels seen early in development in the kidney and neural crest-derived tissues, such as the emerging peripheral nervous system, the thyroid and adrenal glands [4–6]. Consistent with this pattern, the phenotype of ret-null mice has shown us that RET is critical to the development of the kidneys and peripheral nerves, particularly for the formation of the enteric nervous system [5, 6]. The structure of the RET receptor is typical of the receptor tyrosine kinases. It has a large extracellular domain (fig. 1) involved in the recognition and binding

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of its ligands and co-receptors [1, 2]. The specific RET sequences required for these interactions have not yet been determined; however, a large cysteine-rich region, close to the RET transmembrane domain, is crucial to the RET tertiary structure and these interactions [1, 2]. The RET tyrosine kinase domain, required for phosphorylation of the activated RET receptor and stimulation of downstream signalling pathways, lies intracellularly [1, 2]. Terminally, RET receptors contain one of three distinct C-terminal sequences, arising through alternative splicing of 3) exons [7, 8], each of which has a distinct contribution to the nature of the downstream targets activated.

RET Ligand/Co-Receptor Complexes Before examining its involvement in MEN 2, it is worth reviewing the normal mechanisms and the molecules involved in RET activation. The RET receptor is unique among mammalian receptor tyrosine kinases in requiring a multicomponent complex to trigger activation and downstream signalling. This complex involves the interaction of two distinct components, one soluble and the other a non-signalling cell surface-bound molecule (fig. 1). RET-mediated signal transduction cannot occur in the absence of either component. Members of the glial cell line-derived neurotrophic factor (GDNF) family make up the soluble ligand component of the RET signalling complex. Four closely related members of the family, GDNF, neurturin, persephin and artemin, have been characterized to date [9–12]. The members of the GDNF family are distantly related members of the TGF-ß superfamily and retain the 7 conserved, similarly spaced cysteine residues that form the cysteine knot structure characteristic of this family [11]. GDNF family members are all neuronal survival factors and this process has been shown to require signalling through RET [13–17]. GDNF proteins do not bind directly to RET, presumably because they do not have the appropriate conformation. They must first interact with a member of the GDNF receptor alpha (GFR·) family, which comprises a novel group of extracellular proteins attached to the cell membrane by a glycosyl-phosphatidylinositol-linkage [18–20] (fig. 1). The role of the GFR· family members in RET activation is as co-receptors or adapter molecules. GDNF and GFR· form a cell surface-tethered complex which then binds to RET and triggers RET dimerization and autophosphorylation [18–20]. As GFR· family members have no transmembrane domain or cytoplasmic region, they do not act as signalling members of the RET/GDNF/GFR· complexes [18–20]. However, recent studies have suggested that RET-independent mechanisms of GFR· signal transduction may also exist [21, 22]. Four members of the GFR· family have been identified to date, GFR·-1, -2, -3 and -4. Each forms specific interactions with a subset of GDNF

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family members: GFR·-1 binds primarily to GDNF [18–20]; GFR·-2 binds neurturin [14–16, 19, 23]; GFR·-3 binds artemin [12], and GFR·-4 binds persephin [17]. Although each of the GDNF and GFR· family members have distinct expression patterns and are implicated in distinct developmental roles, all of the different GDNF/GFR· complexes have been shown to bind to, and activate, RET.

RET Mutations and MEN 2 Mutations of RET are the underlying cause of each of the three MEN 2 subtypes, MEN 2A, MEN 2B and familial MTC (FMTC), which are described in detail in the chapter by Gimm [this vol.]. In the MEN 2A disease subtype, MTC or its precursor lesion, C cell hyperplasia, is detectable in the majority of cases, while PC occurs in approximately 50% and HPT in 15–30% of cases (reviewed in the chapter by Gimm [this vol.]). Germline mutations are found in the extracellular domain of RET in MEN 2A and disrupt the sequence or relative spacing of cysteine codons in the cysteine-rich domain [24–27] (fig. 2). Missense mutations affecting 1 of 5 codons for cysteine in exons 10 and 11 (amino acids 609, 611, 618, 620, 634) have been identified in more than 98% of MEN 2A cases [26, 27]. The most frequent of these mutations (185%) is at codon 634 [26, 27]. In each case, a cysteine residue involved in intramolecular disulphide bonds is replaced by another amino acid. The result is a RET isoform with an unpaired cysteine available for the formation of intermolecular bonds which can contribute to ligandindependent dimerization and activation of the RET receptor [28–31]. Rarely, MEN 2A has been associated with micro-duplications in exon 11, resulting in the insertion of 2 or 3 amino acids, including a cysteine residue, which is again available for the formation of intermolecular bonds that can lead to RET dimerization [32–34]. Mutations of this same group of cysteine residues are also found in patients with FMTC, which is associated with MTC as its only phenotype [25–27, 35] (fig. 2). However, while MEN2A mutations occur most frequently at codon 634, FMTC-RET mutations are more evenly distributed amongst cysteines 618, 620 and 634, with lower frequencies at cysteines 609, 611 and 630 [26, 27, 36]. Consistent with this pattern, a single report of FMTC associated with a 9-base pair duplication in exon 8, including a cysteine codon [37], suggests that the FMTC phenotype may result from more broadly distributed mutations within the cysteine-rich region than does MEN 2A. In addition to these cysteine residues, mutations have been identified in the RET intracellular domain at codons 768 in exon 13 (E768D), 804 in exon 14 (V804L, V804M) and 891 in exon 15 (S891A) in FMTC or ‘small FMTC’ families

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Fig. 2. Diagram of the RET receptor showing the relative positions of common mutations found in each subtype of MEN 2. The RET protein domains correspond to those indicated in figure 1.

[26, 38–43]. The mechanisms of action of these mutations have not been clearly established; however, it is likely that they increase ATP binding or alter the interaction of RET with normal substrates, resulting in altered activation of downstream pathways [29, 39, 44–46]. Interestingly, mutations of codons 790 (L790F) and 791 (Y791F) in exon 13, within the tyrosine kinase domain, have been identified exclusively in a German population of MEN 2A or FMTC families [47]. The functional mechanisms associated with these variants and the nature of this founder effect remain to be determined. The MEN 2B disease subtype is associated with MTC, with PC in 50% of cases, and with other developmental anomalies (see the chapter by Gimm [this vol.]). Either of two missense mutations of the RET tyrosine kinase domain are found in MEN 2B patients. The majority (95%) of cases are caused by a methionine to threonine change of amino acid 918 (M918T) [48–50]. Mutations of

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codon 883 (exon 15), resulting in an alanine to phenylalanine change (A883F), have now been identified in approximately 3% of patients [51, 52]. In each case, these substitutions lie in the catalytic site of the kinase and appear to alter the recognition of peptide substrates by the RET kinase, thus altering the preferred substrate binding to RET [51–53]. Consequently, these RET isoforms are predicted to recognize and phosphorylate the substrates preferred by cytoplasmic tyrosine kinases, rather than those more usual for receptor tyrosine kinases, with the result that RET activates inappropriate downstream signalling pathways [53, 54].

RET Mutation Genotype and Disease Phenotype As can be seen from the above section, there are distinct mutation patterns associated with each MEN 2 subtype, but there is also some overlap of the mutations observed between subtypes, particularly between MEN 2A and FMTC [26, 27]. The pattern of association between the RET mutation genotype and disease phenotype becomes clearer when we consider genotype relative to the occurrence of specific MEN 2 tumour types rather than the broader category of disease subtype. Several large multicentre and single-centre studies, using the family as unit, have shown a strong association between mutations at RET codon 634 and the occurrence of PC in MEN 2 families [26, 27, 55, 56]. Similarly, mutations of this codon are also associated with the occurrence of HPT [26, 27, 55, 56]. The correlation of these tumour types, which are prevalent in MEN 2A (see the chapter by Gimm [this vol.]), with codon 634 mutations is consistent with the observation that 85% of MEN 2A cases have mutations of this codon [26, 27]. Likewise, a large multicentre study has shown a dearth of C634R mutations in families with FMTC, suggesting that this mutation is a strong predictor of risk for both PC and HPT [26]. A correlation of HPT with a specific TGC→CGC (C634R) substitution at codon 634, found in one large study [35], has been more difficult to confirm, likely due to differences in population definitions, the completeness of the screening data available and interpopulation variation in age and follow-up between studies [26, 27, 35, 55, 56]. Consistent with the original observation however, a recent study, which was unable to confirm the association of C634R with HPT on a family-as-unit basis has shown that there is an increased risk of HPT on an individual basis in families with this specific mutation [55]. All of the above observations have suggested that not all RET mutations are equivalent with respect to their transforming potential. Functional analyses of mutant RET proteins have helped to further clarify these predicted variations. These studies have shown that RET isoforms with mutations in the cysteine-rich region are processed and transported to the cell surface with varying degrees of

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efficiency [57–60]. Thus, significantly more protein is expressed on the cell surface for RET isoforms with codon 634 mutations than for isoforms containing codon 609, 618 or 620 mutations [57]. Further, PC and HPT are highly associated with mutations of codon 634, but are rare in families with codon 609 mutations [26, 27]. These data have suggested a model in which the thyroid C cells are the most sensitive to appropriate levels of RET activation, and even low levels of activated RET on the cell surface, as seen in the presence of the codon 609 mutations, can lead to tumorigenesis. At the other end of the spectrum, the parathyroid is the least sensitive to the presence of activated RET, and hyperplasia occurs primarily in the presence of the maximal amounts of mutant RET on the cell surface, as seen with the codon 634 mutations [35, 59]. To date, mutations of codons 768, 804 and 891 have been associated exclusively with FMTC or ‘small FMTC’ families [38, 39, 41–43]. Functional analyses have shown that variants at these sites have low transforming potential as compared to other RET mutations [45, 46]. These data suggest that such mutations carry a very low risk for PC or HPT and result in RET isoforms which are insufficient for transformation in adrenal or parathyroid tissues. Interestingly, recent characterization of a specific valine to leucine substitution at amino acid 804 (V804L) has suggested that patients with this mutation have a particularly late age of disease onset (160 years of age), also consistent with a mutation having reduced penetrance [43, 61]. This finding will need to be assessed in further families to confirm the less aggressive phenotype associated with the mutation. The strongest genotype/phenotype associations seen in MEN 2 are between the MEN 2B phenotype and mutations of codon 918 in exon 16 or codon 883 in exon 15 [26, 27, 51, 52]. These germline mutations are exclusively associated with the MEN 2B phenotype, and vice versa [26, 27]. To date, there have been no reported differences in the phenotypes of patients with the different MEN 2B mutations.

RET Mutations in Sporadic Tumours As described in the chapter by Gimm [this vol.], the majority of MTCs and PCs occur as sporadic tumours outside the context of MEN 2. A small percentage of these (3–7%) have been shown to represent new MEN 2 cases harbouring de novo germline RET mutations [55, 62, 63]. However, germline RET mutations are absent in the majority of cases. Somatically occurring mutations of RET have been identified in sporadic MTC and PC. Studies have shown that 23–70% of sporadic MTCs have somatic RET mutations, the vast majority of which are M918T mutations, identical to those common in MEN 2B [25, 36, 44, 49, 50, 64–70]. Other mutations are much less frequent and include A883F, E768D,

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exon 10 or 11 cysteine substitutions, mutations of exon 15 and deletions of one or several amino acids in the RET extracellular domain [36, 65, 68–70]. Analyses of subpopulations of cells within MTC tumours, or in multiple metastases from a single patient, have shown that somatic RET mutations affect only a subset of clones, suggesting that these mutations may be later events in tumorigenesis, reflecting progressional rather than initiating events [71, 72]. Interestingly, recent studies have shown that a rare C→T variant at base pair 2439 of RET (c.2439C→T; S836S) is over-represented in sporadic MTC tumours with codon 918 mutations [73]. Further studies are required to determine whether this variant may represent a predisposing susceptibility allele for ‘sporadic’ MTC. Somatic RET mutations are less frequent in sporadic PC, being found in 10–15% of cases. Again, the most frequent mutation is M918T, although in this instance, it makes up only about one half of detected mutations [44, 74–76]. In addition, missense mutations of exon 10 and 11 cysteines and deletions in the cysteine-rich domain have been seen in smaller numbers of PCs [44, 75]. It seems apparent that, in keeping with the predictions on tissue-specific thresholds for RET activity described above, RET activation is not as transforming in the adrenal gland as in the thyroid, and thus, does not contribute to as large a proportion of these tumours. As RET is normally activated primarily in neural crest-derived cell types, it has been proposed that RET activation might also contribute to other neuroendocrine tumour types. RET mutations have not been detected in sporadic parathyroid tumours despite its involvement in MEN 2 HPT [65, 74, 77, 78]. To date, mutations in RET have not been identified in small cell lung carcinoma, neuroblastoma, malignant melanoma, schwannoma and other endocrine tumours [74, 79, 80], suggesting that RET may not play a major role in tumorigenesis outside of the MEN 2-type tumours.

RET Downstream Signalling Activation of RET, either through normal ligand-co-receptor binding or MEN 2 mutations, results in autophosphorylation, which in turn stimulates a cascade of intracellular protein interactions. RET can trigger growth, differentiation or survival responses through different downstream signalling pathways, depending on the cell type or developmental stage at which it is activated [81, 82]. RET has 16 intracellular tyrosine residues with two additional tyrosines that are found in only one of the RET C-terminal isoforms (Y1090, Y1096) [1, 8, 83]. To date, phosphorylation of 9 of the 18 intracellular tyrosines of RET has been shown to result from RET activation, suggesting an array of possible downstream interactions [83]. A number of adapter proteins containing SH2 domains have been

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Fig. 3. Summary of the downstream signalling pathways stimulated by activation of the RET kinase. Proteins known to interact with specific RET tyrosines are shown. Predicted or demonstrated downstream interactions with these adapter proteins are indicated. The specific interactions and the phenotypic effects they may mediate are discussed more fully in the text.

shown to interact with these phosphorylated residues, including GRB7 and -10, which bind tyrosine 905 (Y905), phospholipase C-Á (PLCÁ) (Y1015), SHC (Y1062), GRB2 (Y1096), src, Crk and Nck [84–94]. RET activates a number of downstream signalling pathways through these various associated adapter proteins (fig. 3).

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One of the most important residues in RET activation is the multifunctional docking site at Y1062 [94]. This amino acid acts as a phosphorylation-dependent binding site for the SHC adapter protein, which subsequently acts, through a series of other associated molecules, to activate several different downstream signalling pathways [87, 94]. Interestingly, Y1062 also interacts in a phosphorylation-independent manner with enigma, a cytoplasmic, membrane-anchored, LIM domain protein which may play a role in positioning the RET receptor at the cell surface [92, 95]. Both SHC and enigma have roles in mitogenesis and transformation associated with RET activation [92, 95]. RET has been shown to stimulate RAS-mediated activation of MAP kinase, which is required for neuronal survival and differentiation [81, 96–101]. The RAS-MAP kinase cascade is activated through SHC and GRB2 (Y1062), or directly through GRB2 binding (Y1096) to RET. RET-mediated activation of phosphatidylinositol 3 (PI3)-kinase likely occurs indirectly, through SHC, GRB2 and GAB1 (Y1062), which can recruit p85, the PI3-kinase regulatory subunit [54,100,102]. Through PI3-kinase, RET activation influences cell motility, leading to lamellipodia formation or membrane ruffling, as well as phosphorylation of proteins linked to the formation of focal adhesions, including paxillin, focal adhesion kinase and the Crk-associated substrate p130cas, and to increased expression of the gap junction protein connexin 43 [81, 82, 90, 102–105]. Similarly, RET acts through PI3-kinase to activate protein kinase B/AKT signalling, which is important for cell survival and proliferation and has been shown to be crucial to the oncogenic potential of RET [54, 102, 106]. RET has also been shown to activate the c-jun N-terminal kinase (JNK) pathway through a cdc42/RAC1 small GTPase [107, 108]. This pathway has been demonstrated to play a role in establishing the neoplastic phenotype [107, 108]. Although the interactions linking this pathway with RET have not yet been clarified, JNKs have been shown to lie downstream of PI3-kinase in other systems [109, 110]. PLCÁ interaction with Y1015 is required for mitogenesis and transformation associated with RET activation [86, 100]. While the mechanisms involved have not yet been elucidated, this may occur by the triggering of changes in the levels of intracellular free Ca2+ [111]. Interestingly, different tyrosine residues appear to be significant for transformation induced by different MEN 2-RET mutations. Although MEN 2B-RET mutations do not alter a tyrosine residue or the crucial sequences surrounding a tyrosine, they alter the substrate specificity of the RET kinase, perturbing its normal interactions [53]. Thus, tyrosines 864 and 952 are phosphorylated and crucial to transformation associated with the MEN 2B mutation of codon 918, while Y905 is essential to transformation in the context of MEN 2A mutations [83, 112]. Further, it appears that pathways commonly stimulated by all RET forms

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may be activated to varying extents in the presence of different RET mutations. For example, higher levels of signalling through PI3-kinase may be associated with RET containing MEN 2B-type mutations [54, 90, 108]. However, decreased phosphorylation of Y1096 in these same mutants suggests lesser interaction with GRB2 [83]. It is interesting to note that tyrosine 1062, which has been shown to bind SHC and enigma and is thought to stimulate the PI3-kinase pathway, is required for transformation in all mutation contexts [88, 94, 98, 113]. Splicing variants of RET 3) exons may also impact on the activation of RET downstream signalling pathways. Alternative splicing downstream of RET exon 19 results in three protein isoforms with 9 (RET 9), 43 (RET 43) or 51 (RET 51) distinct C-terminal amino acids [1, 7, 8]. As the last amino acid common to all isoforms is Y1062, this residue lies in three different amino acid contexts in the three isoforms. As a result, adapter interactions with RET, and thus downstream signalling, may differ. For example, in the context of RET 9, Y1062 provides a docking site for the SH2 domain of SHC, while in the RET 51 isoform, the preferred binding is with the SHC PTB domain [88, 114, 115]. Consistent with these differences, the three RET isoforms have been shown to have distinct transforming potentials and to stimulate the differentiation of neural cell types to different extents in vitro [29, 114, 115]. These variants may also have distinct developmental expression patterns in vivo, suggesting differential contributions to normal RET-mediated developmental processes [116].

RET-Associated Diseases It is impossible to discuss the role of RET in human disease without touching on other phenotypes associated with mutations of this gene which have also contributed to our understanding of the normal functions of RET. Hirschsprung Disease In contrast to the activating mutations found in MEN 2, inactivating RET mutations are found in patients with Hirschsprung disease (HSCR) [117–120]. HSCR is a congenital abnormality characterized by the absence or depletion of enteric ganglia of the hindgut [121]. In normal development, neural crest-derived cells migrate out of the vagal neural crest and along defined pathways to reach the developing intestinal tract [122]. These cells migrate into and populate the gut wall, where they proliferate and eventually differentiate to form the enteric nerve plexus [5, 6, 122, 123]. RET is thought to act as a survival factor for these immature neural crest cells and to act in targeting their migration [5, 6]. In the absence of RET, these cells are unable to populate the gut and the enteric nerves and ganglia do not form [5, 6].

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The genetics of HSCR is very complex and has only recently begun to be dissected. The first breakthrough in this area came with the discovery of mutations in RET, including deletions, insertions and point mutations, in familial and sporadic HSCR patients [for reviews, see ref. 124, 125]. These changes have been demonstrated to reduce the functional RET protein on the cell surface [57, 60, 126, 127]. Thus, it appears that a minimal threshold amount of RET is critical for the normal development of the enteric nervous system. HSCR patients with RET mutations have approximately 50% reduction in functional RET protein and, therefore, have insufficient RET protein for enteric neurogenesis to proceed appropriately. The observation that MEN 2A and HSCR can co-segregate has further clarified just how sensitive normal development of the enteric ganglia may be. In a few families, MEN 2A and HSCR can be associated with the same mutation, generally a cysteine to arginine change in exon 10 [119, 128, 129]. As described above, these variant RET forms are expressed and functional, but are not efficiently transported to the cell surface, although once there they do show ligand-independent activation [57–60]. Together, these data suggest that HSCR can occur in individuals with 60–70% of the normal levels of functional RET protein, and that even this partial reduction in RET protein is sufficient to prevent normal neurogenesis. Although initial studies suggested that RET was the major contributing gene in HSCR, further data have indicated that RET mutations account for less than 40% of familial disease and only 3–7% of sporadic HSCR [130, 131]. Recent studies, however, have demonstrated an association of the HSCR phenotype with specific polymorphic alleles of RET codons 45 (exon 2), 769 (exon 13) and 904 (exon 15) in sporadic HSCR [132, 133], suggesting the involvement of low-penetrance RET variants in the expression of the HSCR phenotype, even in the absence of detectable RET mutations. The current favoured model suggests that HSCR is a multigenic disease, with multiple loci contributing to the disease phenotype in each family or individual [130, 134, 135]. Consistent with this model, mutations of the RET ligands GDNF and neurturin have also been found in HSCR patients, frequently associated with RET mutations [136–139]. Mutations of the endothelin B receptor have also been found to co-occur with HSCR-RET mutations in HSCR families [140, 141]. In addition to the above genes, other loci on chromosomes 9q31 [134] and 22q11 [135] are strongly implicated in HSCR. We await the identification and characterization of the relevant genes in these regions to determine whether these also act as modifiers of HSCR-RET mutations and phenotype. Papillary Thyroid Carcinoma Somatic RET mutations have also been identified in another form of thyroid cancer, sporadic papillary thyroid carcinoma (PTC), which arises from the thy-

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roid follicular cells. Unlike MEN 2-RET mutations, these variants result from chromosomal translocations or inversions involving the RET locus and one of several other loci. In each case, the 3) sequences of the RET gene, spanning the tyrosine kinase domain, become juxtaposed with 5) sequences from any of several other loci (H4, R1·, ELE1, RFG5, HTIF1), all of which appear to function in dimerization of the chimeric fusion protein [for reviews, see ref. 142, 143]. As a result, RET signalling pathways are activated in a cell type that does not normally express RET and tumorigenesis occurs [113, 144, 145]. RET/PTC rearrangements are detected in 25–40% of PTC cases; however, the incidence is much higher where radiation exposure has occurred, such as in the Chernobyl region and in the area of the Semipalatinsk nuclear testing site [146–150]. Rearrangements involving RET and ELE1 (RET/PTC3) are the most prevalent in radiation-induced PTC in children [146–148], although these variants have low frequency in sporadic PTC. Interestingly, RET/H4 rearrangements (RET/PTC1) may be more common in adult PTC after radiation exposure [151]. Despite its role in sporadic PTC, analyses of familial forms of PTC have not identified linkage or germline mutations of the RET gene [152–154]. Other Pathologies and Beyond The RET gene has not been directly implicated in other human pathologies. However, its role as a receptor for neurotrophic factors has made it a target of interest in therapies for diseases of nerve degeneration and nerve injury [9–12]. The GDNF family members have been shown to be potent survival factors for dopaminergic neurons and have been investigated as therapeutic factors for Parkinson’s disease [155–157]. Similarly, activation of the RET signalling complex has been considered as a potential mechanism for stimulating nerve regeneration after physical injury [158, and references therein]. Recent work has even suggested that RET activation may be a viable target for therapy in male infertility [159]. While these applications are a long way from realization in any clinical setting, it is clear that the information gleaned from characterizing RET and its interactions and functions in the model provided by MEN 2 may have even broader implications in the future.

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87 Borrello MG, Pelicci G, Arighi E, DeFilippis L, Greco A, Bongarzone I, Rizzetti MG, Pelicci PG, Pierotti MA: The oncogenic versions of the ret and trk tyrosine kinases bind Shc and Grb2 adaptor proteins. Oncogene 1994;9:1661–1668. 88 Arighi E, Alberti L, Torriti F, Ghizzoni S, Rizzetti MG, Pelicci G, Pasini B, Bongarzone I, Piutti C, Pierotti MA, Borrello MG: Identification of Shc docking site on Ret tyrosine kinase. Oncogene 1997;14:773–782. 89 Alberti L, Borrello MG, Ghizzoni S, Torriti F, Rizzetti MG, Pierotti MA: Grb2 binding to the different isoforms of Ret tyrosine kinase. Oncogene 1998;17:1079–1087. 90 Bocciardi R, Mograbi B, Pasini B, Borrello MG, Pierotti MA, Bourget I, Fischer S, Romeo G, Rossi B: The multiple endocrine neoplasia type 2B mutation switches the specificity of the Ret tyrosine kinase towards cellular substrates that are susceptible to interact with Crk and Nck. Oncogene 1997;15:2257–2265. 91 Lorenzo MJ, Gish GD, Houghton C, Stonehouse TJ, Pawson T, Ponder BAJ, Smith DP: RET alternative splicing influences the interaction of activated RET with the SH2 and PTB domains of Shc, and the SH2 domain of Grb2. Oncogene 1997;14:763–771. 92 Durick K, Wu RY, Gill GN, Taylor SS: Mitogenic signaling by Ret/ptc2 requires association with enigma via a LIM domain. J Biol Chem 1996;271:12691–12694. 93 Melillo RM, Barone MV, Lupoli G, Cirafici AM, Carlomagno F, Visconti R, Matoskova B, Di Fiore PP, Vecchio G, Fusco A, Santoro M: Ret-mediated mitogenesis requires Src kinase activity. Cancer Res 1999;59:1120–1126. 94 Asai N, Murakami H, Iwashita T, Takahashi M: A mutation at tyrosine 1062 in MEN2A-Ret and MEN2B-Ret impairs their transforming activity and association with shc adaptor proteins. J Biol Chem 1996;271:17644–17649. 95 Durick K, Gill GN, Taylor SS: Shc and Enigma are both required for mitogenic signaling by Ret/ ptc2. Mol Cell Biol 1998;18:2298–2308. 96 Califano D, Monaco C, De Vita G, D’Alessio A, Dathan NA, Possenti R, Vecchio G, Fusco A, Santoro M, de Franciscis V: Activated RET/PTC oncogene elicits immediate early and delayed response genes in PC12 cells. Oncogene 1995;11:107–112. 97 van Puijenbroek AA, van Weering DH, van den Brink CE, Bos JL, van der Saag PT, de Laat SW, den Hertog J: Cell scattering of SK-N-MC neuroepithelioma cells in response to Ret and FGF receptor tyrosine kinase activation is correlated with sustained ERK2 activation. Oncogene 1997;14:1147–1157. 98 Ohiwa M, Murakami H, Iwashita T, Asai N, Iwata Y, Imai T, Funahashi H, Takagi H, Takahashi M: Characterization of Ret-Shc-Grb2 complex induced by GDNF, MEN 2A, and MEN 2B mutations. Biochem Biophys Res Commun 1997;237:747–751. 99 Xing S, Furminger TL, Tong Q, Jhiang SM: Signal transduction pathways activated by RET oncoproteins in PC12 pheochromocytoma cells. J Biol Chem 1998;273:4909–4914. 100 Santoro M, Wong WT, Aroca P, Santos E, Matoskova B, Grieco M, Fusco A, di Fiore PP: An epidermal growth factor receptor/ret chimera generates mitogenic and transforming signals: Evidence for a ret-specific signaling pathway. Mol Cell Biol 1994;14:663–675. 101 van Weering DH, Medema JP, van Puijenbroek A, Burgering BM, Baas PD, Bos JL: Ret receptor tyrosine kinase activates extracellular signal-regulated kinase 2 in SK-N-MC cells. Oncogene 1995;11:2207–2214. 102 Segouffin-Cariou C, Billaud M: Transforming ability of MEN2A-RET requires activation of the phosphatidylinositol 3-kinase/AKT signaling pathway. J Biol Chem 2000;275:3568–3576. 103 Murakami H, Iwashita T, Asai N, Iwata Y, Narumiya S, Takahashi M: Rho-dependent and -independent tyrosine phosphorylation of focal adhesion kinase, paxillin and p130Cas mediated by Ret kinase. Oncogene 1999;18:1975–1982. 104 Romano A, Wong WT, Santoro M, Wirth PJ, Thorgeirsson SS, DiFiore PP: The high transforming potency of erbB-2 and ret is associated with phosphorylation of paxillin and a 23 kDa protein. Oncogene 1994;9:2923–2933. 105 van Weering DH, de Rooij J, Marte B, Downward J, Bos JL, Burgering BM: Protein kinase B activation and lamellipodium formation are independent phosphoinositide 3-kinase-mediated events differentially regulated by endogenous Ras. Mol Cell Biol 1998;18:1802–1811.

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106 Soler RM, Dolcet X, Encinas M, Egea J, Bayascas JR, Comella JX: Receptors of the glial cell linederived neurotrophic factor family of neurotrophic factors signal cell survival through the phosphatidylinositol 3-kinase pathway in spinal cord motoneurons. J Neurosci 1999;19:9160–9169. 107 Chiariello M, Visconti R, Carlomagno F, Melillo RM, Bucci C, de Franciscis V, Fox GM, Jing S, Coso OA, Gutkind JS, Fusco A, Santoro M: Signalling of the Ret receptor tyrosine kinase through the c-Jun NH2-terminal protein kinases (JNKS): Evidence for a divergence of the ERKs and JNKs pathways induced by Ret. Oncogene 1998;16:2435–2445. 108 Marshall GM, Peaston AE, Hocker JE, Smith SA, Hansford LM, Tobias V, Norris MD, Haber M, Smith DP, Lorenzo MJ, Ponder BAJ, Hancock JF: Expression of multiple endocrine neoplasia 2B RET in neuroblastoma cells alters cell adhesion in vitro, enhances metastatic behavior in vivo and activates Jun kinase. Cancer Res 1997;57:5399–5405. 109 Logan SK, Falasca M, Hu P, Schlessinger J: Phosphatidylinositol 3-kinase mediates epidermal growth factor-induced activation of the c-Jun N-terminal kinase signaling pathway. Mol Cell Biol 1997;17:5784–5790. 110 Rodrigues GA, Park M, Schlessinger J: Activation of the JNK pathway is essential for transformation by the Met oncogene. EMBO J 1997;16:2634–2645. 111 Airaksinen MS, Titievsky A, Saarma M: GDNF family neurotrophic factor signaling: Four masters, one servant? Mol Cell Neurosci 1999;13:313–325. 112 Iwashita T, Asai N, Murakami H, Matsuyama M, Takahashi M: Identification of tyrosine residues that are essential for transforming activity of the ret proto-oncogene with MEN2A or MEN2B mutation. Oncogene 1996;12:481–487. 113 Durick K, Yao VJ, Borrello MG, Bongarzone I, Pierotti MA, Taylor SS: Tyrosines outside the kinase core and dimerization domain are required for the mitogenic activity of RET/ptc2. J Biol Chem 1995;270:24642–24645. 114 Lorenzo MJ, Eng C, Mulligan LM, Stonehouse TJ, Healey CS, Ponder BAJ, Smith DP: Multiple mRNA isoforms of the human RET proto-oncogene generated by alternative splicing. Oncogene 1995;10:1377–1383. 115 Ishiguro Y, Iwashita T, Murakami H, Asai N, Iida K, Goto H, Hayakawa T, Takahashi M: The role of amino acids surrounding tyrosine 1062 in ret in specific binding of the shc phosphotyrosinebinding domain. Endocrinology 1999;140:3992–3998. 116 Ivanchuk SM, Myers SM, Mulligan LM: Expression of RET 3) splicing variants during human kidney development. Oncogene 1998;16:991–996. 117 Angrist M, Bolk S, Thiel B, Puffenberger EG, Hofstra RM, Buys CHCM, Cass DT, Chakravarti A: Mutation analysis of the RET receptor tyrosine kinase in Hirschsprung disease. Hum Mol Genet 1995;4:821–830. 118 Romeo G, Ronchetto P, Luo Y, Barone V, Seri M, Ceccherini I, Pasini B, Bocciardi R, Lerone M, Kääriäinen H, Martucciello G: Point mutations affecting the tyrosine kinase domain of the RET proto-oncogene in Hirschsprung’s disease. Nature 1994;367:377–378. 119 Attié T, Pelet A, Edery P, Eng C, Mulligan LM, Amiel J, Boutran L, Beldjord C, Nihoul-Fékété C, Munnich A, Ponder BAJ, Lyonnet S: Diversity of RET proto-oncogene mutations in familial and sporadic Hirschsprung disease. Hum Mol Genet 1995;4:1381–1386. 120 Edery P, Lyonnet S, Mulligan LM, Pelet A, Dow E, Abel L, Holder S, Nihoul-Fékété C, Ponder BAJ, Munnich A: Mutations of the RET proto-oncogene in Hirschsprung’s disease. Nature 1994;367: 378–380. 121 Passarge E: The genetics of Hirschsprung’s disease. Evidence for heterogeneous etiology and a study of sixty-three families. N Engl J Med 1967;276:138–143. 122 Le Douarin NM, Teillet MA: The migration of neural crest cells to the wall of the digestive tract in avian embryo. J Embryol Exp Morphol 1973;30:31–48. 123 Natarajan D, Grigoriou M, Marcos-Gutierrez CV, Atkins C, Pachnis V: Multipotential progenitors of the mammalian enteric nervous system capable of colonising aganglionic bowel in organ culture. Development 1999;126:157–168. 124 Chakravarti A: Endothelin receptor-mediated signaling in Hirschsprung disease. Hum Mol Genet 1996;5:303–307.

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125 Eng C, Mulligan LM: Mutations of the RET proto-oncogene in the multiple endocrine neoplasia type 2 syndromes, related sporadic tumours and Hirschsprung disease. Hum Mutat 1997;9:97– 109. 126 Carlomagno F, De Vita G, Berlingieri MT, de Franciscis V, Melillo RM, Colantuoni V, Kraus MH, Di Fiore PP, Fusco A, Santoro M: Molecular heterogeneity of RET loss of function in Hirschsprung’s disease. EMBO J 1996;15:2717–2725. 127 Iwashita T, Murakami H, Asai N, Takahashi M: Mechanisms of Ret dysfunction by Hirschsprung mutations affecting its extracellular domain. Hum Mol Genet 1996;5:1577–1580. 128 Mulligan LM, Eng C, Attié T, Lyonnet S, Marsh DJ, Hyland VJ, Robinson BG, Frilling A, VerellenDumoulin C, Safar A, Venter DJ, Munnich A, Ponder BAJ: Diverse phenotypes associated with exon 10 mutations of the RET proto-oncogene. Hum Mol Genet 1994;3:2163–2167. 129 Borst MJ, VanCamp JM, Peacock ML, Decker RA: Mutational analysis of multiple endocrine neoplasia type 2A associated with Hirschsprung’s disease. Surgery 1995;117:386–391. 130 Sancandi M, Ceccherini I, Costa M, Fava M, Chen B, Wu Y, Hofstra R, Laurie T, Griffths M, Burge D, Tam PK: Incidence of RET mutations in patients with Hirschsprung’s disease. J Pediatr Surg 2000;35:139–142. 131 Svensson PJ, Molander ML, Eng C, Anvret M, Nordenskjold A: Low frequency of RET mutations in Hirschsprung disease in Sweden. Clin Genet 1998;54:39–44. 132 Borrego S, Saez ME, Ruiz A, Gimm O, Lopez-Alonso M, Antinolo G, Eng C: Specific polymorphisms in the RET proto-oncogene are over-represented in patients with Hirschsprung disease and may represent loci modifying phenotypic expression. J Med Genet 1999;36:771–774. 133 Fitze G, Schreiber M, Kuhlisch E, Schackert HK, Roesner D: Association of RET protooncogene codon 45 polymorphism with Hirschsprung disease. Am J Hum Genet 1999;65:1469–1473. 134 Bolk S, Pelet A, Hofstra RM, Angrist M, Salomon R, Croaker D, Buys CH, Lyonnet S, Chakravarti A: A human model for multigenic inheritance: Phenotypic expression in Hirschsprung disease requires both the RET gene and a new 9q31 locus. Proc Natl Acad Sci USA 2000;97:268–273. 135 Kerstjens-Frederikse WS, Hofstra RM, van Essen AJ, Meijers JH, Buys CH: A Hirschsprung disease locus at 22q11? J Med Genet 1999;36:221–224. 136 Angrist M, Bolk S, Halushka M, Lapchak PA, Chakravarti A: Germline mutations in glial cell line-derived neurotrophic factor (GDNF) and RET in a Hirschsprung disease patient. Nat Genet 1996;14:341–343. 137 Doray B, Salomon R, Amiel J, Pelet A, Touraine R, Billaud M, Attié T, Bachy B, Munnich A, Lyonnet S: Mutation of the RET ligand, neurturin, supports multigenic inheritance in Hirschsprung disease. Hum Mol Genet 1998;7:1449–1452. 138 Ivanchuk SM, Myers SM, Eng C, Mulligan LM: De novo mutation of GDNF, ligand of the RET/ GDNFR-· receptor complex in Hirschsprung disease. Hum Mol Genet 1996;5:2023–2026. 139 Salomon R, Attié T, Pelet A, Bidaud C, Eng C, Amiel J, Sarnacki S, Goulet O, Ricour C, NihoulFékété C, Munnich A, Lyonnet S: Germline mutations of the RET ligand, GDNF, are not sufficient to cause Hirschsprung disease. Nat Genet 1996;14:345–347. 140 Auricchio A, Griseri P, Carpentieri ML, Betsos N, Staiano A, Tozzi A, Priolo M, Thompson H, Bocciardi R, Romeo G, Ballabio A, Ceccherini I: Double heterozygosity for a RET substitution interfering with splicing and an EDNRB missense mutation in Hirschsprung disease. Am J Hum Genet 1999;64:1216–1221. 141 Svensson PJ, Anvret M, Molander ML, Nordenskjold A: Phenotypic variation in a family with mutations in two Hirschsprung-related genes (RET and endothelin receptor B). Hum Genet 1998;103:145–148. 142 Jhiang S, Mazzaferri E: The ret/PTC oncogene in papillary thyroid carcinoma. J Lab Clin Med 1994;123:331–337. 143 Klugbauer S, Rabes HM: The transcription coactivator HTIF1 and a related protein are fused to the RET receptor tyrosine kinase in childhood papillary thyroid carcinomas. Oncogene 1999;18:4388– 4393. 144 Klugbauer S, Demidchik EP, Lengfelder E, Rabes HM: Molecular analysis of new subtypes of ELE/ RET rearrangements, their reciprocal transcripts and breakpoints in papillary thyroid carcinomas of children after Chernobyl. Oncogene 1998;16:671–675.

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145 Tong Q, Xing S, Jhiang SM: Leucine zipper-mediated dimerization is essential for the PTC1 oncogenic activity. J Biol Chem 1997;272:9043–9047. 146 Fugazzola L, Pierotti MA, Vigano E, Pacini F, Vorontsova TV, Bongarzone I: Molecular and biochemical analysis of RET/PTC4, a novel oncogenic rearrangement between RET and ELE1 genes, in a post-Chernobyl papillary thyroid cancer. Oncogene 1996;13:1093–1097. 147 Klugbauer S, Lengfelder E, Demidchik EP, Rabes HM: High prevalence of RET rearrangement in thyroid tumors of children from Belarus after the Chernobyl reactor accident. Oncogene 1995;11:2459–2467. 148 Nikiforov YE, Rowland JM, Bove KE, Monforte-Munoz H, Fagin JA: Distinct pattern of ret oncogene rearrangements in morphological variants of radiation-induced and sporadic thyroid papillary carcinomas in children. Cancer Res 1997;57:1690–1694. 149 Alipov G, Ito M, Prouglo Y, Takamura N, Yamashita S: Ret proto-oncogene rearrangement in thyroid cancer around Semipalatinsk nuclear testing site. Lancet 1999;354:1528–1529. 150 Nikiforov YE, Koshoffer A, Nikiforova M, Stringer J, Fagin JA: Chromosomal breakpoint positions suggest a direct role for radiation in inducing illegitimate recombination between the ELE1 and RET genes in radiation-induced thyroid carcinomas. Oncogene 1999;18:6330–6334. 151 Smida J, Salassidis K, Hieber L, Zitzelsberger H, Kellerer AM, Demidchik EP, Negele T, Spelsberg F, Lengfelder E, Werner M, Bauchinger M: Distinct frequency of ret rearrangements in papillary thyroid carcinomas of children and adults from Belarus. Int J Cancer 1999;80:32–38. 152 Canzian F, Amati P, Harach HR, Kraimps JL, Lesueur F, Barbier J, Levillain P, Romeo G, Bonneau D: A gene predisposing to familial thyroid tumors with cell oxyphilia maps to chromosome 19p13.2. Am J Hum Genet 1998;63:1743–1748. 153 Decker RA: Expression of papillary thyroid carcinoma in multiple endocrine neoplasia type 2A. Surgery 1993;114:1059–1063. 154 Kogon MD, Green JS, Kwan A, Morris-Larkin C, Kaiser S, Myers SM, Mulligan LM: Genetic analyses of a family with familial medullary and papillary thyroid carcinoma. J Endocr Genet 1999;1:27–32. 155 Gash DM, Zhang Z, Ovadia A, Cass WA, Yi A, Simmerman L, Russell D, Martin D, Lapchak PA, Collins F, Hoffer BJ, Gerhardt GA: Functional recovery in parkinsonian monkeys treated with GDNF. Nature 1996;380:252–255. 156 Bilang-Bleuel A, Revah F, Colin P, Locquet I, Robert J-J, Mallet J, Horellou P: Intrastriatal injection of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminergic neuron degeneration and behavioral impairment in a rat model of Parkinson disease. Proc Natl Acad Sci USA 1997;94:8818–8823. 157 Walker DG, Beach TG, Xu R, Lile J, Beck KD, McGeer EG, McGeer PL: Expression of the protooncogene Ret, a component of the GDNF receptor complex, persists in human substantia nigra neurons in Parkinson’s disease. Brain Res 1998;792:207–217. 158 Olson L: Regeneration in the adult central nervous system: Experimental repair strategies. Nat Med 1997;3:1329–1335. 159 Meng X, Lindahl M, Hyvonen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M, Pichel JG, Westphal H, Saarma M, Sariola H: Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 2000;287:1489–1493.

Dr. Lois M. Mulligan, Department of Paediatrics, Queen’s University 20 Barrie Street, Kingston, Ontario K7L 3N6 (Canada) Tel. +1 613 533 6310, Fax +1 613 548 1348, E-Mail [email protected]

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Dahia PLM, Eng C (eds): Genetic Disorders of Endocrine Neoplasia. Front Horm Res. Basel, Karger, 2001, vol 28, pp 103–130

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Multiple Endocrine Neoplasia Type 2: Clinical Aspects Oliver Gimm Department of General Surgery, Martin-Luther-University, Halle-Wittenberg, Germany; Human Cancer Genetics Program, The Ohio State University, Columbus, Ohio, USA

Contents 104 104 107 108 108 108 110 111 113 113 113 115 115 115 116 117 118 119 119 120 120 121 121 122 122 123 123 123 124 124

Introduction Molecular Etiology of MEN 2 and Genotype-Phenotype Correlation Medullary Thyroid Carcinoma Diagnosis of MTC Patients with Apparently Sporadic MTC The Index Patient Further Diagnostic/Imaging Techniques Screening Treatment of MTC Primary Tumor and Locoregional Lymph Nodes Prophylactic Thyroidectomy MTC in MEN 2B Distant Metastases Nonsurgical Treatment Modalities Follow-Up of MTC Pheochromocytoma Diagnosis Treatment Nonsurgical Treatment Modalities Follow-Up Patients with Apparently Sporadic Pheochromocytoma MEN 2A-Specific Features Diagnosis of Hyperparathyroidism Treatment of Hyperparathyroidism Cutaneous Lichen Amyloidosis MEN 2B-Specific Disorders Specific Issues Patients with Suspected MEN 2 but No RET Mutation Future Aspects References

Introduction In 1968, Steiner et al. [1] suggested that the entity characterized by the occurrence of pheochromocytoma, medullary thyroid carcinoma (MTC) and parathyroid tumors be designated ‘multiple endocrine neoplasia, type 2’. Its abbreviation, MEN 2, is nowadays widely used. MEN has also been named multiple endocrine adenomatosis [2], but this term is rarely used today, since the consensus nomenclature for MEN has been accepted since the Second International Workshop on Multiple Endocrine Neoplasia Type 2 Syndromes, Cambridge, UK, 1987 [3]. The familial occurrence of pheochromocytoma was already noticed in the early 20th century. However, the significant proportion of patients with both MTC and pheochromocytoma was not reported until the mid-1960s [4–7]. This is partly due to the fact that MTC was not identified as an entity until 1959 [8], even though some characteristic features of MTC were described in earlier reports [9, 10]. Based on clinical findings, three subtypes of familial disease have been classified: MEN 2A, MEN 2B and familial MTC (FMTC) (table 1). In 1987, the MEN 2 locus was linked to chromosome 10 [11, 12]. In 1993, germline mutations of the proto-oncogene RET, which is located on chromosome subband 10q11.2, were found in patients with FMTC and MEN 2A [13, 14] (see the chapter by Mulligan [this vol.]). Subsequently, in 1994, germline mutations in a different codon of RET were also identified in MEN 2B [15–17]. To date, germline RET mutations have been found in more than 95% of patients with MEN 2. Prior to the identification of the susceptibility gene for MEN 2, patients at risk had to undergo repeated, unpleasant screening procedures with intravenous injection of calcitonin-provocative agents in order to be identified as being at risk for MEN 2, with the attendant false-positive and false-negative results [18–22]. The identification of RET as the disease-causing gene offers clinicians a powerful tool, and MEN 2 became the paradigm of molecular medicine. However, many genetic, biochemical, diagnostic, therapeutic and psychological issues are still unresolved.

Molecular Etiology of MEN 2 and Genotype-Phenotype Correlation Given that RET has been identified as the susceptibility gene for MEN 2, the knowledge of any existing genotype-phenotype correlation would be of great value. If a specific germline RET mutation were clearly associated with a particular phenotype, e.g. MEN 2B with its seemingly very aggressive form of MTC, patients identified as having this mutation might benefit from an earlier diagnosis. From the clinical but also the economic point of view, it would be important to know whether specific mutations never lead to a particular phenotype, e.g.

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Table 1. Phenotypes of FMTC, MEN 2A and MEN 2B

Age at diagnosis1 (mean), years Male:female ratio MTC Pheochromocytoma3 pHPT3 Ganglioneuromatosis Multiple neuromas Marfanoid habitus Thickened corneal fibers

FMTC

MEN 2A

MEN 2B

45–55 1:1 100%2 – – – – – –

25–35 1:1 100%2 50% 10–30% – – – –

10–20 1:1.5 100%2 50% – + + + +

– = Disease/finding absent or frequency observed not higher than in the general population; + = disease/finding present in most cases but neither required nor pathognomonic. 1 The age of diagnosis has become younger since the identification of RET. 2 Since the identification of RET, many patients undergo surgery before MTC occurs. 3 If patients present with pheochromocytoma and/or hyperparathyroidism they have MEN 2A.

pheochromocytoma. In these instances, burdensome and expensive follow-up procedures could be abandoned. Much effort has been invested to answer these questions. For instance, studies of the International RET Mutation Consortium have shown that any given mutation in codon 634 is significantly associated with pheochromocytoma and hyperparathyroidism [23, 24]. While 85% of MEN 2A patients have a codon 634 mutation, only about 30% of FMTC patients have a mutation affecting codon 634. In general, it appears that FMTC RET mutations are more evenly distributed among the cysteine codons. A simplified genotypephenotype association is shown in table 2. Obviously, there is no 100% correlation in many instances and a broad overlap between MEN 2A and FMTC is observed. Large families with ‘FMTC only’ have been reported. Interestingly, affected members of these families often harbor germline RET mutations which are also found in MEN 2A patients. Hence, other genetic or epigenetic factors must play an important role in the pathogenesis of accompanying diseases, e.g. pheochromocytomas. Since these factors remain to be identified, patients harboring one of these mutations cannot be excluded from follow-up screening procedures with regard to pheochromocytoma and hyperparathyroidism. In contrast, some mutations, e.g. E768D, V804M, Y791F and A891S, have so far only been seen in patients with FMTC only (FMTC-specific RET mutation). Very recently, 2 patients with pheochromocytoma were identified in a family with the V804L mutation, which

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Table 2. Correlation between germline RET mutation (genotype) and phenotype Phenotype Exon 10

codon

11 13

14

FMTC

MEN 2A

609 611 618 620

609 611 618 620

630 634

634

768 790 791

790 791

804

(8041)

15

MEN 2B

(8042) 883

891 16

918

1

Based on one report with MTC and adrenal and extraadrenal pheochromocytoma [25]. 2 Based on one report with an additional Y806C germline mutation in cis (see text) [26].

had been thought to be associated with FMTC only [25]. Of course, the occurrence of both pheochromocytomas in this family might be coincidental. It is doubtful, however, whether our current knowledge is justification enough to exclude patients with these presumed ‘FMTC-specific’ RET mutations from follow-up with regard to the other organ systems that may possibly be affected. The germline mutations M918T and A883F have thus far been seen exclusively in patients with MEN 2B, which is thought to be the most aggressive form of MEN 2. Hence, these patients could theoretically be identified at a young age and targeted for increased clinical surveillance and early surgical intervention. Recently, a report from Japan found a V804M mutation together with a Y806C mutation in cis in a patient with features suggestive of MEN 2B [26]. Due to the diagnosis of MTC, bumpy lips, ‘multiple nodules on her tongue’ and medullary hyperplasia of the adrenal glands [diagnosed by computed tomography (CT)], this patient was classified as having MEN 2B. It is worth noting that neither mucosal neuromas nor thickening of the corneal nerves are pathognomonic for MEN 2B

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[27, 28]. It was further intriguing that this patient showed a return of elevated serum calcitonin levels to normal after total thyroidectomy with bilateral modified neck dissection indicating no residual tumor. This finding is highly unusual for a classic MEN 2B patient diagnosed at the age of 23 years, when metastatic spread is almost always present and biochemical cure impossible. It is therefore questionable whether this patient really has classic MEN 2B. Functional analysis will be required to prove that this double mutation is capable of causing the clinical MEN 2B phenotype, although this link might be impossible to prove. This Japanese case most likely represents an FMTC case with germline V804M mutation, normally associated with older age of onset [29], modulated by the Y806C mutation, which is postulated to modify the age of onset in a downwards direction and perhaps the other manifestations as well. Interestingly, some germline RET mutations (affecting either RET codon 609, 618 or 620) have been observed in patients with both MEN 2 and Hirschsprung disease. This finding is intriguing, since MEN 2 RET mutations usually cause gain of function of RET, while RET mutations leading to Hirschsprung disease are usually associated with loss of function. Hence, the same mutation seems to cause both gain and loss of function [30] (see the chapter by Mulligan [this vol.]). Overall, patients with a MEN 2 RET mutation harbor a high lifetime risk of developing MTC (table 1).

Medullary Thyroid Carcinoma MTC is a rare disease. While thyroid cancer accounts for about 1% of all malignancies, MTC comprises about 5–10% of all thyroid malignancies. Of these, about 25% have a hereditary background [31]. The only known hereditary syndromes with an increased risk of MTC are FMTC, MEN 2A and MEN 2B. Anecdotally, in the original description of the Birt-Hogg-Dube syndrome, a hereditary syndrome with multiple fibrofolliculomas with trichodiscomas and acrochordons, 6 out of 9 patients had MTC [32]. However, in later reports, MTC has never again been described and it therefore remains questionable whether this family also had MEN 2-related MTC. MTC derives from the parafollicular C cells which are part of the amine precursor uptake and decarboxylation system described by Pearse [33] and is the sine qua non of FMTC/MEN 2. Thyroid C cells are derived from the neural ectoderm. The cells come from the vagal neural crest and migrate to the caudal portion of the fourth branchial arch. From there, they migrate into the central and upper portion of each thyroid lobe.

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In hereditary MTC, C cell hyperplasia (CCH) seems to precede the development of C cell carcinoma (i.e. MTC), and, seemingly, all patients develop morphological changes of the C cells [34, 35]. The definition of CCH, i.e. an increased number of C cells, varies, ranging from 50 to greater than 200 C cells in at least one low-power (!100) field [36, 37]. These morphological changes are neither specific for hereditary MTC nor are they essential findings. They have also been observed in sporadic MTC and in normal thyroid glands [38, 39]. Other criteria for C cell abnormality include the pattern of C cell arrangement, the position of the C cells in relation to the follicle and cytological atypia. From the clinical point of view, one should be aware of the possibility of dealing with a hereditary type of MTC if the histological appearance is suggestive of hereditary MTC but no MEN 2-associated RET mutation has been found (about 5% of all hereditary MTCs). However, the diagnosis of hereditary MTC cannot be made solely on the basis of the appearance of the C cells.

Diagnosis of MTC Patients with Apparently Sporadic MTC Sporadic MTC is usually seen in individuals older than 40 years. Patients who are younger, have a family history of MTC or present with other signs or symptoms that accompany MEN 2, are obviously candidates for having a hereditary form of MTC. Even in the absence of obvious signs, RET mutation analysis has identified patients as having hereditary MTC even in the seventh decade [29]. Subsequent RET mutation analysis in family members may enable the identification of early-stage MTC with a high chance of cure [29]. This is the rationale for demanding RET mutation analysis in every patient with MTC, and enables the distinction between a patient with sporadic MTC and an ‘index patient’. The Index Patient The so-called ‘index patient’ is the first person in a given MEN 2 family in whom MTC is diagnosed. In this patient, the strategy to diagnose MTC does not differ from the strategy applied to patients with sporadic MTC. Once the diagnosis of MTC is made, RET mutation analysis should be performed in the setting of clinical cancer genetic consultation. Once the MEN 2-specific RET mutation is found in a given individual, the diagnosis of MEN 2 can be made and subsequent investigation of other potentially affected endocrine organ systems as well as screening of first-degree relatives (see below) should be undertaken. In contrast to most patients who are being actively clinically screened for MEN 2, the index patient almost always presents with a thyroid nodule. The great majority of thyroid nodules are benign; thyroid nodules can be very common,

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particularly in iodine-deficient areas. However, it should be standard practice to rule out malignancy before operating on a thyroid nodule. This is even more crucial since it has been shown that patients undergoing reoperation for MTC are less likely to be ‘biochemically cured’ than those patients undergoing primary operation [40]. The thyroid nodule might be palpable during physical examination but almost never causes any functional disorder of the thyroid gland. Similar to patients with sporadic MTC, most (50–80%) index patients with hereditary MTC have lymph node metastases at the time of diagnosis. Enlarged lymph nodes may thus be the initial symptom. Two diagnostic techniques have been shown to be helpful in making the preoperative diagnosis of MTC. Fine-needle aspiration cytology is an inexpensive, safe procedure, and the cytomorphologic appearance of MTC is highly distinctive [41]. However, some experience is required in order to provide high sensitivity. Ultrasound guidance should be used to show that the tip of the needle is within the thyroid nodule. Since only a small fraction of patients with thyroid nodules will turn out to have hereditary MTC, general preoperative RET mutation analysis cannot be recommended. However, depending on the technique, 30–70% of sporadic MTCs carry a somatic RET mutation, almost exclusively the MEN 2Bspecific mutation M918T [42]. Interestingly, additional somatic RET mutations (in particular the M918T mutation) have also been found in MTC in patients with germline RET mutations [43]. While the role of somatic mutations in the pathogenesis of MTC is unknown, it has been shown that these somatic mutations can be diagnosed preoperatively using cytological specimens [44, 45]. One has to keep in mind that a negative result, i.e. the absence of a M918T mutation, from the analysis of DNA obtained in this manner does not rule out the existence of MTC, either hereditary or sporadic. Nor does the finding of a positive result (i.e. a RET mutation in the cytological specimen) allow a definitive diagnosis of hereditary MTC. The same mutation would have to be found in DNA extracted from blood leukocytes to prove that this was a germline mutation. Hence, the identification of a RET mutation in a specimen obtained by fine-needle aspiration cytology may only be regarded as an additional aid in making the diagnosis of MTC, and does not permit determination of whether it is sporadic or hereditary. Others have shown that the detection of gene expression on the mRNA level of RET, the calcitonin gene, and the gene for carcinoembryonic antigen (CEA), at the transcript level, is highly specific for MTC and has not been observed in the normal thyroid gland [46]. It is worth noting that as a result of intra- or interchromosomal rearrangement, RET can also be expressed in papillary thyroid carcinoma [47, 48]. Preoperative measurement of serum calcitonin is another highly sensitive method to establish the diagnosis of MTC. Besides measurement of the basal level, calcitonin secretion should be stimulated with either calcium (2 mg/kg body weight of 10% Ca2+ injected i.v. in 1 min) or, preferably, pentagastrin (0.5 Ìg/kg

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body weight, diluted in 5–10 ml of sterile saline and injected i.v. in 5–15 s) or a combination of both. Pentagastrin appears to be the more provocative reagent, but recently it seems to be in short supply, at least in the USA. Its unpleasant side effects (including flushing, warmth, nausea and esophageal spasm) are often mild and transient. Calcitonin levels should be measured immediately before and 1, 2, 5 and 10 min after injection of the provocative agent. Immunoradiometric assays are highly sensitive (2.5 pg/ml). The normal basal level of calcitonin is lower than 10 pg/ml [49]. It is of note that elevated basal calcitonin levels can also be found in a variety of other conditions, such as pregnancy, use of the contraceptive pill, renal failure, liver disease and various tumors. An increase in calcitonin after injection (3 ! basal level or more) of a provocative reagent, however, seems to be specific for MTC [34, 49]. Women are found to have lower levels than men, and premenopausal women show higher levels of plasma calcitonin than postmenopausal women [50]. Further Diagnostic/Imaging Techniques Ultrasonographic examination of the thyroid tumor, its relation to the trachea and/or esophagus as well as evaluation of enlarged cervical lymph nodes should be performed preoperatively to identify extraglandular invasion, involvement of vital structures and lymph node metastases. If invasion of the trachea or the esophagus is suspected, CT or, preferably, magnetic resonance imaging (MRI) should be performed. In advanced cases, endoscopy can be helpful to confirm this suspicion. If metastatic disease is suspected (basal calcitonin levels 1 400 pg/ml, after stimulation 1 1,000 pg/ml – the threshold may vary based on the assay used), further attempts should be undertaken to localize the tumor. In patients with high calcitonin levels, technetium-99m-sestamibi scans are more sensitive than CT scans in detecting tumors within the neck and chest, but CT seems to be more sensitive for imaging hepatic lesions [51]. Bone scans are generally better for imaging bone lesions. Several other imaging techniques, e.g. thallium-201 thallous chloride, technetium-99m pentavalent dimercaptosuccinic acid and technetium-99m-tetrofosmin, have been analyzed [52, 53] and were found to be complementary, often having a high sensitivity but a low specificity. The method using dimercaptosuccinic acid seems to be superior to the other techniques [53, 54]. Selective venous sampling catheterization is probably the single most valuable technique to identify occult disease. When a local/peripheral calcitonin gradient of more than 2.5 is used, selective venous sampling catheterization has been shown to identify lesions and/or tumor within the liver and neck with high accuracy. While selective venous sampling catheterization is rarely used as a diagnostic tool in primary therapy, it can be of tremendous help in recurrent disease. However, anatomic changes due to previous operations can make evaluation of the findings difficult. With regard to the identification of liver metastases, laparosco-

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py has been shown to produce comparable accuracy [55]. Octreotide scans have been shown to be quite sensitive in detecting lung metastases; however, this technique fails to visualize tumors less than 1 cm in size. More recently, 131I-anti-CEA monoclonal antibodies have been used successfully in detecting occult disease [56, 57]. Since CEA production and secretion is required, this technique is more sensitive in detecting recurrent disease. Also, fluorine-18-deoxyglucose positron emission tomography has recently been described as an accurate, noninvasive technique for staging purposes and visualizing tumor spread [58, 59]. Screening Once MTC has been diagnosed, every patient should undergo RET mutation analysis regardless of the age at diagnosis, the presence or absence of a family history or other manifestations suggestive of MEN 2A/MEN 2B. In some instances, the index patient him/herself may not benefit from this strategy, but every first-degree relative (children, parents, siblings) who is a potential mutation carrier might very well do so. Hence, every single potential mutation carrier needs to be screened genetically in order to establish whether he/she harbors the familyspecific RET mutation. Mutation analysis is usually performed by analyzing DNA extracted from peripheral blood leukocytes. A variety of different techniques (e.g. sequencing, restriction endonuclease analysis, single-stranded conformational polymorphism, denaturing gradient gel electrophoresis and denaturing high-performance liquid chromatography) have been used successfully with a high sensitivity and specificity (almost 100%). For MEN 2A and FMTC, RET mutation analysis should begin with exons 11 and 10, where most mutations are found. Exons 13, 14 and 15 need to be analyzed if no mutation was identified in exons 11 or 10. If MEN 2B is suspected, only germline analysis of exons 16 and 15 seems to be indicated. If MEN 2B is not suspected, mutation analysis of exon 16 does not seem to be required. However, the MEN 2B-specific phenotype may be very mild in a very young (!2 years) mutation carrier. The ease of identifying these mutations (M918T, A883F) using restriction enzymes speaks in favor of a more liberal analysis of these two mutations together, since the failure to identify a MEN 2B patient might have fatal consequences. Interestingly, germline RET mutations in patients with MEN 2B seem to occur more often de novo; up to 40% of MEN 2B cases are isolated cases. In a family with a known RET mutation, at-risk individuals only need to be screened for that family-specific mutation. In an ideal world, no clinical decision should be made based on the result of only one mutation analysis, i.e. the result – positive or negative – should be confirmed with a second, independently obtained sample. This ‘ideal’ stance is historical, in a sense. Clinical RET mutation testing was begun in Europe within 6 months after the appearance of the report that RET

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was the MEN 2A susceptibility gene [60], and in many instances, the research laboratories that first described the mutations actually performed the ‘diagnostic’ mutation analysis, hence the caution [61]. In the USA, clinical RET testing is always performed in a CLIA-certified laboratory, which guarantees the accuracy of the test. Further, most, if not all, CLIA laboratories will replicate their results at least three times, albeit on a single sample. Further, it is conceivable that thirdparty payers (e.g. health insurance companies), especially in the USA, might be reluctant to cover the cost of a confirmation test based on a second separate phlebotomy. While, from the medical point of view, it seems justified to screen patients with MTC and relatives of mutation carriers for germline RET mutations, the psychological impact is often neglected. The findings of a study from France on the psychological impact of genetic testing for FMTC suggest a high level of frustration and latent discontent [62]. This discontent is either related to the management of the genetic information given by the clinicians and its psychological consequences or simply to the knowledge of the genetic risk of cancer. It should be noted, however, that this sort of psychological outcome might not be similar among other countries and other cultures. Nonetheless, more effort should be put into the interaction between clinicians and the potentially affected individuals. This underscores the vital importance of performing all cancer genetic testing with the input of a clinical cancer genetics team, which at a minimum comprises a genetic counselor familiar with cancer genetics and a physician specifically trained in clinical cancer genetics. It would also be of tremendous help if further investigation could develop a more reliable genotype-phenotype prediction system. Since germline RET mutations have been identified as causing MEN 2, the use of additional measurement of calcitonin is questionable. However, calcitonin levels may be helpful in determining the extent of disease and, hence, the extent of surgery required. It has been suggested that patients younger than 10 years with normal calcitonin levels after stimulation might not have to undergo central lymph node dissection, since lymph node involvement is unlikely [21]. However, in a recent study summarizing the results reported in the literature, only 12 out of 139 patients (8.6%) with MEN 2 who underwent preventive total thyroidectomy had lymph node metastases. Two out of these 12 patients (17%) had normal calcitonin levels [22]. Therefore, it remains to be clarified in a larger series whether any recommendation regarding the extent of surgery can be made based on calcitonin levels. Imaging techniques are generally not required in patients identified as RET mutation carriers at a young age (!5 years). However, if patients are older or calcitonin levels are suggestive of metastatic spread, the same recommendations as in sporadic cases (see above) apply. If there is any doubt, at least ultrasonographic examination of the thyroid gland and the neck should be performed.

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Treatment of MTC Primary Tumor and Locoregional Lymph Nodes The primary therapy for MTC is surgical. Total thyroidectomy (since 80– 90% of hereditary MTCs are bilateral/multifocal) and lymphadenectomy of the cervicocentral compartment (since 50–80% of nonscreening individuals will have lymph node metastases) are widely accepted as the treatment of choice. Since lymph node metastases of MTC can be very small and unable to be identified preor intraoperatively, a systematic lymphadenectomy (i.e. compartment-oriented dissection of lymph nodes and connective adipose tissue together while preserving nerves, vessels and muscles if not infiltrated by MTC) is recommended if cure is intended [63, 64]. However, surgical therapy extending beyond this is often controversial, since prospective long-term follow-up studies are still missing. There is agreement that lymph node dissection should be extended to compartments with proved lymph node metastases. If lymph node metastases are found within the cervicocentral compartment, both cervicolateral compartments should also be dissected. The size of the primary tumor does not correlate with the presence of lymph node metastases and should therefore not be relied on in making a decision. Prophylactic Thyroidectomy The term ‘prophylactic thyroidectomy’ theoretically implies removal of the entire thyroid gland when no malignant C cells can be found histologically, i.e. the pathologist finds either a normal thyroid gland or CCH only. This is, of course, a very strict use of the term. Prophylactic thyroidectomy has been used in the past to describe patients who have been identified as germline RET mutation carriers and in whom thyroidectomy with curative intent, sometimes undertaken in patients as young as 3 years, has been performed [21]. Naturally, it is often impossible to determine preoperatively whether these patients already have C cell carcinoma or ‘just’ CCH. Worldwide, more than 300 children and adolescents have been operated with curative intent after germline RET mutations had been identified. Pathological analysis of the C cells in thyroid glands after prophylactic thyroidectomy almost always revealed morphological changes. In the largest published series to date, from Germany and Austria, 61% of the children and adolescents undergoing ‘prophylactic’ thyroidectomy for mutation carrier status were found to already have MTC [21]. All thyroid specimens showed at least CCH. In rare instances, however, no pathological changes were noted [65, 66], calling into question whether this regimen (germline RET mutation as an indication to operate) is justified. According to epidemiological data, however, about 70% of germline RET mutation carriers develop clinically significant MTC by the age of 70 years [67]. This is the rationale to perform a

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prophylactic thyroidectomy in patients with MEN 2-specific RET germline mutations. Hence, such ‘negative’ findings are difficult to interpret. For instance, could some of these be examples of low-penetrance mutation cases such that CCH might not have become manifest in an obvious manner at the time of prophylactic surgery (patients with the V804M mutation might present as isolated cases in their seventies [29])? Technical reasons such as the sampling of tissue to be histopathologically examined might be another plausible explanation. These issues are important because clinicians should always keep in mind that prophylactic thyroidectomy is not without some risks, e.g. hypoparathyroidism and paresis of the recurrent laryngeal nerve. However, among 209 patients who underwent total thyroidectomy based on the presence of a germline RET mutation, only 3.4% were classified histologically as ‘normal’ [66]. Also, the risk of not identifying a patient with MTC if the indication to operate is based solely on calcitonin levels has to be taken into account. All in all, therefore, it would appear that RET mutation testing is highly sensitive and, generally, no prophylactic operation should be considered without a mutation-positive result on predictive testing. Even though lymph node metastases are rare in children and adolescents, they have been found in children as young as 5 years (MEN 2A) and 3 years (MEN 2B). Calcitonin levels and age might be good indicators for lymph node involvement and can be used to modify the extent of lymph node dissection [21]. However, lymph node metastases have been found despite normal stimulated calcitonin levels [22]. Hence, it is generally recommended to dissect the central lymph node compartment when performing a prophylactic total thyroidectomy. For certain RET mutations, an age-specific penetrance has been shown [29, 68]. If further data can be obtained for age-specific penetrance, it may be shown that a specific mutation may lead to MTC after the age of 20 years and, hence, would not require early operation, thus avoiding the risk of complications from neck dissection in infants and young children. Although current data are suggestive, they cannot be used to justify such an approach for patient management as yet. There is also some controversy about when to operate, since some mutation carriers theoretically may be very young. RET mutation analysis could be performed at birth on an at-risk individual. Most surgeons recommend that thyroidectomy be performed on children between the age of 4 and 6 years [21, 65, 69]. Some investigators do not believe in performing prophylactic thyroidectomy at a given age but rather recommend surgical intervention when calcitonin levels become pathologic [70]. However, basal and stimulated calcitonin levels can still be normal even in the presence of C cell carcinoma (false-negative result) [21, 22]. Furthermore, this strategy requires repeated unpleasant injection of a provocative reagent.

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Complications, in particular hypoparathyroidism and paresis of the recurrent laryngeal nerve, are not to be neglected, and since thyroid surgery is uncommon in young children, such operations should be performed in a specialized center by an experienced endocrine surgeon. MTC in MEN 2B Even though unequivocal evidence is missing, MEN 2B MTC is considered to be the most aggressive form of MTC. Morphologic C cell changes have been reported in patients within a couple of months after birth and MTC has been reported to occur as early as 3 years of age. If not diagnosed during infancy, most patients are incurable by the time of diagnosis due to metastatic disease. Therefore, the approach to MTC is more aggressive and prophylactic thyroidectomy is recommended within the first years after birth. In addition, cervicocentral and bilateral cervicolateral lymphadenectomy is recommended. Distant Metastases MTC often metastasizes to the lung, liver and bones. Once MTC has spread beyond the locoregional lymph node compartments, biochemical cure (indicated by normal calcitonin levels, both basal and after stimulation) cannot be achieved. However, long-term survival is still possible. Nonsurgical Treatment Modalities Radioiodine. Since MTC does not derive from follicular cells, there is no uptake of radioiodine. In one study, however, radioimmunotherapy with radioiodine-labeled anti-CEA monoclonal antibodies was able to show antitumor effects in 7 out of 14 patients (50%), demonstrated by decreasing levels of tumor marker [71]. The additional application of chemotherapeutic agents showed superior results (85%) in a nude mouse model if they were given with bone marrow support [72]. External Radiation. External radiation should be avoided as long as possible, and there is no need for ‘prophylactic’ radiation. The long-term side effects of cough and dryness can be very unpleasant. In addition, scarring of the neck may make evaluation of local recurrence difficult, both clinically and using imaging techniques, as well as potentially complicating reoperation. Surgery should be performed whenever feasible. However, external radiation might be indicated for palliation if the tumor (cervical and also distant metastases, e.g. bone) is not amenable to surgery. Chemotherapy. The use of chemotherapy in the treatment of MTC is limited. Partial responses and/or long-term stable disease have only been reported in rare instances. Various combinations (e.g. doxorubicin and cisplatin; 5-fluorouracil and streptozocin; cyclophosphamide and vincristine, and dacarbazine) have been

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investigated. Combined radiochemotherapy has been shown to improve treatment outcome in a nude mouse model, but clinical experience is still missing [72]. Other seemingly promising anecdotal responses have been obtained with small cell lung carcinoma-type regimens, based on the belief that MTC and small cell lung cancer are of neural crest origin. These regimens include platinum-based compounds and VP-16 (etoposide), ifosfamide, carboplatin, etoposide and taxol and paclitaxel-based regimens. Taxane-based regimens are extrapolated from the ongoing phase II trial of paclitaxel in carcinoids (MD Anderson Cancer Hospital), another tumor of neural crest derivation. Octreotide. Octreotide has been shown to be useful in the diagnosis of MTC. Its therapeutic effect, however, is low, but octreotide might alleviate the symptoms (e.g. diarrhea) caused by excessive levels of serum calcitonin [73]. In a more recent study, symptomatic patients with advanced MTC were treated with a combination of recombinant interferon ·-2b and octreotide [74]. Preexisting diarrhea improved significantly in 4 and flushing in 1 out of 8 patients. However, the therapy was stopped in 2 patients because of diarrhea and toxicity of the drugs used. While calcitonin levels showed a maximum decrease after 1–3 months, CEA decreased continuously during the treatment period of 12 months. However, no significant changes in the size of metastases were observed.

Follow-Up of MTC The outcome of patients with MTC is unpredictable. It has been shown that besides tumor stage, early (3–7 days after surgery) calcitonin levels are the most important prognostic factor [75, 76]. It has also been described that calcitonin levels can rise again during follow-up despite normal levels postoperatively. Many other prognostic factors (e.g. DNA aneuploidy, serum CEA levels and CEA immunoreactivity, microvessel count, somatic RET M918T mutation) have been analyzed [77–81]; however, none of them is of clinical use at this time. The outcome of patients with hereditary MTC is often believed to be better than that of patients with sporadic MTC. There is, however, no study which has compared only index patients with hereditary MTC and patients with sporadic MTC. It is almost certain that the introduction of routine clinical/biochemical screening procedures following the discovery of RET as the disease-causing gene has greatly improved the outcome of patients with hereditary MTC due to the identification of mutation carriers at an asymptomatic stage. The follow-up strategy in patients after surgery depends on the knowledge of residual tumor burden. If present, calcitonin levels will almost always remain elevated in this case. Hence, repeated measurements of calcitonin levels, in par-

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ticular in combination with stimulating substances that have an unpleasant side effect, should be avoided. Calcitonin is a very sensitive marker and should be measured yearly unless medical management of the patient is not altered in response to calcitonin levels. In a given individual, the comparison of the preoperative and early postoperative calcitonin level correlates very well with the remaining tumor burden [82]. However, over time, calcitonin levels may vary and do not necessarily correlate with tumor burden [83]. Actually, decreasing calcitonin levels despite known persistent tumor may indicate dedifferentiation of the tumor rather than decrease of actual tumor mass. The measurement of CEA may be very helpful in these instances. Once recurrent disease has been found, reoperation should be considered. Since all patients undergo total thyroidectomy, lifelong replacement of levothyroxine is mandatory. The dose should be adjusted depending on measurements of serum thyrotropin, which should be within the normal range.

Pheochromocytoma Pheochromocytoma derives from large pleiomorphic chromaffin cells, most commonly arising from the adrenal medulla. Rarely, pheochromocytomas are extra-adrenal, most often located within the abdomen. Most pheochromocytomas are sporadic, but about 10% are thought to have a familial background and may then be found in association with the MEN 2 syndromes. Other syndromes associated with pheochromocytoma are von Hippel-Lindau syndrome, type 1 neurofibromatosis and tuberous sclerosis, although pheochromocytoma rarely occurs in type 1 neurofibromatosis (1% of cases) or tuberous sclerosis (!1%) [84]. Isolated familial forms of pheochromocytoma without other associated clinical features occur even less commonly [85]. Patients with MEN 2A and MEN 2B have an expected 50% lifetime risk of developing pheochromocytoma [34, 86]. The risk of developing a contralateral tumor following unilateral adrenalectomy is approximately 50% [87]. As a component of MEN 2, pheochromocytomas are almost always benign (90–95%), but tend to be bilateral in 50–80% of cases. Extra-adrenal pheochromocytomas have rarely been observed in MEN 2. Whether extraadrenal pheochromocytomas have a greater malignant potential than adrenal pheochromocytomas remains controversial [88–90]. However, if properly diagnosed and treated, most patients are likely curable. Pheochromocytomas can be present at the time MTC is diagnosed (synchronous occurrence) or may develop in up to 40–50% of patients during follow-up (metachronous occurrence). In MEN 2, the development of pheochromocytoma rarely precedes MTC [91]. Pheochromocytoma is almost never present in children and adolescents diagnosed by screening.

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Diagnosis Pheochromocytomas produce and secrete catecholamines and, hence, can cause hypertension. However, only 0.1–0.5% of all hypertensive patients will be found to have a pheochromocytoma. Even though pheochromocytomas are rarely diagnosed prior to MTC, they have to be excluded before surgery for MTC to avoid intraoperative life-threatening hypertensive crisis. Hypertension caused by pheochromocytoma can be paroxysmal or permanent. Other symptoms are headache, tachycardia, palpitation, sweating and even severe anxiety. It is worth noting that not all patients with pheochromocytomas present with hypertension. In addition, orthostatic hypotension is not a rare finding. However, since pheochromocytoma as part of MEN 2 is often preceded by the diagnosis of MTC, its diagnosis is often made at an early stage without the clinical presence of the above-mentioned symptoms. In addition, pheochromocytoma may cosecrete other hormones, such as adrenocorticotropic hormone, hence resembling Cushing’s syndrome, which may confuse the interpretation of the clinical picture [92]. The biochemical diagnosis can be made by measurement of elevated 24-hour urinary excretion of free catecholamines (epinephrine, norepinephrine) or their metabolites (e.g. vanillylmandelic acid). Plasma metanephrines, however, seem to be more sensitive (97%) and specific (96%) [93]. Patients at risk of developing a pheochromocytoma, i.e. after identification of a MEN 2-specific RET mutation, should undergo yearly assessment in this manner. Some exponents also measure serum catecholamine levels and serum chromogranin A. The latter is less specific, as it may rise in any condition associated with a neuroendocrine tumor, such as MTC. Once the biochemical diagnosis of pheochromocytoma has been made, its localization and extent needs to be determined. If the patient is already symptomatic, enlarged adrenal glands can almost always be found and, hence, can be detected by CT and/or MRI (sensitivity of up to 100% for both). It should be emphasized that MRI should always include both T2 and T1 weighted images of the abdomen through the pelvis, as the ratio of density on T2 to T1 weighted images can help clarify the diagnosis of pheochromocytoma. However, neither CT nor MRI is very specific (about 70%), and they may fail to detect a pheochromocytoma at an early stage. Also, extra-adrenal pheochromocytomas and or metastases in unusual areas are not very likely to be found using these imaging techniques. Instead, 131I-meta-iodobenzylguanidine, which has a sensitivity of about 80% and a specificity of nearly 100%, may be very helpful. Rarely, vena cava catheterization with selective venous sampling for catecholamines is indicated.

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Treatment Surgical resection is the treatment of choice for pheochromocytoma. Before surgery, medical management should be initiated using a drug with alpha-adrenergic blocking activity for a few weeks to reach maximal effect. Unpleasant side effects, such as nasal congestion, may be treated by the local application of drugs stimulating alpha-adrenergic receptors. In the presence of tachyarrhythmias, low doses of a beta-blocker should be used as well (beta-blockers should never be given prior to alpha-adrenergic blockade in patients suspected of having pheochromocytoma) [94]. Whereas total adrenalectomy has been the treatment of choice for many years, cortical-sparing adrenalectomy has recently gained more acceptance [95, 96]. It is of particular importance to conserve functioning adrenal tissue, since patients with MEN 2 are at risk of developing bilateral pheochromocytoma. The cortical-sparing technique may enable the avoidance of chronic replacement of steroid hormones and lower the risk of an Addisonian crisis [97]. However, longterm follow-up data do not exist yet, but will be necessary to evaluate the risk of recurrent disease. Despite the fact that about 50% of patients develop a contralateral pheochromocytoma after adrenalectomy, prophylactic (subtotal) adrenalectomy is not recommended. An experienced anesthesiologist is as important as an experienced surgeon to ensure an uneventful intra- and postoperative period. Several anesthetic methods have been proposed, but one prospective randomized study showed that the choice of the anesthetic technique is not a crucial factor in determining the patient outcome. Intra- and postoperative hypotension is very common and can usually be best treated with volume expansion. Accordingly, postoperative hypertension should be treated with diuretics, which should not be used preoperatively. One has to keep in mind that postoperative hypertension can be indicative of residual tumor. Biochemical and imaging assessment is recommended to rule out this possibility. Nonsurgical Treatment Modalities Long-term nonsurgical treatment modalities should only be considered if the tumor is unresectable. Alpha- and beta-adrenergic blockers or calcium antagonists can be continued on a long-term basis. In more refractory cases, alpha-methyl-para-tyrosine is a very effective drug which inhibits catecholamine synthesis [98]. This drug has been shown to decrease circulating catecholamine levels by 80% and can alleviate many disease-related symptoms. Despite the observation that pheochromocytomas possess specific somatostatin-binding sites, octreotide had no antisecretory effect in patients with pheochromocytoma in a placebo-controlled trial [99].

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In malignant disease, external radiation therapy may be indicated for the palliation of local complications due to unresectable local tumor. Also, external radiation therapy has been used successfully to achieve palliation of painful bone metastases. Despite its successful application in the detection of pheochromocytomas, 131I-meta-iodobenzylguanidine has met with limited success with respect to treatment with targeted radiation therapy. There is no evidence that chemotherapy contributes to improved patient survival. It should therefore only be used to alleviate symptoms. The most active chemotherapy regimen appears to be the combination of cyclophosphamide, vincristine and dacarbazine. This combination has been shown to produce partial remission of moderate duration in symptomatic patients [100].

Follow-Up Following surgery, patients should undergo regular clinical and biochemical assessments to detect persistent or recurrent disease. If no symptoms are present, yearly assessment seems to be sufficient. Once biochemical analyses indicate recurrent disease, imaging studies have to be performed to identify the location of increased catecholamine production. Depending on the previous operation, recurrent disease can be due to incomplete resection of tumor or due to the development of contralateral pheochromocytoma. Only very rarely is recurrent disease due to extra-adrenal or metastatic tumor.

Patients with Apparently Sporadic Pheochromocytoma Historically, about 10% of pheochromocytomas are believed to be inherited. Surprisingly, in a recent study, more than 20% of the 82 unselected pheochromocytomas investigated turned out to be inherited [101]. Because of the location of the institution involved in that study (the Black Forest region in southern Germany), where a particular VHL founder mutation is common [102], and because this mutation is associated with the development of pheochromocytoma [103, 104], there is little doubt that this percentage is artificially elevated due to geographic reasons. In contrast, a series of 48 unselected European patients presenting with apparently sporadic pheochromocytoma revealed an occult germline VHL mutation frequency of 4% and an occult germline RET mutation frequency of 2% [85]. So, this molecular-based study confirms the findings from clinical epidemiologic studies that 5–10% of all pheochromocytomas are hereditary. Interestingly, it is the apparently sporadic bilateral pheochromocytoma patients that tend to harbor occult germline VHL mutations [85, 103], although the number of bilateral cases

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in the available studies is relatively small. In some centers, particularly those where VHL founder mutations exist, it is accepted to screen patients with apparently sporadic pheochromocytoma for germline mutations, at least in RET and VHL. In other centers (the majority of institutions around the world), if a complete and careful patient history, family history and physical examination are performed, it is probably unnecessary to perform RET mutation analysis in apparently sporadic presentations of unilateral pheochromocytomas. However, apparently sporadic bilateral pheochromocytoma presentations should be subjected to germline VHL mutation testing at this time. It will be the task of clinical cancer geneticists, genetic counselors and researchers to prove that this testing strategy is not only beneficial to the patients but also cost efficient, so that third-party payers can be convinced to cover the costs as well.

MEN 2A-Specific Features MEN 2A has also been termed Sipple syndrome since Sipple described the frequent occurrence of thyroid carcinoma and pheochromocytoma in several kindreds in 1961 [105]. Besides MTC and pheochromocytoma, patients with MEN 2A may develop primary hyperparathyroidism (pHPT) (table 1). Hyperparathyroidism is caused by hyperplasia of the chief cells of the parathyroid gland. Actually, the pathologic changes vary from focal hyperplasia to true adenoma. There is no correlation between the histopathological changes and serum calcium and/or parathyroid hormone levels. In contrast to the histopathological changes found in parathyroid glands removed from patients with MEN 2A, clinical evidence is much less frequent and usually mild.

Diagnosis of Hyperparathyroidism Since hyperparathyroidism was first described in 1925, the symptoms have become known as ‘moans, groans, stones and bones’. Although most people with pHPT, and especially those with MEN 2-related pHPT, claim to feel well when the diagnosis is made, the majority of these will actually say they feel better after the disease has been treated. The most accurate and definitive way to diagnose pHPT is by showing an elevated parathyroid hormone level in the face of elevated serum calcium. The elevated secretion of parathyroid hormone is genetically determined and is not a response to the elevated calcitonin levels due to MTC. If the diagnosis is made, the indication to operate is always present, since the risks of untreated pHPT are diverse [severe osteoporosis and osteopenia, bone fractures (‘bones’), kidney

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‘stones’, peptic ulcers, pancreatitis and nervous system complaints (‘moans and groans’)]. There is generally no need to use imaging techniques. However, if hyperparathyroidism persists after primary surgery, sestamibi scanning is the preferred way in which to localize remaining parathyroid tissue [106]. This technique has a high sensitivity (90%) and specificity (98–100%).

Treatment of Hyperparathyroidism There is no need to perform prophylactic parathyroidectomy in patients with MEN 2A. Firstly, clinical hyperparathyroidism will only develop in about 10– 30% of patients [24, 107]. Secondly, when compared to sporadic hyperparathyroidism or hyperparathyroidism as part of MEN 1 (Wermer’s syndrome), hyperparathyroidism as part of MEN 2A seems to be milder. Hence, surgical management should focus on the preservation of parathyroid function. Due to the genetic determination, every single parathyroid gland has the potential to become adenomatous. Hence, when operating on a patient with MEN 2A-associated hyperparathyroidism, all four glands have to be identified. Up to 20% of patients do have additional glands, very often found in the thymus. Therefore, the thymus should be removed as part of the parathyroidectomy to avoid reoperation. It is recommended to resect all enlarged parathyroid glands. In the rare event that all parathyroid glands are enlarged, the least enlarged gland or the least enlarged portion of one gland should be preserved. There is no uniform recommendation regarding the nonenlarged glands. Some authors recommend that these glands should remain in situ (subtotal parathyroidectomy) [65]. Others recommend an autotransplantation of these glands either into the sternocleidoid muscle or the forearm [69]. It seems that similar short-term results can be achieved. Our current knowledge, in particular on the natural course of the parathyroid glands in RET mutation carriers after total thyroidectomy, is limited. Hence, final conclusions await more long-term follow-up studies.

Cutaneous Lichen Amyloidosis In some MEN 2A families, skin amyloidosis has been observed [108]. Mutation screening in familial cutaneous lichen did not reveal any RET mutations, indicating that the skin amyloidosis found in some MEN 2A families and familial cutaneous lichen amyloidosis are different conditions [109].

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MEN 2B-Specific Disorders MEN 2B has also been termed Wagenmann-Froboese syndrome and MEN 3 [110, 111]. Since the molecular etiology of this syndrome is similar to that for MEN 2A and FMTC and it is not associated with another gene, the term MEN 2B seems to be more correct than MEN 3. In contrast to MEN 2A, clinical hyperparathyroidism does not seem to be a part of MEN 2B (table 1), even though mild chief cell hyperplasia can often be found [112]. Besides MTC and pheochromocytoma, which are also part of MEN 2A, patients with MEN 2B present with multiple mucosal neuromas, a marfanoid habitus, gastrointestinal ganglioneuromatosis with resulting megacolon and thickened corneal nerves. Unfortunately, none of these signs in and of themselves is pathognomonic for MEN 2B; i.e., they have been observed in patients without MTC and without characteristic MEN 2Bspecific RET mutation. The mucosal neuromas may manifest as thickened, lumpy and enlarged lips. They may also be seen as nodular protuberances of the margin of the tongue. There is generally no treatment required and if so, for example, due to a hyperactive bowel, treatment is symptomatic. It is of note that MEN 2B patients often do not have a family history, as observed in MEN 2A patients. Slightly less than half of these patients present as isolated cases (see above). The high aggressiveness of the disease and the low number of individuals who survive the disease beyond the reproductive age explain this, at least in part. Screening studies and improved cure rates might alter the proportion of ‘de novo’ MEN 2B cases in prospective studies.

Specific Issues Patients with Suspected MEN 2 but No RET Mutation In contrast to many other hereditary syndromes, clinicians dealing with MEN 2 patients do have a big advantage. A single disease-causing gene has been identified, and in more than 95% of cases, germline mutations have been found. This advantage can easily turn into a disadvantage when a clinician is faced with a RET mutation-negative MEN 2 family. Since the majority of MEN 2 cases without RET mutation have FMTC, these patients are most likely members of a small family with FMTC. Further, since MTC is rare, even the occurrence of two family members with MTC is most likely not coincidental. It also remains questionable whether FMTC is a true entity or rather belongs to MEN 2A, i.e. a given FMTC family might actually be a MEN 2A family just ‘waiting’ for pheochromocytoma/ hyperparathyroidism to develop. Genetic heterogeneity, i.e. linkage of a given family without germline RET mutation to a different locus, has not been reported

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yet. If the RET mutation-negative MEN 2 family is large, i.e. several affected members, then linkage analysis [11, 12] might be considered. Mutation-negative families that are not amenable to linkage analysis should still be managed like MEN 2 families prior to the era of DNA-based testing.

Future Aspects Many issues remain unresolved and require future investigation, for example, why does the same germline mutation lead to different phenotypes? Probably, our focused view on single so-called high-penetrance mutations may blind us to looking at polymorphic sequence variants that may act as low-penetrance mutations. Hence, future research will have to take into account the ‘whole’ genotype instead of just focussing on single-sequence variants [113, 114]. Also, other RETinteracting proteins [e.g. glial cell line-derived neurotrophic factor (GDNF), neurturin, artemin, persephin and GDNF receptor-·1–4] may very well play a role. Only GDNF has been looked at genetically and no mutations have been found [115]. How do we explain the different age of onset even in members of a given family harboring the same RET mutation and even the same haplotype? Do lowpenetrance mutations and unlinked modifier loci play a role? Clinical anticipation has been noticed but not very well investigated. How can we improve our diagnostic techniques, which very often fail to identify the usually small metastases of MTC? One of the most important issues will be the improvement of the treatment of locally advanced and metastatic MTC, and there is much to do in this regard. Current treatment strategies are all noncurative. Although much has been learned from the intense research into MEN 2 over the course of the last decade, many questions remain, and more are generated as new research findings are unearthed each week. The MEN 2 community will doubtless be kept very busy for decades more to come.

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102 Brauch H, Kishida T, Glavac D, Chen F, Pausch F, Hofler H, Latif F, Lerman MI, Zbar B, Neumann HP: Von Hippel-Lindau (VHL) disease with pheochromocytoma in the Black Forest region of Germany: Evidence for a founder effect. Hum Genet 1995;95:551–556. 103 Woodward ER, Eng C, McMahon R, Voutilainen R, Affara NA, Ponder BA, Maher ER: Genetic predisposition to phaeochromocytoma: Analysis of candidate genes GDNF, RET and VHL. Hum Mol Genet 1997;6:1051–1056. 104 Zbar B, Kishida T, Chen F, Schmidt L, Maher ER, Richards FM, et al: Germline mutations in the Von Hippel-Lindau disease (VHL) gene in families from North America, Europe, and Japan. Hum Mutat 1996;8:348–357. 105 Sipple JH: The association of pheochromocytoma with carcinoma of the thyroid gland. Am J Med 1961;31:163–166. 106 Weber CJ, Vansant J, Alazraki N, Christy J, Watts N, Phillips LS, Mansour K, Sewell W, McGarity WC: Value of technetium 99m sestamibi iodine 123 imaging in reoperative parathyroid surgery. Surgery 1993;114:1011–1018. 107 Gagel RF, Tashjian AH Jr, Cummings T, Papathanasopoulos N, Kaplan MM, DeLellis RA, Wolfe HJ, Reichlin S: The clinical outcome of prospective screening for multiple endocrine neoplasia type 2a. An 18-year experience. N Engl J Med 1988;318:478–484. 108 Gagel RF, Levy ML, Donovan DT, Alford BR, Wheeler T, Tschen JA: Multiple endocrine neoplasia type 2a associated with cutaneous lichen amyloidosis. Ann Intern Med 1989;111:802–806. 109 Hofstra RM, Sijmons RH, Stelwagen T, Stulp RP, Kousseff BG, Lips CJ, Steijlen PM, Van Voorst Vader PC, Buys CH: RET mutation screening in familial cutaneous lichen amyloidosis and in skin amyloidosis associated with multiple endocrine neoplasia. J Invest Dermatol 1996;107:215–218. 110 Froboese C: Das aus markhaltigen Nervenfasern bestehende ganglienzellenlose echte Neurom in Rankenform – zugleich ein Beitrag zu den nervösen Geschwulsten der Zunge und des Augenlides. Virchows Arch Pathol Anat 1923;240:312–327. 111 Wagenmann A: Multiple Neurome des Auges und der Zunge. Ber Dtsch Ophthalmol Ges 1922;43: 282–285. 112 Carney JA, Roth SI, Heath H 3d, Sizemore GW, Hayles AB: The parathyroid glands in multiple endocrine neoplasia type 2b. Am J Pathol 1980;99:387–398. 113 Borrego S, Saez ME, Ruiz A, Gimm O, Lopez-Alonso M, Antinolo G, Eng C: Specific polymorphisms in the RET proto-oncogene are over-represented in patients with Hirschsprung disease and may represent loci modifying phenotypic expression. J Med Genet 1999;36:771–774. 114 Gimm O, Neuberg DS, Marsh DJ, Dahia PL, Hoang-Vu C, Raue F, Hinze R, Dralle H, Eng C: Over-representation of a germline RET sequence variant in patients with sporadic medullary thyroid carcinoma and somatic RET codon 918 mutation. Oncogene 1999;18:1369–1373. 115 Marsh DJ, Zheng Z, Arnold A, Andrew SD, Learoyd D, Frilling A, Komminoth P, Neumann HP, Ponder BA, Rollins BJ, Shapiro GI, Robinson BG, Mulligan LM, Eng C: Mutation analysis of glial cell line-derived neurotrophic factor, a ligand for an RET/coreceptor complex, in multiple endocrine neoplasia type 2 and sporadic neuroendocrine tumors. J Clin Endocrinol Metab 1997;82: 3025–3028.

Oliver Gimm, MD, Department of General Surgery, Martin-Luther-University Halle-Wittenberg, Ernst-Grube-Strasse 40, D–06097 Halle (Germany) Tel. +49 345 557 2314, Fax +49 345 557 2551, E-Mail [email protected]

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Von Hippel-Lindau Disease: Genetic and Clinical Observations Othon Iliopoulos MGH Cancer Center, Harvard Medical School, Boston, Mass., USA

Contents 131 132 132 134 134 136 137 137 138 140 140 142 143 148 150 154 155 155

Introduction Clinical Features CNS Hemangioblastomas Renal Cysts and Clear Cell Renal Carcinomas Pheochromocytoma Other VHL Disease Manifestations The VHL Gene Structure and Expression of the VHL Gene VHL Gene Mutations in Familial and Sporadic Tumors Functional Analysis of pVHL Identification of Endogenous pVHL Isoforms pVHL Functions as a Gatekeeper Tumor Suppressor Protein pVHL Is the Receptor of a Ubiquitin Ligase (E3) Multiprotein Complex Substrates of SCFVHL Ubiquitin Ligase Physiology of the VHL–/– Cell Future Directions and Therapeutic Potential Acknowledgement References

Introduction Familial pheochromocytoma alone, or in combination with other lesions, may be a clinical manifestation of von Hippel-Lindau (VHL) disease (OMIM 193300). VHL disease is an inherited cancer disorder. Affected individuals are at risk of developing histologically characteristic lesions of the central nervous system (CNS), including the retina, called hemangioblastomas, renal cell carcinomas

(RCCs), pheochromocytomas and other lesions to be described below [1]. Treacher Collins [2] was the first to report on two siblings suffering from what is known today as retinal hemangioblastoma. The German ophthalmologist Eugene von Hippel [3] described the occurrence of retinal angiomas in two unrelated kindreds in 1904. The association between retinal angiomatous lesions and ‘larger angiomatous lesions of the CNS’, as well as cysts of the kidney and pancreas, was underlined by the Swedish ophthalmologist Arvid Lindau [4] in 1927. The term ‘von Hippel-Lindau syndrome’ was introduced by Melmon and Rosen [5] in 1964, in a comprehensive review of the literature. The familial nature of this disease was fully appreciated by neurophysiologist John Fulton [6], in 1920. VHL syndrome has been loosely classified among the phacomatoses because rarely reported cutaneous lesions (hemangioblastomas) occur in the context of CNS abnormalities. Major advances in the understanding of the molecular abnormalities underlying the disease were made feasible by the identification and cloning of the gene responsible for it, the VHL gene, in 1992. This chapter describes the known clinical features of the disease to date, the current concept of the biochemical functions of the VHL protein and the molecular physiology of VHL–/– cells. Clinical and molecular studies on VHL-associated pheochromocytomas are also examined more closely.

Clinical Features CNS Hemangioblastomas Hemangioblastomas are nonmetastatic, noninvading tumors of the CNS, including the optic nerve and the retina. Retinal hemangioblastomas are synonymous with the lesion referred to initially as retinal angiomas [7]. They are the commonest, earliest and most characteristic lesions of VHL disease, occurring in 60–80% of VHL patients [8, 9]. In some clinical series, hemangioblastoma was the presenting manifestation of VHL disease in approximately 40% of the patients [8–11]. Histologically, hemangioblastomas consist of ‘stromal’ cells embedded within a particularly rich network of capillary vessels [12]. The cell responsible for the generation of this lesion is the ‘stromal cell’ of ill-defined origin. As described below, biallelic inactivation of the VHL gene occurs within this ‘stromal’ cell, signaling the proliferation of capillary vessels. Immunohistochemical and electron microscopy studies indicate that stromal cells are most likely of mesenchymal origin [12, 13]. Currently, it is believed that stromal cells share a common ancestor with endothelial cells, but it is still unclear at which stage stromal and endothelial cells become committed to a distinct differentiation program [7, 12].

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Macroscopically, a typical hemangioblastoma presents as a cystic lesion harboring a solid component attached to its wall (85%). The solid to cystic ratio varies. Purely solid (10%) or cystic (5%) lesions do exist [14]. Spinal hemangioblastomas are usually solid or mixed tumors [14]. VHL-associated hemangioblastomas occur typically in multiple sites, either synchronously or metachronously. They involve the cerebellar hemispheres (75%), spinal cord (20%) and brain stem (5%). Spinal hemangioblastomas occur more often in the cervical and lumbar area, are intradural and are often associated with syringomyelia [14, 15]. Rare cases of supratentorial, nerve root, peripheral nerve, skin and hepatic hemangioblastomas, as well as spinal leptomeningeal hemangioblastosis, have been reported [16–20]. Sporadic, non-VHL-associated hemangioblastomas occur usually at single sites, rarely involve the spinal cord and they recur, after surgical excision, less frequently than their VHL-associated counterparts [21, 22]. Retinal hemangioblastomas occur also in multiple sites and bilaterally, mostly in the temporal or nasal periphery of the retina (90%), and to a lesser extent in the macular retina, the papilla (8%) or the optic nerve (1%) [23]. Despite the fact that hemangioblastomas are nonmetastatic lesions, they contribute to the morbidity and mortality of VHL disease as much as renal cancer does [24, 25]. Their space-occupying nature generates symptoms which develop once the tumor reaches a size critical for its location, i.e. headache, vomiting and cerebellar ataxia (infratentorial lesions with increased intracranial pressure), orthostatic hypotension (brain stem lesions), spinal motor and sensory disturbances (spinal cord lesions) and pain (peripheral nerve lesions). Retinal hemangioblastomas cause retinal exudates, hemorrhage and, ultimately, glaucoma and retinal detachment, leading to blindness [23]. Decreased visual acuity in a VHL patient may also be the result of retinal pathology or of optic nerve atrophy secondary to hemangioblastoma-mediated chronic obstructive hydrocephalus. The stromal cell of the hemangioblastoma may inappropriately produce and secrete erythropoietin, causing secondary paraneoplastic erythrocytosis (10–20% of cases) [26, 27]. The standard approach to the treatment of hemangioblastoma is microsurgical removal [14]. Despite recent advances in neurosurgical care, repeated surgery, due to the multiple and recurrent nature of the lesions, increases the likelihood of postoperative morbidity and mortality. Lesions located in the brain stem and the spine present a particular challenge. Stereotactic radiosurgery offers the possibility of treating multiple lesions simultaneously without craniotomy [28–30]. This increasingly popular technique is recommended, therefore, for small lesions (!2.5 cm), with tissue-proven diagnosis, located at a safe distance from sensitive normal tissues such as the brain stem, spine or optic nerve and with a minimal cystic component that does not contribute significantly to the symptoms [14]. With the advantage of molecular diagnosis of VHL disease (see below), early

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radiologic (MRI) detection of hemangioblastomas in surveyed individuals at risk is feasible. The optimal timing for the treatment of such asymptomatic lesions becomes an important question for clinical investigation. Renal Cysts and Clear Cell Renal Carcinomas RCC, along with hemangioblastoma, are the major cause of morbidity and mortality in VHL disease [8, 9, 31]. Between 60 and 85% of VHL patients will develop hundreds of bilateral renal cysts during their lifetime [9, 10, 32]. Although occasionally cysts may regress, most of them will grow slowly over a period of years, remaining clinically silent. Rarely, a few of them may cause cortical atrophy and impaired renal function [33]. Macroscopically, they appear to be purely cystic, mixed cystic with a solid component or multiloculated [32]. It is currently believed that mixed and multiloculated cysts arise from the proximal renal tubule and that purely cystic ones arise from the distal renal tubule [34]. VHL-associated RCCs are multicentric and bilateral and may arise from the epithelium of a preexisting cyst or ‘de novo’ from the epithelium lining the proximal renal tubule [35]. The risk of developing malignancy does not correlate with the size of the cyst [36]. These tumors are almost exclusively of clear cell histology and they appear synchronously to each other or metachronously [8, 31, 35]. Nowadays, radiographic surveillance of VHL patients at risk leads to an increasing rate of diagnosis of localized RCC [32]. Surgical removal of such a localized lesion is the mainstream of treatment [37]. Loss of renal parenchyma as a result of total or successive partial nephrectomies may lead to chronic renal failure and subsequent morbidity and mortality. Therefore, recent efforts have focused on the development of tumor enucleation or nephron-sparing surgical techniques that will minimize the loss of normal tissue without exposing the patient to the risk of metastasis [38–40]. To this end, the optimal timing of surgery is under investigation. Retrospective studies suggest that delaying nephronsparing surgery in VHL patients until RCC tumors reach 3 cm is a reasonably safe practice that may maximize the protection of renal parenchyma [41]. Pheochromocytoma Pheochromocytomas arise from the chromaffin cells of the neural crest located in the adrenal medulla and typically secrete large amounts of catecholamines. Extra-adrenal chromaffin cells may persist developmentally along the spinal nerve roots or the aorta and give rise to tumors histologically similar to pheocromocytomas, called paragangliomas. Pheochromocytomas are the main neural crest tumors encountered in VHL patients. Isolated cases of extra-adrenal paragangliomas have also been reported in VHL patients [42]. VHL is a phenotypically heterogeneous disease, divided into types I and II, according to the clinical risk of developing pheochromocytomas [43–45]. VHL

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type I manifestations include RCC, CNS hemangioblastomas and other VHLassociated lesions (see below), but not pheochromocytomas. VHL type II patients are predisposed, in addition to the lesions listed for type I disease, to pheochromocytomas. Type II families are subdivided into low (type IIA) or high (type IIB) risk for RCC. A type IIC disease phenotype has also been recently identified [46–49]. Type IIC patients present with a history of familial occurrence of pheochromocytoma without any other stigmata of VHL disease, even after detailed physical and radiologic examination (e.g. absence of hemangioblastomas or cystic lesions). The correlation of these phenotypes to specific VHL mutations is discussed below (VHL Gene Mutations in Familial and Sporadic Tumors). It is therefore possible that familial occurrence of pheochromocytoma may be the only manifestation of VHL. Woodward et al. [50] examined 8 kindreds with hereditary pheochromocytoma and 2 patients with isolated bilateral pheochromocytoma without a family history of the disease. They detected germline VHL mutations in 3 out of the 8 kindreds with hereditary pheochromocytoma and in 1 of the 2 patients with bilateral disease [50]. Gross et al. [47] reported on a 25-member VHL kindred with familial pheochromocytoma as the only manifestation of the disease (type IIC), with the exception of 1 member who developed CNS hemangioblastoma 25 years after the initial pheochromocytoma diagnosis. Similarly, Ritter et al. [48] reported familial pheochromocytoma as the only manifestation of the disease among three generations of a VHL family. Thus, familial pheochromocytoma may be a manifestation of either VHL, multiple endocrine neoplasia type 2 (MEN 2) or NF 1 disease. It appears that among these, germline mutations in the VHL gene are the most frequent cause of hereditary pheochromocytoma. Neumann et al. [51] examined the frequency and clinical characteristics of familial pheochromocytomas (MEN 2 and VHL) in an unselected group of 82 consecutive patients with documented pheochromocytoma; 19% of the patients had VHL and 4% had MEN 2 disease. Compared to those with sporadic pheochromocytomas, VHL patients had multifocal and bilateral lesions, were diagnosed at an earlier age and were less likely to have histologically malignant disease [51]. Screening of family members at risk revealed the presence of pheochromocytoma in 46% of the cases [51]. Patients included in this study were from the Black Forest region, in Germany, where one of the founder VHL mutations originated. This might have created a bias towards a higher frequency of VHL mutations in unselected pheochromocytomas from that specific geographic region [52]. Despite these findings, hereditary pheochromocytoma-only syndrome may also represent a distinct entity, for which the genetic basis is still unknown, as seen with kindreds where no germline VHL mutations were found [50].

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Many VHL-associated pheochromocytomas are asymptomatic [53]. When symptoms occur, they include sweating, palpitations, anxiety and headaches due to catecholamine hypersecretion. If left untreated, they may result in sequelae of uncontrolled hypertension such as stroke or myocardial infarction [53]. Adrenalsparing surgery, when feasible, is the preferred method of treating symptomatic pheochromocytomas [54, 55], although this strategy has not yet been routinely employed by many centers. The diagnosis of pheochromocytoma is made with imaging studies (CT, MRI, ultrasound, MIBG scintigraphy) and measurement of serum and urine catecholamines. Periodic surveillance may lead to the early detection of biochemical activity in clinically ‘silent’ pheochromocytomas. Eisenhofer et al. [56] studied 26 patients with VHL-associated and 9 patients with MEN 2-associated pheochromocytomas. The measurement of serum metanephrine and normetanephrine prior to surgical excision was 97% sensitive and 96% specific for diagnosis of pheochromocytoma. These tests compared favorably to measurements of serum and urinary levels of epinephrine, norepinephrine and vanillylmandelic acid, which are more standard diagnostic tests for pheochromocytoma. Moreover, VHL patients were characterized by almost exclusively elevated levels of normetanephrine only, whereas MEN 2 patients had elevated levels of metanephrine only [56]. Preoperative screening to exclude asymptomatic pheochromocytoma is warranted in every VHL patient undergoing surgery. Other VHL Disease Manifestations Pancreatic lesions may also be a sign of VHL disease, albeit causing less morbidity and mortality than hemangioblastomas and RCC. The most common lesions are pancreatic cysts, microcystic serous adenomas (cystadenomas) and islet cell tumors [57–59]. Their estimated frequency varies from 10 to 60% among various series, depending primarily on the mode of detection (clinical, radiological, pathological) [8, 60]. Simple cysts, ranging from a few millimeters to more than 10 cm in size, may occur throughout the pancreas without predilection for a specific site. Serous cystadenomas are benign clusters of grape-like multicystic lesions, and are usually asymptomatic [17, 61]. Rarely, excessive local growth of cysts or cystadenomas may cause biliary duct obstruction, pancreatitis and, in rare cases, exocrine and endocrine pancreatic insufficiency due to cystic replacement of the parenchyma [61]. VHL patients do not appear to be at risk for classic adenocarcinoma of the pancreas [17]. Islet cell tumors are unrelated to pancreatic cystic disease. Islet cells are of neural crest origin and occur more frequently in patients with pheochromocytoma [60]. In most cases, these tumors grow slowly and remain asymptomatic. More rarely, though, they may exhibit more aggressive biology, manifested by rapid growth and metastasis coupled with the secretion of biologically active peptides, such as vasoactive intestinal peptide, calcitonin, insulin, glucagon, gastrin or somatostatin [17]. In the lat-

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ter case, the type of symptoms depends on the endocrine function of the hypersecreted peptide. Endolymphatic sac tumors are nonmetastatic, locally invasive, papillary adenocarcinomas. They arise from an ectodermal extension of the membranous labyrinth of the internal ear, called the endolymphatic sac. Early symptoms consist of gradual onset of decreased auditory acuity, tinnitus and vertigo [62]. Cases of acute onset have been reported. Progressive tumor growth and invasion of local structures may, if left untreated, result in facial paresis and anesthesia, vocal cord paralysis manifesting as hoarseness and sternocleidomastoid muscle atrophy [63]. Prospective audiologic evaluation of VHL patients at the NCI revealed that 65% of them had pure tone threshold abnormalities, which were bilateral in half of the cases [62]. It is possible that early auditory abnormalities herald a microscopic development of endolymphatic sac tumor, although many VHL patients have hearing loss without developing a radiographically detectable endolymphatic sac tumor, at least at the time of examination. Adnexal papillary cystadenomas of mesonephric origin have recently been identified as part of VHL disease. They derive from the anatomical structures developmentally related to the embryonic mesonephric duct. Up to 60% of male VHL patients develop papillary cystadenomas of the ductus deferens or the epididymis [64]. These are nonmetastatic tumors ranging in size from 1 to 5 cm. They are mostly asymptomatic unless they inflame, rupture or cause infertility in the case of bilateral tumors. Simple epididymal cysts are common in the general population and do not raise suspicion of VHL disease. Papillary cystadenomas, however, are rare in the general population and, if bilateral, are considered almost diagnostic for VHL disease. Tumors may also develop at any site along the mesonephric duct, in the adnexal area of female patients [65, 66]. The incidence of this entity is still unknown.

The VHL Gene Structure and Expression of the VHL Gene Using genetic linkage analysis, the VHL gene was mapped to human chromosome band 3p25 [67–69]. A consortium of investigators led by Drs. Marston Linenhan, Berton Zbar, Michael Lerman and Eamon Maher cloned the gene in 1993 [70]. The gene consists of three exons and generates two mRNA species: the larger (4.6 kb) corresponds to exons 1, 2 and 3, and the smaller to an alternative spliced form comprised of exons 1 and 3 only [71]. The VHL promoter contains several putative transcription factor binding sites, namely Sp1, AP-2, PAX, nuclear respiratory factor-1 and retinoic acid receptor, but not TATA or CCAAT boxes [72]. Transcription is initiated around a putative Sp1 site located 60 bp

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upstream of the first AUG codon in the VHL mRNA. The 3.6-kb full-length 3) UTR of the VHL gene has also been isolated and found to be rich in Alu repeat elements [73]. The VHL gene is expressed in every adult human tissue examined so far and in all three germ layers during human and mouse embryonic development [74– 76]. In adults, the VHL gene is strongly expressed in tissues which develop VHLtype tumors (such as the renal proximal tubular epithelium and the CNS), as well as in tissues not known to be targets of tumor development in VHL disease, such as the testis, lung and liver [75, 77]. The mouse and rat VHL genes have high homology to the human gene [78, 79]. VHL homologs have also been identified in Caenorhabditis elegans [80], Drosophila [81] and sea urchin [70]. No clear yeast homologs have been reported so far. VHL Gene Mutations in Familial and Sporadic Tumors Genetic analysis of the VHL gene indicates that both alleles are inactivated in VHL-associated tumors, suggesting that VHL acts as a tumor suppressor gene [82]. Germline mutations of one VHL allele can be detected in almost 100% of the patients presenting with VHL disease, defined by clinical criteria [83]. VHL-associated hemangioblastomas [84], RCCs [82] and islet cell tumors of the pancreas [60] show loss of heterozygosity (LOH) in the VHL locus, resulting from deletion of the wild-type allele. Lubensky et al. [85] and Zhuang et al. [86] reported such a biallelic inactivation in the endothelial cells lining not only atypical renal cysts from VHL patients but also what are pathologically defined as benign renal cysts. These findings indicate that biallelic VHL inactivation occurs early during tumor development, similarly to APC gene inactivation in the early stages of colon cancer [87]. Both VHL alleles are also frequently inactivated in the sporadic counterparts of VHL-associated tumors; 50–60% of sporadic, clear cell RCCs manifest LOH in the VHL locus with simultaneous mutational inactivation of the remaining allele [71, 88–90]. Inactivation of the VHL gene is specific for the clear cell type; no LOH and/or mutations were detected in papillary RCC, a histological type not encountered in VHL families. In an additional 25% of sporadic clear cell RCCs, LOH at the VHL locus was combined with silencing of the remaining VHL gene by methylation [91]. In total, both copies of the VHL gene are inactivated in 75% of sporadic clear cell RCCs. Mutational inactivation of both VHL alleles was also found in 50–60% of sporadic hemangioblastomas [92–95]. Silencing of the remaining VHL allele by hypermethylation has been reported so far in VHL-associated but not sporadic hemangioblastomas [96]. Lastly, in one study, LOH was reported in 7 out of 10 sporadic pancreatic papillary cystadenomas [97]. In contrast to the VHL inactivation described above, no clear LOH or silencing by methylation has been reported in VHL-associated pheochromocytomas.

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Furthermore, it appears that mutational inactivation of VHL occurs with low frequency in sporadic pheochromocytomas. So far, only two documented cases have been reported [49, 98]. However, the frequency of VHL methylation in sporadic pheochromocytomas has not been examined in a large number of such tumors. Interestingly, while VHL has been shown to play a prominent role in VHL pheochromocytoma, sporadic disease seems, in contrast, to be associated with genetic defects in the 1p and 22q [99]. Taken together, these observations raise the possibility that VHL genetics with regards to sporadic or VHL-associated pheochromocytomas may differ from those of RCC, hemangioblastomas or pancreatic cystadenomas. There are differences between the type and distribution of VHL mutations encountered in familial and sporadic tumors. Approximately 60% of all germline VHL mutations include large deletions, microdeletions (1–18 bp), microinsertions (1–8 bp) and nonsense point mutations. The rest (40%) are missense mutations [44, 100–106]. Almost all intragenic germline point mutations reported so far map downstream of the codon corresponding to amino acid Met 54 and they exhibit a bimodal distribution: they cluster at the 3) end of exon 1 and the 5) end of exon 3. In comparison, VHL mutations in sporadic RCC are more frequently clustered in exon 2 and affect slightly different codons than do germline mutations [71]. One possible explanation links this different mutational pattern to exposure to various renal carcinogens. Brauch et al. [107] reported on VHL mutations identified in clear RCC from patients significantly exposed to the chemical carcinogen trichloroethylene; tumor specimens in 33 out of 44 RCC patients harbored LOH at the VHL locus along with mutation of the remaining VHL allele. There is a correlation between the type of germline mutations and the clinical phenotype of VHL disease. More than 96% of type II patients have missense-only mutations, whereas large deletions, microdeletions/microinsertions and nonsense mutations correlate with type I disease [44, 45, 102, 103, 106]. Missense mutations frequently associated with various subtypes of type II disease are presented in table 1. A possible explanation of this genotype-phenotype correlation emerged from studying the crystal structure of the VHL protein (pVHL) (for details, see pVHL Is the Receptor of a Ubiquitin Ligase (E3) Multiprotein Complex below) [108]. Point mutations corresponding to type I disease frequently map to the hydrophobic core of the protein and they predict a complete unraveling of the pVHL structure with a consequent total loss of its function. In contrast, most missense mutations corresponding to type II disease predict mostly local defects in the protein structure, likely affecting protein-protein interaction [108]. This observation, along with a paucity of gross deletion and insertion mutations in type II disease, indicates that the development of pheochromocytoma may involve some dominant negative function of the mutant pVHL or necessitate some residual pVHL function required for the viability of the pheochromocytoma precursor cell [108].

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Table 1. Genotype-phenotype correlations Classification

Phenotype

Germline mutations

Type I

H + RCC

Type II IIA IIB IIC

PHEO + H + RCC (low) PHEO + H + RCC (high) PHEO only

46% deletions 10% nonsense 44% missense N78H/S/T L184P/R S111R S80R/I 96% missense Y98H Y112H V74G R161G R167W/Q/G L178P/Q/V L188V G114S F119S V84L V166F S68W

PHEO = Occurrence of pheochromocytoma; RCC = occurrence of RCC with (low) or (high) frequency; H = occurrence of hemangioblastoma. Frequent germline missense mutations corresponding to either type I or to each type II disease subtype are listed.

No LOH and/or VHL mutations have been detected in tumors other than the ones encountered as part of VHL disease or their sporadic counterparts [71]. This is even the case for tumors suspected of harboring inactivation of a putative tumor suppressor gene located in chromosome 3p, such as small cell lung cancer, squamous cancer of the head and neck and esophageal tumors [109–111].

Functional Analysis of pVHL Identification of Endogenous pVHL Isoforms The human VHL gene encodes two isoforms (fig. 1). The longest isoform comprises 213 amino acids and is encoded by the entire putative open reading frame of the gene. This isoform migrates with an apparent molecular weight of 28–30 kD in SDS-PAGE gels, and is thus termed pVHL30 [78, 112]. The shorter isoform migrates with a molecular weight corresponding to 19 kD (pVHL19). Pulse chase experiments and mutational analysis suggest that pVHL19 is the product of internal translation initiated at methionine 54 [113–115]. Both isoforms are simultaneously present in every cell line expressing wild-type pVHL and both are biologically active in functional studies presented below. It appears that the inactivation of both isoforms is necessary for tumor formation, since almost all known tumor-associated point mutations map downstream of methionine 54 (fig. 1). In the remainder of this chapter, we will collectively refer to both isoforms as pVHL, unless stated otherwise. Immunoprecipitation of cellular extracts followed by Western blot analysis with monoclonal anti-VHL antibodies

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Fig. 1. Structure of the VHL gene and its products. a The frequency of germline VHL gene mutations. The arrows indicate the missense mutations, the vertical bars show the frameshift mutations and the dots represent the stop codons. D = Microdeletions. The shaded area represents exon 2. Amino acid numbers define exon borders. b The crystal structure of pVHL isoforms. The · and ß domains are indicated in gray and green, respectively. The yellow box indicates the ‘BC box’ within the · domain. c A high degree of homology exists between the human, mouse and rat VHL genes. The percentages indicate the degree of amino acid homology with the corresponding area of the human gene.

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indicates that additional pVHL isoforms may exist [116]. In addition, Northern blot analysis of several cell lines detected the presence of an alternatively spliced VHL mRNA, consisting of exons 1 and 3 [71], but no endogenous pVHL isoform assigned to this alternatively spliced mRNA has been reported yet. Several techniques have been employed to study the cellular localization of endogenous and ectopically expressed pVHL: (1) immunocytochemistry of human normal and neoplastic tissue; (2) biochemical fractionation and immunofluorescence of cells growing exponentially in culture, and (3) ectopic expression of pVHL fused to green fluorescent protein. Despite the limitations and critical pitfalls that any of these techniques may harbor, they consistently indicate that pVHL resides primarily in the cytoplasm and, to a lesser extent, in the nucleus [77, 112]. In the cytoplasm, pVHL30 has been detected in the soluble cytosolic fraction as well as the fraction enriched for membranes, including the Golgi apparatus, endoplasmic reticulum (ER) and mitochondria [112]. pVHL19 is detectable in the cytosol, but not, at least measurably, in the membranous fractions [113, 114]. This pattern of cellular distribution is, at least in part, the steady state of a continuous nucleocytoplasmic shuttling [117]. Lee et al. [117] observed, in a series of elegant studies, that pVHL shuttles between the nucleus and the cytoplasm. In vitro and in vivo studies have indicated that the nuclear export of pVHL is an active process requiring RNA polymerase II-mediated transcription, the formation of polyadenylated mRNA and ATP hydrolysis. Nuclear export requires the presence of the GTP-binding protein Ran [118]. pVHL nucleocytoplasmic shuttling depends on protein sequences included in the second exon [117]. The physiologic stimuli that regulate this ‘shuttling’ are currently unknown. The potential importance of these observations is underscored by the fact that disruption of pVHL nucleocytoplasmic shuttling (by mutations within the second exon or fusion of pVHL to nuclear export signals that would ‘stably’ localize pVHL in the cytoplasm and therefore disrupt the shuttling) results in loss of its function [117] with regards to regulating hypoxia-inducible genes (see Physiology of the VHL–/– Cell below). In asynchronously growing cells, pVHL30, but not pVHL19, is phosphorylated on serine residues. The sites of phosphorylation, the relevant kinase(s) and the functional consequences of pVHL phosphorylation are currently unknown [Iliopoulos and Kaelin, unpubl. observations]. pVHL Functions as a Gatekeeper Tumor Suppressor Protein Molecular studies in human colon cancer led to the notion that the formation of a fully malignant phenotype is an orderly multistage process. A sequence of mutations which is unique for every tumor type leads to the formation of a malignant clone with growth advantage over nonmalignant cells [119]. The earliest among these mutations is the inactivation of the so-called ‘gatekeeper’ tumor sup-

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pressor genes, such as APC for colon cancer, WT-1 for Wilms’ tumor and Rb for retinoblastoma. ‘Gatekeeper’ tumor suppressor genes directly control cell growth, and reconstitution of the activity of these tumor suppressor genes in the cell that lost their function leads to suppression of the malignant phenotype. In contrast, reconstitution of gene functions lost in subsequent steps of malignant transformation may not be sufficient to surmount the effect of accumulated mutations and suppress tumor formation [120]. Such tumor suppressor genes may survey the genome stability, with MSH2 and MLH1 providing classic examples; they are called ‘caretaker’ genes. Restoration of a ‘caretaker’ function to a cancer cell will not inhibit its growth. Clinical and genetic evidence indicates that VHL is a tumor suppressor gene inactivated at an early stage of RCC formation. Several investigators have studied the effect of reintroduction of pVHL in VHL–/– epithelial cell lines derived from clear cell RCC. They generated either clones stably expressing wild-type or naturally occurring mutants of pVHL or they created polyclonal cell populations by retroviral infection of VHL–/– cell lines with the corresponding constructs. With the exception of one report [121], reintroduction of wild-type pVHL did not alter the ability of these cell lines to proliferate in vitro, to arrest at the same cell density or to form colonies in soft agar compared to their VHL–/– counterparts. In contrast to these in vitro studies, wild-type but not mutant pVHL drastically suppressed the ability of these cell lines to form tumors when injected subcutaneously into nude mice [112, 114, 122]. These initial observations led to the hypothesis that pVHL may induce tumor suppression in a nonautonomous manner, through mechanisms regulating cell to cell or cell to extracellular matrix interactions and/ or tumor angiogenesis [112, 116]. Subsequent to these observations, Pause et al. [123] revisited the in vitro effect of pVHL on cellular proliferation. When cultured strictly at a certain density, RCC clones expressing wild-type pVHL exited the cell cycle upon serum withdrawal, while their VHL–/– counterparts continued to cycle. The critical importance of cell density in this phenomenon is unclear, and although it presents an experimental restriction, it may underscore the importance of cell to microenvironment communication in the mechanism of tumor suppression by pVHL. The experiments described above indicate that VHL is a gatekeeper tumor suppressor gene. The observation that reconstitution of the function of pVHL in an established VHL–/– cell line leads to tumor suppression has obvious therapeutic potential. pVHL Is the Receptor of a Ubiquitin Ligase (E3) Multiprotein Complex Analysis of the primary sequence of pVHL showed no homology to other known proteins and therefore revealed no indication regarding its potential biochemical function. To gain insight into such function(s), several groups have

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sought to identify cellular proteins stably interacting with pVHL. The first proteins reported to form stable endogenous complexes with pVHL were the Elongins C and B [124–126]. Elongins C, B and A were initially cloned, by Garrett et al. [127, 128] and Conaway et al. [129], as subunits of a nuclear tripartide enzymatic complex (Elongins/SIII) that enhances the rate of in vitro RNA polymerase II-mediated transcription (elongation) off artificial DNA templates, thus their names. This in vitro enzymatic activity resides on Elongin A and is increased by the presence of Elongins C and B [124, 130]. pVHL binds directly to Elongin C, and this interaction is further stabilized by the binding of Elongin B to Elongin C [124, 125]. The domains of Elongin C and Elongin A required for mutual interaction and in vitro promotion of transcriptional elongation have been mapped [130, 131]. The Elongin C-binding domain of pVHL was initially mapped to amino acids 157–172, a ‘hot spot’ for naturally occurring, tumor-associated mutations, supporting the notion that Elongin binding is connected to the ability of pVHL to act as a tumor suppressor [126]. pVHL amino acids 157–172 contain an ElonginC/B-binding motif (initially termed ‘BC box’), also present in Elongin A: (T,S)Lxxx(C,S) xxxV(L,I) [130, 132]. Binding of Elongin C to Elongin A or pVHL appears to be mutually exclusive [133]. This series of initial observations led to the hypothesis that pVHL competes with Elongin A with regards to its interaction with Elongins C/B, and by doing so, pVHL regulates the transcriptional elongation of yet unidentified cellular genes. This concept motivated a quest for such genes and, along with a series of clinical observations, led to the connection of pVHL function with the regulation of hypoxia-inducible genes (see below, Physiology of the VHL–/– Cell). Subsequent observations, however, challenged the ‘competition’ model: (1) Elongins C/B appear to be in excess of pVHL and Elongin A [134], rendering the ‘competition’ model improbable, unless posttranslational modifications specify a subset of pVHL-binding elongins, and (2) a significant fraction of Elongins C/B reside primarily in the cytoplasm, raising the possibility that they may play a role in processes other than transcriptional elongation [132, 133]. Finally, the identification of human Cullin-2 (Cul-2) and Rbx-1 as partners of the pVHL/ElonginsC/B complex facilitated the formation and direct testing of an alternative model for the function of this multiprotein complex. Cul-2 is one of the six members of the human Cullin proteins family, identified as orthologs of the C. elegans Cullin-1 [135]. The latter was cloned through a random mutagenesis screen as the gene that, when inactivated, blocks cell cycle exit and results in hyperplastic and elongated larvae [135]. Mammalian cullins and their yeast homologs serve as ‘docking’ proteins for the assembly of multiprotein complexes that function as ubiquitin ligases. Insights into the potential function of cullin-containing complexes were provided by the observation that human Cullin-1 (Cul-1) bears a high degree of homology to yeast cdc53 [135]. In Saccha-

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romyces cerevisiae, cdc53 serves as a scaffold protein for the assembly of SCF complexes (for Skip-1, Cdc53/cullin and F-box receptor). The reader is referred to a recent review for detailed studies on the structure and function of yeast and mammalian SCF complexes [136]. In brief, the yeast SCF multiprotein complexes contain Skp1p, directly interacting with the N-terminus of Cdc53, Hrtp1 bound to a midregion of cdc53 highly conserved across species and referred to as ‘the cullin homology domain’ (CHD), and the ubiquitin conjugase cdc34, recruited to the complex through interaction with Hrtp1 (fig. 2a ). Skp1p bridges the complex to one of the F-box-containing proteins. The latter are polypeptides with two or more domains mediating protein-protein interaction. The F-box (so named after its initial identification in F-cyclins) mediates direct interaction with Skp1. A second domain, different in each protein, mediates the direct interaction with a ‘substrate’ protein. Substrate proteins contacting the complex undergo ubiquitination and destruction by the proteasome. Thus, it appears that F-box proteins function as substrate receptors, conferring specificity to the SCF complex with regards to substrate selection. SCF complexes containing the F-box protein Grr1 bind and degrade yeast G1 cyclin cln2, whereas complexes containing the F-box receptor Cdc4 bind and degrade, among other substrates, the clb5/cdc28 inhibitor p40/sic1. Orderly activation of these yeast complexes is necessary for passage from G1 to the S phase of the cell cycle [137, 138 and reviewed in ref. 139]. A structurally similar multiprotein complex (anaphase-promoting complex; APC) guides the progression through mitosis [140]. One of its subunits, APC2, bears C-terminal homology to Cdc53 and is regarded as the equivalent E3 scaffold ligase. In mammalian cells, at least cullins-1, -2 and -3 appear to participate in the formation of similar SCF complexes with E3 ubiquitin ligase activity. Human cullin-1 binds to SKP1 through its N-terminus [141, 142] and to Rbx-1/ROC1, the human orthologs of Hrtp1, through its CHD (fig. 2b) [143, 144]. Rbx-1 recruits the mammalian Cdc34 ubiquitin ligase to the complex. SKP1 binds to either of the F-box substrate receptors SKP2 or Trcp. SKP2, through its leucinerich region domain, binds to either E2F1, p27, p21 or Cyclin D and recruits them to the complex (fig. 2b), where they undergo ubiquitination and subsequently proteosomal degradation [145–147]. By doing so, SKP2-containing SCF complexes (SCFSKP2) regulate critical steps in cell cycle entry and progression through the G1 phase. Overexpression of SKP2 leads to cell cycle arrest [148]. Through its second substrate receptor Trcp, the SCFTrcp complex has the ability to bind and degrade ß-Catenin or IÎB, thus regulating critical transcription factors implicated in cellular responses to cell adhesion and inflammatory stimuli [143, 149, 150]. Structure-function analysis of these mammalian SCF complexes and studies of the molecular events that temporally and spatially regulate their activation are currently emerging. We provide below three examples of such studies. Firstly, in

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the case of E2F1, p27 or IÎB, it appears that phosphorylation of the substrate at specific residue(s) regulates its recognition by the SCF receptor and therefore leads to proteasomal degradation and instability of these substrates. It is still unknown if and how the substrates compete for recognition by the same SCF complex or if posttranslational modifications other than phosphorylation are critical for this recognition. Secondly, experiments reconstituting in vivo the ubiquitination of a substrate by recombinant proteins of its cognate SCF complex helped to gain insights into the regulation of SCF complex activity. The N-terminus and the CHD of cullin-1 recruit the substrate and the Rbx-1/ubiquitin conjugase ubiquitin ligase correspondingly, and they were both shown to be necessary for ubiquitination of the substrate [151]. Similar structural requirements are also met in the case of yeast Cdc53 [136]. Thirdly, it is now appreciated that yeast cdc53 and all mammalian cullins are modified in vivo by conjugation to yeast Rub-1 or its mammalian ortholog Nedd8. These molecules belong to the growing family of ubiquitin-like proteins by virtue of their N-terminus homology to ubiquitin [152– 154]. Neddylation (conjugation to Nedd8) of Cul-1 enhances its function in vitro and may be required for SCF complex localization in vivo [155–157]. In conclusion, it is likely that the temporal and spatial regulation of SCF functions may be regulated at several critical levels. Recent experiments have provided compelling evidence that pVHL functions as a substrate receptor of an SCF complex containing Cul-2 and Elongins C and B (SCFVHL) (fig. 2c). Mammalian Cul-2 has homology to Cul-1, especially at the CHD [135]. Elongin C has homology to SKP1 [142, 158] and binds to the N-terminus of Cul-2. Rbx-1 binds to the CHD of both Cul-2 and Cul-1 [159, 160]. The subsequent resolution of the crystal structure of pVHL in complex with Elongins C and B, by the Pavlevitch group [108], provided strong evidence that pVHL may function as the substrate receptor of SCFVHL. Two major domains of pVHL form molecular patches available for protein-protein interaction. The alpha domain (residues 193–204) consists of three alpha helices (H1, H2 and H3) forming a groove. A fourth helix from Elongin C enters this groove and makes multiple contacts with critical residues mediating the binding of pVHL to Elongin C. The

Fig. 2. Structure of yeast and mammalian SCF complexes. a Structure of the yeast Cdc53-based SCFs. A fraction of Cdc53 conjugates with the ubiquitin-like molecule Rub-1 (R). Cdc34 transfers ubiquitin (U) to the substrates, such as Cln2 and Sic1. Skp1 interacts directly with the F-box (F) of the substrate receptors. b Structure of mammalian Cullin1-based SCFs. A fraction of human Cullin-2 conjugates with the ubiquitin-like molecule Nedd8 (N8). Human Cdc34 transfers ubiquitin (U) to the substrates, such as E2F1 and IÎB. c Structure of the mammalian Cullin-2-based SCFs. A fraction of human Cullin-2 is modified by Nedd8. The yellow box in pVHL and SOCS-1 corresponds to the ‘BC box’. The · and ß domains of pVHL are marked.

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previously identified ‘BC box’ motif of pVHL resides within helix H1 and mediates extensive contact with Elongin C. The beta domain of pVHL (residues 63–154 and 192–294) consists of seven beta strands packed together in a ‘sandwich’ forming a hydrophobic core. The outer surface of the beta domain forms a patch available for protein-protein interactions. Elongin B binds to Elongin C and appears to stabilize the complex. The overall structure of the complex resembles the crystal structure of SKP1/SKP2 encountered in the SCFSKP2 [108]. In keeping with this model, the Elongin C-binding area of pVHL (‘BC box’) presents loose homology to the F-box [161]. It was thus hypothesized that the ß domain of pVHL mediates contact with the substrate(s) to be ubiquitinated by SCFVHL. Biochemical experiments have further supported the hypothesis that pVHL is part of an E3 ubiquitin ligase complex. Cellular immunoprecipitates of ectopically expressed wild-type pVHL manifested E3 ligase activity when reconstituted in appropriate buffer and supplemented with ubiquitin, the ubiquitin-activating enzyme E1 and the E2 ubiquitin conjugase Ubc5 [161]. This in vitro activity could be enhanced by the addition of exogenous Rbx-1. In contrast, immunoprecipitates of pVHL mutants unable to enter ElonginC/B/Cul-2 complexes contained no ligase activity [161]. Further studies on the role of pVHL as a member of a ubiquitin protein ligase were made feasible by the identification of the first substrate of the SCFVHL complex. Substrates of SCFVHL Ubiquitin Ligase The notion that pVHL is the receptor of an E3 ubiquitin ligase complex was consolidated by the recent identification of hypoxia-inducible factor 1-alpha (HIF1·) and 2-alpha (HIF2·) as the first substrates specifically recognized and ubiquitinated by SCFVHL [162]. HIF1· is the prototypic member of a family of basic helix-loop-helix-PAScontaining cellular transcription factors [163]. The HIF1 complex was purified by Wang and Semenza [164] using the hypoxia-responsive enhancer of erythropoietin gene in DNA-affinity chromatography. Under normoxic conditions (21% ambient O2 tension), cellular HIF1· is virtually undetectable due to its ubiquitination and rapid degradation by the proteasome (half-life !5 min). When cells are exposed to hypoxia (0.5–2% ambient oxygen tension), HIF1· becomes stabilized and is detected in the nucleus within 30 min. The oxygen-dependent degradation domain of HIF1· was initially mapped to amino acids 401–603 [165, 166]. Recent analysis showed that amino acids 557–571 are necessary and sufficient for conferring stabilization by hypoxia [167]. Stabilized HIF1· heterodimerizes with constitutively expressed HIF1ß (Arnt), binds to specific promoter sequences, termed hypoxia-responsive elements, and transactivates a family of HIF1-inducible genes [168]. The DNA binding, heterodimerization and transactivation functions of HIF1· were mapped to distinct domains [163]. A series of observations

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indicate that HIF1 activity may be implicated in tumor establishment and growth: (1) HIF1· is overexpressed in a variety of human tumors, as studied by immunocytochemistry, but not in the normal corresponding tissue [169]; (2) HIF1·–/– mice embryonic cells exhibit reduced capacity to form teratocarcinoma tumors when injected into syngeneic animals, as compared to HIF1+/+ counterparts [170], and (3) wild-type p53 promotes HIF1· degradation and inhibits HIF1·-mediated transcription [171, 172]. Maxwell et al. [162] showed that HIF1· enters complexes containing pVHL, and they drew several parallels between the effect of hypoxia on HIF1· and the absence of pVHL function; they observed that (1) VHL–/– cells contain a constitutively stabilized HIF1· (or HIF2·) and markedly increased HIF1 promoter activity, (2) pVHL coimmunoprecipitates with HIF1·/2· in hypoxic cell extracts and is found in the HIF1 DNA-binding complex, and (3) hypoxia mimetics, such as iron chelators or cobaltous irons, disrupt complex formation between pVHL and HIF1·, leading to HIF1· stabilization. Recent in vitro studies further support these observations and provide mechanistic insights into how pVHL regulates HIF1·/2· stability; in vitro ubiquitination of HIF1· is supported by S100 cellular extracts obtained from VHL+/+ cells, whereas extracts from isogenic cell lines expressing mutant or no pVHL do not [173, 174]. In vitro reconstitution of the VEC complex by coexpression of recombinant pVHL, Elongin C, Elongin B, Cullin-2 and Rbx-1 is sufficient for HIF1· ubiquitination in the presence of ubiquitin, E1 and ATP [175]. The pVHL-HIF1·/2· interaction occurs directly between the · domain of pVHL and the oxygen-dependent degradation domain of HIF1·/2· [173, 174]. This interaction is abrogated by tumor-derived VHL mutations of the ß domain. These observations confirmed the role of pVHL as the receptor of the SCFVHL complex and provided, at least in part, a mechanistic link between loss of pVHL function and deregulation of hypoxia-inducible genes (see Physiology of the VHL–/– Cell below). Overexpressed pVHL has also been reported to form complexes with the atypical protein kinase C (PKC) forms PKC˙, PKCÏ and PKC‰, as well as the Sp1 transcription factor [176, 177]. PKCs are involved in cellular processes such as proliferation, transformation, differentiation and apoptosis. Sp1 transactivates, among others, the vascular endothelial growth factor gene. Binding of pVHL to PKC˙ inhibits the association of the latter with Sp1 and the phosphorylation of Sp1, leading to decreased Sp1 activity [178, 179]. Okuda et al. [177] showed that the ß domain of pVHL interacts with PKC˙ and Ï. The interaction of pVHL with atypical PKCs or Sp1 is not interrupted by the tumor-associated mutations tested so far. These molecules may serve as substrates of SCFVHL-mediated ubiquitination, although their expression in VHL–/– and VHL+/+ cell lines, as well as their potentially pVHL-dependent ubiquitination in vitro or in vivo is currently unknown.

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As is the case for Cul-1-containing SCF complexes, Cul-2/Elongin C complexes use alternate substrate receptors, other than pVHL, for substrate selection. The ‘BC box’ motif, through which Elongin C binds to pVHL, is also present in the suppressor of cytokine signaling (SOCS) superfamily of proteins [132]. Members of this superfamily are therefore candidate receptors for substrate proteins targeted for ubiquitination in a non-VHL-dependent manner. SCF complexes containing SOCS-1/Elongin C/B/Cul-2 (SCFSOCS-1) were shown to regulate the stability of vav-1 [180]. Physiology of the VHL–/– Cell pVHL Inactivation Results in Inappropriate Overexpression of Hypoxia-Inducible Proteins. The initial implication of pVHL in transcriptional elongation stimulated an interest in identifying genes differentially expressed between VHL+/+ and VHL–/– cells. A series of observations shaped this initial quest: (1) VHLassociated or sporadic RCC and hemangioblastoma are hypervascular and overexpress the angiogenic polypeptide vascular endothelial growth factor [181–183]; (2) a subset of patients with RCC, hemangioblastoma or pheochromocytoma develop paraneoplastic erythrocytosis due to inappropriate secretion of erythropoietin by the tumor cells [26, 184, 185], and (3) cellular assays of pVHL function (see pVHL Functions as a Gatekeeper Tumor Suppressor Protein above) supported the notion that pVHL may mediate tumor suppression by modulating angiogenesis or cell to cell/extracellular matrix communication. These observations raised the hypothesis that pVHL is implicated in biochemical pathways sensing ambient oxygen tension and regulating polypeptides induced by hypoxia. Exposure of a normal cell to hypoxia (1% ambient oxygen tension) results in marked alteration, direct or indirect, of a series of intracellular and secreted proteins [186, 187]. These hypoxia-inducible proteins have diverse functions aimed at restoring cellular and tissue oxygenation while adapting the cellular behavior to the temporary hypoxic environment. To this end, hypoxia-inducible proteins promote angiogenesis directly by stimulating endothelial cell proliferation (e.g. vascular endothelial growth factor, platelet-derived growth factor) or indirectly by remodeling the extracellular matrix [transforming growth factor (TGF)-ß, metalloproteinases], altering entry into and progression through the cell cycle (e.g. p53) and shifting cell metabolism to energy-conserving modes (e.g. Glut-1, several glycolytic enzymes). In most cases, hypoxia leads to marked elevation of these polypeptides. The mechanism(s) underlying these changes appear to involve transcriptional activation [188], marked posttranscriptional stabilization of mRNA [189] and posttranslational protein modification. A typical and most studied example of posttranscriptional protein modification is the stabilization of the transcription factor HIF1, which binds to the promoter of and transactivates several of the hypoxiainducible proteins [163, 190], as well as the stabilization of p53 [191].

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Several groups have showed that VHL–/– cells inappropriately overexpress hypoxia-inducible proteins under normoxic conditions. The reintroduction of wild-type pVHL into these cells downregulates the expression of the hypoxiainducible genes under normoxic conditions without affecting their normal upregulation by hypoxia; thus, it restores the normal cellular response to ambient oxygen tension [122, 192, 193]. Regulation of hypoxia-inducible gene mRNAs occurs at the transcriptional and posttranscriptional level. Transcriptional regulation involves transactivation by HIF1 [194]. Posttranscriptional regulation of mRNA stability is a complex process involving several mRNA-binding proteins that facilitate cytoplasmic mRNA transport, poly A tail shortening, uncapping and nuclease digestion [195, 196]. The effect of pVHL on the hypoxia-inducible peptides appears to have a strong posttranscriptional component [122, 192, 197, 198] and depends on the ability of pVHL to enter the SCFVHL complex. pVHL mutants unable to bind Elongin C and, therefore, to serve as a substrate receptor of the SCFVHL ligase activity fail to inhibit the expression of hypoxia-inducible genes [158]. It is therefore possible that critical mRNA-binding and -stabilizing proteins, still to be identified, are substrates degraded by SCFVHL. Degradation of these proteins by pVHL may account for the posttranslational component of hypoxia-inducible mRNA inhibition, while the degradation of HIF1 may mediate the effect of pVHL on the transcription of these mRNAs. Alternatively, HIF1 may directly or indirectly positively regulate the levels of mRNA-stabilizing proteins, and, therefore, HIF1 regulation by pVHL may alone account for both the transcriptional and posttranscriptional regulation of hypoxia-inducible genes. A list of the hypoxia-inducible polypeptides regulated by pVHL is included, along with other pVHL-regulated genes, in table 2. Several of these polypeptides may not only serve the cellular adaptation to a hypoxic environment, but they may also have direct oncogenic potential. Examples include platelet-derived growth factor-B, a cellular proto-oncogene, TGF· [199], a ligand of the tyrosine kinase EGF receptor, and TGFß [200]. The latter is a growth factor reported to have pro- and antiproliferative functions [201]. With regard to renal epithelial cells, it appears that TGFß promotes RCC tumorigenicity in vivo through a paracrine mechanism [200]. Systemic administration of anti-TGFß antibodies inhibits tumor formation by VHL–/– cells in the mouse xenograft assay [200]. It is therefore possible that the ‘hypoxia mimicry’ provided by pVHL inactivation is sufficient to set in motion a cellular program ultimately leading to tumorigenesis. VHL–/– Cells Have Impaired Extracellular Milieu. The regulation of tumor angiogenesis is one example of cell-extracellular environment communication regulated by pVHL. Additional examples of how pVHL may influence aspects of the cell-extracellular environment communication are provided by studies on extracellular fibronectin matrix assembly and carbonic anhydrase activity ob-

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Table 2. Genes regulated by pVHL Gene VEGF PDGF-B GLUT-1 TGF-· TGF-ß AK-3 ALD-A PGK-1 PFK-L LDH-A CA 9 CA 12 MMP-2 MMP-9 TIMP-1 TIMP-2 uPA PAI-1

Expression vascular endothelial growth factor1 platelet-derived growth factor1 glucose transporter-11 transforming growth factor-· transforming growth factor-ß adenylate kinase 31 aldolase-A1 phosphoglyceratekinase 11 phosphofructokinase-L1 lactose dehydrogenase-A1 carbonic anhydrase 9 carbonic anhydrase 12 matrix metalloproteinase 22 matrix metalloproteinase 92 tissue inhibitor metalloproteinase 1 tissue inhibitor metalloproteinase 2 urokinase-type plasminogen activator plasminogen activator inhibitor1

D D D D D D D D D D D D D D I I I D

The expression of these genes is decreased (D) or increased (I) in VHL+/+ cells compared to VHL–/– cells, under culture conditions of 21% ambient oxygen tension. 1 Genes directly transactivated by HIF1. 2 Genes induced by hypoxia; direct transactivation by HIF unknown.

served in cells lacking normal pVHL function. VHL–/– and VHL+/+ cells produce and secrete comparable levels of extracellular fibronectin. In contrast to this, only VHL+/+ cells manifest the ability to support the formation of a mature extracellular fibronectin matrix [202]. This effect is specific for fibronectin, since other components of the extracellular matrix such as collagen and laminin seem to be unaffected [202]. The effect of pVHL on fibronectin may be directly linked to its tumor suppressor function; fibronectin has been shown to have antiproliferative and antimetastatic effects [203–205], and specific reorganization of the extracellular matrix is required for tumor neoangiogenesis [206]. The mechanism(s) by which pVHL regulates fibronectin assembly is under investigation. VHL–/– cells possibly secrete immature/misfolded fibronectin that fails to assemble into extracellular matrix. This is an attractive hypothesis given that a fraction of pVHL30

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colocalizes with fibronectin in the ER [202]. Cellular proteins targeted for secretion enter the ER and fold into their mature state with the help of the endoluminal chaperons. The accuracy and completion of this process is actively surveyed in the ER. Unfolded or misfolded proteins are specifically recognized and actively exported in the cytosol, ubiquitinated and degraded by the proteasome [207, 208]. Accumulation of misfolded proteins in the ER may be the result of environmental stress stimuli and, in turn, may signal to the nucleus and cytosol in order to activate a gene expression program aimed at correcting or adapting to the environmental stress [209]. It is therefore possible that pVHL – as a ubiquitin ligase receptor – participates in this surveillance mechanism, and that lack of pVHL function may lead to the secretion of immature proteins such as fibronectin. A non-mutually exclusive mechanism may invoke pVHL in the regulation of the expression and assembly of integrins at the cell surface; the latter directly contribute to extracellular fibronectin assembly [203, 210]. Evidence for a potential role of pVHL in an ER stress signaling pathway was provided by Gorospe et al. [211]. They showed that glucose deprivation induced greater cytotoxicity in VHL–/– cells than in their isogenic VHL+/+ counterparts, due primarily to the inability of VHL–/– cells to handle abnormally glycosylated and therefore misprocessed proteins. Chemical stimuli directly affecting protein folding and maturation had a much greater cytotoxic effect on VHL-deficient cells [211]. Inactivation of pVHL function also results in posttranscriptional mRNA stabilization and overexpression of the transmembrane proteins carbonic anhydrases 9 and 12 [212]. These enzymes regulate extracellular pH and the activity of certain cell membrane ion channels [213]. There is evidence that extracellular pH may affect the invasiveness and metastatic behavior of carcinoma cells [214]. Since pH and ion channel regulation may affect H20 distribution across cellular membranes, it could be conceivable that the regulation of carbonic anhydrases 9 and 12 may, at least in part, link pVHL inactivation to the cystic nature of VHLassociated lesions. VHL–/– Cells Exhibit a Less Differentiated Phenotype. Lack of differentiation is a cardinal feature of the neoplastic cell. Histological features of differentiated renal epithelial cells include basoapical polarization, basolateral desmosomal formation and tubulogenesis under certain culture conditions [215, 216]. The three-dimensional growth of cells as multicellular spheroids may recapitulate some aspects of cell to extracellular environment communication and the potentially resulting differentiation. Cultured under such conditions, the 786-O, VHL–/–, RCC cell line forms highly cohesive spheroids, with little intercellular space and no fibronectin deposition. Reintroduction of wild-type, but not mutant, pVHL in these VHL–/– cells induces some salient futures of differentiation; the grown spheroids are loose, they form a network of tubular and trabecular structures, they contain intercellular fibronectin and, when examined under electron

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microscopy, they present features of differentiation such as basolateral desmosomal junctions and apical villi [217]. Stimulation of various epithelial (including renal) cells with the mesenchymal cytokine hepatocyte growth factor/steel factor (HGF/SF), the ligand of c-met tyrosine kinase receptor, results in increased proliferation, dissociation/motility (‘scattering’) and invasiveness of the extracellular matrix, along with the induction of cell polarity and tubulogenesis [218]. In epithelial organs, this complex program results in the formation of tubular structures, known as ‘branching morphogenesis’, and has been connected to developmentally programmed organogenesis. These functional outcomes of c-met receptor activation can be genetically dissociable [219]. On the other hand, simple overexpression of oncogenic c-met confers an invasive and metastatic phenotype to tumor cells [220]. pVHL inhibits the invasive program triggered by HGF/SF. VHL–/– clones have been shown to exhibit marked branching morphogenesis in response to HGF/SF, while isogenic clones expressing wild-type pVHL did not respond [221]. The morphologic changes following HGF stimulation of VHL–/– cells correlated with increased secretion of matrix metalloproteinases 2 and 9 and reduction of tissue inhibitor of matrix metalloproteinases 1 and 2 [221]. It is possible that the effect of pVHL on signal transduction by c-met impinges on pathways specifically regulating the invasive and metastatic potential triggered by c-met than its effect on differentiation. Lastly, the expression of pVHL was studied during the induction of differentiation of rat pluripotential neural progenitor cells. When cultured under specific conditions, these progenitors may be induced to differentiate into cells with neuronal or glial features [222]. Undifferentiated progenitor cells appear to contain low levels of pVHL, as assessed by immunocytochemistry. Marked pVHL elevation correlates with acquisition of a neuronal, but not glial, phenotype [222]. Whether pVHL induction is causally linked to igniting a differentiation program is currently unknown. In summary, it appears that pVHL may be involved, directly or indirectly, in pathways executing a differentiation program. Whether the potential involvement of pVHL in differentiation is genetically dissociable from its ability to regulate hypoxia-inducible gene expression and fibronectin matrix assembly is presently not known.

Future Directions and Therapeutic Potential We have just begun to understand how pVHL-inactivating mutations may lead to tumor formation. The cloning of the VHL gene and the establishment of an extensive international database of germline mutations are expected, from the

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clinical standpoint, to facilitate (1) an appreciation of a broader clinical spectrum of the disease, (2) an early surveillance of individuals harboring germline mutations and at risk for developing the disease, and (3) a more accurate prediction of the expected clinical phenotype corresponding to specific mutations. A more comprehensive understanding of the mechanisms employed by pVHL for tumor suppression may lead to the rational design of therapeutic interventions based on specific molecular targets. Examples of such strategies may already be emerging, based on the knowledge that the absence of pVHL results in HIF1·/2· stabilization and inappropriate overexpression of hypoxia-inducible proteins. Small molecules aimed at inhibiting HIF1·/2· transcriptional activity, destabilizing the inappropriately overexpressed hypoxia-inducible mRNAs or blocking the cognate receptors of the secreted hypoxia-inducible polypeptides may prove useful in combating VHL-associated tumors. In addition, the involvement of patients with VHL-associated tumors in current clinical trials employing antiangiogenic agents may be a logical priority. Efforts towards the development of such rational therapies will be greatly enhanced by further understanding the molecular basis of VHL disease and answering significant questions such as: (1) Is inhibition of HIF1 transcriptional activity necessary and/or sufficient for tumor suppression by pVHL?; (2) Which substrates, other than HIF1, are targeted for degradation by pVHL, and which of these substrates are critical for tumor suppression?; (3) What is the molecular mechanism of hypoxia-inducible mRNA stabilization in VHL–/– cells?; (4) Which genes are differentially expressed between VHL–/– and VHL+/+ cells?, and (5) Which molecular events underlie the genotype-phenotype correlation of VHL disease? Mapping of the human genome and the exponentially growing supportive information technology are expected to greatly facilitate answering such questions.

Acknowledgement The author would like to thank Drs. Daniel Haber and Spyros Artavanis-Tsakonas for critically reading the chapter.

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Othon Iliopoulos, MD, Assistant Professor of Medicine Massachusetts General Hospital Cancer Center and Harvard Medical School Building 149, Room 7405, Street 13th, Charlestown, MA 02129 (USA) Tel. +1 617 724 3404, Fax +1 617 724 9648, E-Mail [email protected]

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Dahia PLM, Eng C (eds): Genetic Disorders of Endocrine Neoplasia. Front Horm Res. Basel, Karger, 2001, vol 28, pp 167–213

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Hamartoma and Lentiginosis Syndromes: Clinical and Molecular Aspects Deborah J. Marsh a, Constantine A. Stratakis b a

b

Cancer Genetics, Kolling Institute of Medical Research, Royal North Shore Hospital, and Sydney University, Sydney, Australia; Unit on Genetics and Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md., USA

Contents 168 170 170 170 171 175 179 181 181 183 183 184 185 185 186 186 186 188 188

Introduction Cowden Syndrome Clinical Features Mapping the CS Gene – A Historical Perspective PTEN Structure, a Pseudogene and the Homologues PTEN Signalling Germline PTEN Mutations in CS and Hereditary Associated Malignancies Genotype-Phenotype Correlations in CS PTEN in Sporadic Component Tumours of CS Bannayan-Riley-Ruvalcaba Syndrome Clinical Features PTEN, the CS Susceptibility Gene, Is Also Mutated in BRR CS and BRR – Different Presentations of the Same Syndrome Genotype-Phenotype Correlations in BRR Juvenile Polyposis Syndrome Clinical Features Mapping of Putative JPS Loci – A Historical Perspective SMAD4 (DPC4) – A Cytoplasmic Mediator in the TGF-ß Signalling Pathway SMAD4 in Sporadic Tumours

Both authors contributed equally to this work.

188 Peutz-Jeghers Syndrome 188 Clinical Features 189 Mapping the PJS Gene – A Historical Perspective 190 STK11 (LKB1) – A Serine-Threonine Kinase 191 STK11 in Sporadic Component Tumours of PJS 191 Carney Complex 191 Clinical Features 195 Identification of the CNC Genetic Loci 196 Germline PRKAR1A Mutations in CNC 197 Genetic Heterogeneity in CNC 198 The Related Syndromes – LEOPARD Syndrome, Proteus Syndrome and Cronkhite-Canada Syndrome 199 Summary 200 Acknowledgements 200 References

Introduction Cowden syndrome (CS; OMIM No. 158350), Bannayan-Riley-Ruvalcaba syndrome (BRR; OMIM No. 153480), Peutz-Jeghers syndrome (PJS; No. 175200) and juvenile polyposis syndrome (JPS; OMIM No. 174900) constitute the immediate members of a group of apparently rare, autosomal dominant inherited conditions classified as the hamartoma syndromes. Hamartomas are defined as benign, tumour-like overgrowths of tissue that are developmentally disorganised in structure. Whilst hamartomas, specifically those of the gastrointestinal tract, group these syndromes, distinct findings and the degree of phenotypic and genetic overlap vary between these disorders. For example, the endocrine tumours of CS and PJS, which could classify these disorders as variant types of multiple endocrine neoplasias, are not present in JPS and BRR. In addition to hamartomas, malignancy is an accepted feature of CS, PJS and JPS, yet it is controversial as to whether malignancy is a true component of BRR. Furthermore, the hamartomatous gastrointestinal polyps observed in PJS are histologically distinct from those seen in CS, BRR and JPS. PJS, and to a lesser degree CS and BRR, share a number of tumours with another condition, which is more clearly a type of multiple endocrine neoplasia: Carney complex (CNC; OMIM No. 160980). CNC, PJS and BRR also share the clinical manifestation of lentigines, which are pigmented spots of the skin and mucosae. These disorders, along with a number of other, lesser known genetic diseases, are described as the ‘lentiginoses’. Prior to 1997, little was understood in regards to the underlying genetic defects causing the hamartoma syndromes. However, subsequent to this time

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Table 1. The hamartoma syndromes and their susceptibility genes or loci Hamartoma syndrome

Susceptibility gene

Chromosomal location

CS BRR syndrome JPS

PTEN/MMAC1/TEP1 PTEN/MMAC1/TEP1 SMAD4/DPC4 PTEN/MMAC1/TEP1 ? ? STK11/LKB1 ?

10q23.3 10q23.3 18q21.1 10q23.3 chromosome 6q 19p13.3 (major) 19q13.4 (minor)

Mixed polyposis syndrome PJS

point, susceptibility genes functioning as tumour suppressors have been identified for most of these conditions. Whilst a single locus has been identified for the majority of CS and BRR families, at least two loci have been identified for PJS and CNC; JPS may also be genetically heterogeneous. The first of the hamartoma syndrome genes to be identified was PTEN (also named MMAC1 or TEP1), encoding a dual-specificity phosphatase mapped to 10q23.3, which was mutated in the germline of up to 80% of patients with CS and up to 60% of patients with BRR. Following rapidly was the discovery of the PJS gene, LKB1 (alternatively named STK11), encoding a serine-threonine kinase mapped to 19p13.3 and mutated in the germline of up to 90% of PJS patients. Germline mutations in SMAD4 (SMA- and MAD-related protein 4), also known as DPC4 (homozygously deleted in pancreatic cancer, locus 4), a member of the SMAD gene family mapped to 18q21.1 and encoding a cytoplasmic mediator in the transforming growth factor-ß (TGF-ß) signalling pathway, have been identified in a subset of families with JPS. The hamartoma syndromes and their susceptibility genes, or in some instances loci, are summarised in table 1. Somewhat controversially, germline PTEN mutations have also been reported in JPS. Additionally, somatic mutations of most of these genes have been identified in a number of sporadic component tumours of these syndromes. This chapter summarises the current state of knowledge of the clinical and molecular aspects of CS, BRR, JPS, PJS, CNC and related disorders, i.e. the LEOPARD syndrome (OMIM No. 151100), Proteus syndrome (PS; OMIM No. 176920) and Cronkhite-Canada syndrome (CC; OMIM No. 175500). Each syndrome is discussed individually, including its clinical aspects, a historical overview of the mapping of its genetic locus or potential loci, as well as the reported mutations and gene function. Genotype-phenotype correlations are discussed, as well as the putative function of these susceptibility genes and their potential role

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in sporadic component tumours of these hamartoma syndromes, such as those of the breast and thyroid.

Cowden Syndrome Clinical Features CS, alternatively referred to as ‘multiple hamartoma syndrome’, takes its name from the surname of the proposita of the first family described, who subsequently died of breast carcinoma [1, 2]. The syndrome has variable expressivity within families, although it usually presents in the third decade. The estimated frequency of a mutated gene causing this condition is one in a million [3], but because of the broad and often subtle visible features of CS, it is believed that this condition may be under-diagnosed [4]. In an attempt to improve the diagnostic accuracy of CS, an international CS Consortium was formed and operational criteria for the diagnosis of CS defined [3]. In CS, hamartomas occur in multiple organ systems, including the breast, thyroid, skin, central nervous system and gastrointestinal tract, derived from all three germ cell layers [5–9]. The hallmark hamartomas of CS, occurring in 99% of CS patients, are trichilemmomas, benign tumours of the hair follicle infundibulum, and mucocutaneous papules [6–8]. Other frequently occurring hamartomas include breast fibroadenomas (70% of CS females), thyroid adenomas and multinodular goitre (40–60% of CS patients) and gastrointestinal polyps (35–40% of CS patients). The risk of malignancy in CS is not an insignificant one, with breast cancer believed to develop in 25–50% of affected females, rarely in males, and thyroid cancer developing in 3–10% of all affected individuals [7, 9, 10]. Forty percent of cases develop disease of the central nervous system, most often benign but in some cases malignant [9]. The disorder Lhermitte-Duclos disease, which describes dysplastic gangliocytoma of the cerebellum manifesting as seizures, tremors and poor co-ordination, has also been associated with CS [11–14]. Megencephaly or macrocephaly occurs in approximately 38% of CS patients. Genitourinary abnormalities may also be present. Mapping the CS Gene – A Historical Perspective Prior to 1996, a definitive CS locus had not been identified. A complete genome scan of 12 CS families identified 10q22-23 as a likely CS locus; a critical recombinant identified by linkage analysis in one family suggested that this region was 5 cM in length [3]. Further linkage analysis was able to refine the CS locus to less than 1 cM [15]. 10q22-23 is a region that demonstrates allelic loss in a number of sporadic tumours, including follicular thyroid adenomas and carcinomas, glioblastoma (GBM) and prostate cancer, suggesting the likely presence of a tumour

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suppressor [16–19]. Three independent groups were able to identify a tumour suppressor gene at 10q23.3. PTEN (named for phosphatase and tensin homolog deleted on chromosome ten) was identified as the direct result of a search for genes associated with breast cancer using the subtractive hybridisation technique of representational difference analysis followed by physical mapping of homozygous deletions at 10q23 in tumour cell lines and xenografts [20]. Steck et al. [21] identified an identical gene called MMAC1 (named for mutated in multiple advanced cancers) through their interest in identifying a tumour suppressor in GBMs on the long arm of chromosome 10 [22]. Li and Sun [23] were the third group to identify the same gene, calling it TEP1 (for TGF-ß-regulated and epithelial cell-enriched phosphatase). Their isolation of TEP1 was based on the observation that protein tyrosine phosphorylation levels within a cell are regulated by an interaction between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). PTKs add phosphates to tyrosines whilst PTPs remove them. Given that PTKs are often encoded by oncogenes, it was conceivable that PTPs may in fact be encoded by tumour suppressors. Li and Sun [23] employed pairs of degenerate oligonucleotides complementary to the conserved catalytic domain of PTPs as primers to screen human cDNA libraries in conjunction with searching the GenBank expressed sequence tag (EST) database for conserved-sequence PTP motifs. A full-length cDNA was isolated and TEP1 was mapped to 10q23 by fluorescence in situ hybridisation. Thus, a putative tumour suppressor gene, referred to for the remainder of this chapter as PTEN, mapping to the CS locus had been cloned by three independent groups. This gene is unique in that whilst a large number of PTKs had already been shown to have a role as oncogenes in tumourigenesis, PTEN is the first PTP to function as a classic tumour suppressor. Furthermore, this gene had been shown to be mutated in breast cancer cell lines, a counterpart tumour of CS, thus it was an excellent candidate for the CS gene. PTEN was firmly established as the susceptibility gene for CS after mutation analysis of germline DNA revealed four of five unrelated CS families with either missense or nonsense mutations [15]. PTEN Structure, a Pseudogene and the Homologues PTEN contains nine exons and an open reading frame of 1,209 nucleotides encoding a 403-amino acid protein. A consensus for PTEN transcript size has not yet been reached in the literature; however, it is generally agreed that two major transcripts of between 5.0 and 5.5 kb and 2.0 and 2.5 kb are found to be ubiquitously expressed [23–26]. Other minor transcripts have been reported of 8.0, 4.2– 4.5 and 3.0 kb [25, 26], suggesting the possibility of alternative polyadenylation sites. The 5) untranslated region is especially long and contains multiple CpG islands with the potential to be regulated by DNA methylation, although evidence for this remains controversial [23, 24, 26, 27]. Residues 122–132, located in ex-

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Table 2. Putative PTEN homologues Organism

Putative homologues

Location

Human Mouse Caenorhabditis elegans Saccharomyces cerevisiae

PTEN Pten daf-18

10q23.3 chromosome 19 between D19Mit14 and D19Mit12 left arm of chromosome IV between daf-1 and opu-18 Scer-VI Scer-XIV

CDC14p YNL128W

on 5, encode the classic phosphatase core motif: (I/V)HCXXGXXR(S/T)G. The structure of the 179-residue NH2-terminal domain of PTEN (residues 7–185) is suggestive of both enzymatic and cellular localisation activities, given its homology to several phosphatases and in addition, its extensive homology across approximately 175 amino acids to the cytoskeletal proteins tensin and auxillin. Tensin is known to play a role in the maintenance of cellular structure and possibly in signal transduction by binding to actin filaments at focal adhesions and to phosphotyrosine-containing proteins through its actin binding and Src homology-2 domains [28]. Auxillin is a cytoplasmic protein involved in synaptic vesicle transport. Consistent with homology to the cytoskeletal proteins, PTEN has been localised to the cytoplasm [23], yet immunohistochemical studies of both breast and thyroid tissue have strongly suggested a nuclear predominance of PTEN [29, 30]. Given the absence of a nuclear localisation signal, one possibility is that a shuttle molecule assists PTEN into the nucleus, perhaps in a manner not dissimilar to that which is observed between TP53 and the oncoprotein MDM2. Further clarification of the subcellular localisation of PTEN in both normal and tumour tissue is required; however, nuclear localisation of this protein is in agreement with a role for PTEN in cell cycle control. The 166-residue COOH-terminus of PTEN (residues 186–351) contains a potential PDZ-binding domain encoded by the last four amino acids ITKV. PDZ domains are found in a variety of proteins, including membrane-associated guanylate kinases (MAGUKs), which are a family of membrane-associated scaffold proteins [31]. These domains have been shown to have a role in mediating the association of membrane proximal proteins and in the complexing of signalling molecules, suggesting that they may be important for the subcellular localisation and/or substrate interactions of PTEN. Also in the COOH-terminus are three potential tyrosine phosphorylation sites at residues 240, 315 and 336, and two

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Fig. 1. Schematic of the putative functional domains of PTEN. * corresponds to residues 42–52 (exon 2) and residues 163–166 (exon 5/6), which are conserved residues responsible for increased width and depth of the phosphatase-active pocket of PTEN, allowing it to accommodate a Ptd-Ins(3,4,5)P3 substrate.

potential serine phosphorylation sites at residues 335 and 338. The structure of PTEN and its predicted domains is summarised in figure 1. A human processed PTEN pseudogene has been isolated on chromosome band 9p21 lacking the initiating methionine present in PTEN [32–35]. It shares greater than 98% homology with the coding region of PTEN, varying by only 19 nucleotides. Although conflicting evidence has been reported in the literature, it would appear that this pseudogene may be transcribed in a number of tissues. The existence of this pseudogene has the potential to complicate any mutational analysis study performed from cDNA. Thus, it is recommended to perform all PTEN mutation screening with primers designed to intronic sequences. PTEN homologues have been isolated in Caenorhabditis elegans, mouse and yeast [36–40]. Their names and genomic locations are summarised in table 2. The homologue of PTEN from the nematode C. elegans is the dauer formation gene daf-18 [36–38]. DAF-18 is a component of the insulin-like signalling pathway that controls entry into diapause and adult longevity, hinting at a possible role for PTEN in ageing. Other components of this signalling pathway are homologues to phosphotidylinositol 3-kinase and AKT, the cellular homologue of the viral oncogene v-akt (see PTEN Signalling below).

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Three Pten knockout mouse models have been generated that have provided information on both the normal and abnormal developmental patterns of Pten [41–43]. In normal murine development, the Pten protein is expressed at a low level in embryos on days 8, 9 and 10, with expression becoming more pronounced by day 11. On days 13–16, prominent Pten expression was detected in the dorsal portion of the mantle layer of the spinal cord, heart and epidermis and, in addition, a general expression in a variety of tissues [41]. Each of the three knockout mice generated has a slightly different phenotype, possibly explained by the different genetic backgrounds of the mice used or by the fact that each group constructed a unique mutation. In all cases, Pten-null mice were embryonic lethal, either on embryonic day 7.5 [42] or day 9.5 [41, 43], depending on the specific model generated, and showed abnormally patterned and expanded caudal and cephalic regions [43]. It has been postulated that the death of Pten-null embryos occurs at this stage as a result of an imbalance between growth and patterning, which serves to disrupt the timing of developmental events [43]. Podsypanina et al. [41] created frame shift mutations in the core motif of the phosphatase domain in exon 5 to generate their Pten mutant mouse, which showed neoplasia or hyperplasia in many organ systems including the endometrium, liver, prostate, gastrointestinal tract, thyroid and thymus. Thyroid abnormalities included follicular or papillary non-invasive neoplasia and atypical epithelial changes. Multiple polyps were observed in the large and small intestines, as well as adenoma of the colon. The presence of abnormalities of B and T cell distribution in lymphoid aggregates normally found in the colon and small intestine suggested that Pten may be functioning as a ‘landscaper’ [44] in these tissues, as these abnormal aggregates may be sending improper messages to the overlying epithelium, thereby disrupting normal growth. A second Pten+/– model was generated by Suzuki et al. [43], lacking exons 3–5 of Pten. On embryonic day 7.5, in contrast to their wild-type litter mates, Pten+/– embryos were less advanced and their germ cell layers were reminiscent of an earlier embryonic stage. These mice showed a high incidence of lymphoma/ leukaemia of the T cell type associated with loss of heterozygosity (LOH) at the wild-type allele. Other abnormalities observed in these mice included atypical adenomatous hyperplasia in the liver, one case of prostate cancer and microscopic hamartomatous polyps, mainly distributed in the colon. Of considerable interest is that observation of these mice at greater than 6 months of age has revealed a phenotype more similar to the spectrum of neoplasia observed in CS [45]. Specifically, all Pten+/– females had endometrial hyperplasia, with 22% developing endometrial cancer. An increased frequency of gastrointestinal hamartomas was also observed and 50% of females developed breast tumours [45]. A third Pten+/– model was generated by the deletion of exons 4 and 5 [42]. Polyploid lesions were observed in the gastrointestinal tract of these mice, several

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with severe dysplastic changes. Tumours forming in these mice were compatible with adenocarcinomas and gonadostromal tumours as well as leukaemia. When observed at an early age, none of the mice of the Pten+/– models described developed the classic tumours of the skin, breast and brain seen in CS in humans, although the presence of colonic microscopic hamartomatous polyps (reminiscent of the human CS or BRR phenotypes) was reported by all three groups. It will be of considerable interest to observe all three mouse models as they age for the development of tumours more reminiscent of those seen in CS. Given the phenotype of the model already observed at greater than 6 months of age [45], it could be speculated that Pten+/– mice mimic quite well the type and timeline of tumour development observed in human CS. PTEN Signalling Biological evidence highly suggestive of a tumour suppressor function for PTEN was provided by a study of glioma cells. In these cells with endogenous mutant PTEN alleles, the reintroduction of wild-type PTEN caused growth suppression but no effect was observed on cells containing endogenous wild-type PTEN alleles [25]. Answering the question as to how PTEN functions as a tumour suppressor has shed light on a number of complex, and still evolving, signalling pathways. The major endogenous substrate of PTEN has been identified as phosphatidylinositol 3,4,5-triphosphate [Ptd-Ins(3,4,5)P3], an important second messenger in the regulation of cell growth [46]. Ptd-Ins(3,4,5)P3 is a phospholipid in the phosphatidylinositol 3-kinase (PI-3 kinase) pathway with a known function as a mediator of growth factor-induced activation of cell growth signalling. These growth factors, which include insulin, platelet-derived growth factor and fibroblast growth factor, stimulate the catalytic enzyme PI-3 kinase to phosphorylate Ptd-Ins(4,5)P2 to produce Ptd-Ins(3,4,5)P3. In the PI-3 kinase pathway, PTEN may act as a 3-phosphatase to dephosphorylate Ptd-Ins(3,4,5)P3 to Ptd-Ins(4,5)P2 [47]. Mutant or decreased levels of PTEN lead to the accumulation of PtdIns(3,4,5)P3, which in turn activates protein kinase B/AKT, a serine-threonine kinase and a known cell survival factor, to function as an oncogene causing the tumourigenic state [26, 46–51] (fig. 2). There are a number of known downstream targets of AKT with the potential to affect cell growth, including glycogen synthase kinase 3, FK506-binding protein-rapamycin-associated protein and the apoptosis-inducing protein BAD [52]. Phosphorylation of BAD inhibits BAD binding to Bcl-x1 and leads to the suppression of apoptosis [52]. An indirect apoptotic role for PTEN is supported by results from a study showing high levels of phosphorylated AKT in PTEN-deficient immortalised mouse embryo fibroblasts accompanied with increased cell survival [49], as well as in human malignant cell lines.

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Fig. 2. The role of PTEN in a lipid phosphatase pathway. Growth factors, such as insulin, stimulate the catalytic enzyme PI-3 kinase to phosphorylate Ptd-Ins(4,5)P2 to produce Ptd-Ins(3,4,5)P3, an important regulator of cell growth. By acting as a 3-phosphatase, wildtype PTEN in turn dephosphorylates Ptd-Ins(3,4,5)P3 to Ptd-Ins(4,5)P2. Decreased levels of PTEN lead to the accumulation of Ptd-Ins(3,4,5)P3, which activates AKT, an anti-apoptotic gene, leading to cell growth and survival.

The complexities of PTEN signalling become evident as in addition to its lipid phosphatase activity, PTEN has been shown to display in vitro phosphatase activity against protein substrates. Given the ability to dephosphorylate protein and peptide substrates phosphorylated on threonine, serine and tyrosine residues, PTEN is classed as a dual-specificity phosphatase [20, 21, 23, 53]. Given its homology to tensin, it was considered that PTEN may have effects on intracellular integrin-mediated responses, including the tyrosine phosphorylation of focal adhesion kinase (FAK). Overexpression of PTEN has been shown to negatively regulate cell migration, integrin-mediated cell spreading and the formation of focal adhesions [54]. PTEN has been shown to directly interact with FAK, reducing its tyrosine phosphorylation and indirectly reducing tyrosine phosphorylation of p130 Crk-associated substrate, p130Cas, a molecule acting downstream of FAK

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and down-regulating cell migration and invasion [55]. However, the levels of PTEN required to dephosphorylate FAK in vitro raise some questions as to whether this phenomenon also occurs in vivo. Further, PTEN expression has been shown to inhibit Ras activity, affecting the Ras/ERK common MAP kinase pathway for integrin- and growth factor-mediated signalling [56]. Consistent with this role as a negative regulator of cell motility, PTEN transcription is down-regulated by TGF-ß [23]. This finding, however, has not been reproduced widely by independent groups. TGF-ß has been implicated in tumour progression by its effect on cell adhesion properties, including stimulation of extracellular matrix production and enhancement of cell motility. It has been postulated that the reduction of functional PTEN as a result of TGF-ß may increase cell migration during tumour invasion and metastasis [23]. Studies of the crystal structure of PTEN have served to further elucidate its preferred substrates [57]. Of considerable interest, the phosphatase-active site has been shown to be both wider and deeper than that of other dual-specificity phosphatases, such as VHR [57]. This larger pocket size is important for the accommodation of the Ptd-Ins(3,4,5)P3 substrate. The increased size of this active pocket is due to an 11-residue insertion (residues 42–52) and a 4-residue insertion (residues 163–166) not present in other dual-specificity phosphatases (fig. 1). These insertion residues are conserved amongst the PTEN homologues. In addition, this pocket is highly basic given the make-up of its residues, thus its positive charge is consistent with the negative charge of the Ptd-Ins(3,4,5)P3 substrate and with the preference of PTEN for highly acidic polypeptide substrates [53]. The C-terminal domain was shown to have a C2 domain important for the recruitment of PTEN to the phospholipid membrane. Of considerable interest, the phosphatase domain and the C2 domain associate across an extensive interface that create interdomain hydrogen bonds between conserved residues. Thus, it would appear that the C2 domain not only recruits PTEN to the substrate membrane, but also serves to optimally position the phosphatase catalytic domain with respect to the membrane-bound substrate. Mutation of these conserved residues, including serine at position 170, has been reported [58]. The C-terminus of PTEN houses the PDZ-binding motif and has also been shown to bind to membraneassociated guanylate kinase inverted-2 (MAGI-2), a multi-PDZ domain-containing MAGUK protein previously named AIP-1 (atrophin-interacting protein) [31], providing stability for the multiprotein complex and enhancing the efficiency of PTEN signalling. Given that the PDZ domain of PTEN is encoded by the terminal four amino acids, all C-terminal mutants would be expected to disrupt this protein-protein interaction. Thus, it is perhaps not surprising that PTEN mutants failing to bind MAGI-2 show defects in the regulation of AKT [31]. Thus, the C2 domain and the PDZ-binding motif within it contribute to the recruitment of PTEN to the membrane and its stability at this location. Taken together, the crys-

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tal model of PTEN provides evidence that the PTEN substrate of choice is PtdIns(3,4,5)P3 [57]. Whilst apoptosis via the Ptd-Ins(3,4,5)P3/AKT pathway is a likely mechanism for PTEN-induced growth suppression as described above, it would seem that PTEN is also able to cause cell cycle arrest in cells in the G1 phase [51, 59]. It is believed that functional PI-3 kinase is necessary for G1–S progression in cells via modulation of the phosphorylation levels of RB, and that Ptd-Ins(3,4,5)P3activated accumulation of AKT may encourage cells to progress through the cell cycle. Still, the role of RB in the PTEN arrest of cells remains controversial [60, 61]. However, in mutant PTEN glioblastoma cells where wild-type PTEN is reintroduced and overexpressed, the CDK inhibitor p27Kip1 has been shown to be recruited into cyclin E/CDK2 complexes. This leads to a reduction in cyclin E/CDK2 kinase activity and a decrease in phosphorylation levels of endogenous RB [62]. RB is bound to the transcription factor E2F during the G1 phase, and when phosphorylated, it releases E2F, thus activating the transcription of genes required for transition into the S phase. If RB is not phosphorylated, the progression of cells from G0/G1 to the S phase is blocked. One study of cell cycle arrest resulting from PTEN expression in glioma cells did not report phosphorylation changes in RB, suggesting that PTEN-mediated growth suppression may be executed through an alternate pathway to RB [60]. However, a second study reported that the hyperphosphorylation of transfected RB was inhibited in RB-deficient cells when co-transfected with PTEN [61]. This second study concluded that PTEN-induced cell cycle arrest is mediated primarily by the inhibition of PI-3 kinase-dependent signal transduction pathways. In apparent contrast, yet another report has demonstrated that transient PTEN expression by adenoviral delivery induces apoptosis but not cell cycle arrest in breast cancer cell lines [48]. A further report using breast cancer cell lines has shown PTEN to cause growth suppression via down-regulation of PI-3 kinase signalling, attributed initially to G1 arrest followed by the induction of apoptosis, specifically in that sequential order [63]. Thus, whether PTEN-induced growth suppression occurs via an apoptotic or a cell cycle arrest mechanism, or indeed a combination of both, remains controversial. It must be acknowledged that neither mechanism excludes the other, and in fact it has been suggested that perhaps the presence or absence of PTEN may act to modulate apoptosis, as cell death in PTEN-null cells exposed to apoptotic stimuli such as UV irradiation occurs at a reduced level compared with cells heterozygous for a mutant copy of PTEN [49]. In addition, it is possible that PTENinduced growth suppression via different mechanisms may to some degree be tissue specific. In summary, PTEN has a role in the regulation of a number of different cellular processes, including growth and apoptosis, as well as interaction with the extracellular matrix, cell migration and invasion. PTEN would seem to function

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to regulate cell migration/adhesion through its PTP activity and to regulate cellular growth and apoptosis through its lipid phosphatase activity via the PI-3 kinase pathway. Germline PTEN Mutations in CS and Hereditary Associated Malignancies Germline mutations in PTEN have been identified in between 13 and 81% of CS patients and wherever possible have been shown to segregate with the disease phenotype [10, 15, 64–66]. Mutations have been reported to be scattered largely over the entire gene, with the exception of exon 1, and include point missense and nonsense mutations, insertions, deletions, deletions-insertions and splice site mutations (table 3). In addition, LOH at the wild-type allele has been found in hamartomas from a number of PTEN mutation-positive family members, providing further evidence that PTEN functions as a classic tumour suppressor, conforming to Knudson’s two-mutation model [67, 68]. Distinct clusters of mutations occur in exons 5, 7 and 8, with the great majority occurring in exon 5, which is the largest exon of this gene, representing 20% of the coding region, but also contains the phosphatase core motif. Interestingly, the majority of mutations that occur in the core motif are non-truncating, suggesting the importance of the integrity of this functional domain. Whilst exon 5 is undoubtedly the first hot spot for mutation cluster in this gene, hot spots also exist in exons 7 and 8, believed to encode potential tyrosine and serine phosphorylation sites (fig. 1). Interestingly, a key family used in the linkage mapping of PTEN, which was able to refine the localisation of a PTEN candidate gene to less than 1 cM, did not have a detectable PTEN germline mutation [15]. It is possible that affected individuals in this family may have a mutation in the promoter region, or perhaps mutation deep within an intron. Alternatively, it is possible that transcriptional inactivation via germline methylation may mimic the effects of PTEN germline mutation, although there are no good precedents for this in the cancer literature. Whilst an international study of 12 CS families found no evidence for genetic heterogeneity in CS [3], there has been a single report suggesting that genetic heterogeneity does exist in this syndrome [64]. One possibility that was explored was that germline mutations in MINPP1, a multiple inositol phosphatase approximately 1 Mb centromeric of PTEN, may be responsible for the CS phenotype [69]. Despite its functional similarity to PTEN in being a phosphatase with activity towards lipid substrates and its close proximity, PTEN mutation-negative CS cases did not yield any MINPP1 mutants [70]. Given that PTEN was the susceptibility gene for a breast cancer syndrome and was homozygously deleted in breast cancer cell lines and xenografts, it became an obvious candidate for a non-CS-associated breast cancer predisposition gene. However, since germline PTEN mutation is found at a very low fre-

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Table 3. Germline PTEN mutation in CS and CS with Lhermitte-Duclos disease Mutation

Exon/IVS

Phenotype and reported cases (n)

L22X c.83insA c.137ins3 c.158insATAC c.158-159delTA codon 55insTTAC IVS2-2A→G I167R IVS3+5G→A c.244insT IVS4+1G→T Q87X H93Y c.302-304delTCAinsCC c.304insT Q109X Q110X L112P c.347-351delACAAT H123R C124R G129E R130X R130Q R130L L139X E157X G165E c.530-531delAT codon 183insA R188X c.565delA R232X R233X c.723insTT Q245X c.783-784delGA c.791insAT c.800insA c.915del12 Y315X c.981delA V332E** R334X R335X c.1007insA F346L**

2 2 2 2 2 2 IVS2 3 IVS3 4 IVS4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 7 7 7 7 7 7 7 8 8 8 8 8 8 8 9

CS (1) [131] CS (1) [260] CS (1) [64] CS/LDD (1) [10] CS (1) [10] CS (1) [65] CS (1) [10] CS (1) [10] CS/LDD (1) [10] CS (1) [66] CS/LDD (1) [65] CS (1) [10] CS (1) [66] CS (1) [10] CS (1) [10] CS (1) [131] CS (1) [10] CS/LDD (1) [74] CS (1) [10] CS (1) [65] CS (3) [10, 65] CS (2) [10] CS (5) [10, 65, 259] CS (1) [261] CS (1) [10] CS (1) [262] CS/LDD [10, 65] CS (1) [10] CS (1) [66] CS (1) [65] CS (1) [10] CS (1) [10] CS (1) [131] CS (2) [10] CS (1) [10] CS (1) [10] CS/LDD (1) [65] CS (1) [10] CS (1) [10] CS (1) [64] CS (1) [10] CS (1) [10] CS (1) [131] CS (2) [131] CS (1) [10] CS (1) [10] CS (1) [131]

** Occurred in the same family on the same parental chromosome. Mutations in the phosphatase core motif are shown in bold. LDD = Lhermitte-Duclos disease; IVS = intervening sequences.

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quency in these families, PTEN is not thought to play a major role in the development of familial breast cancer outside of CS [71, 72]. The identification of PTEN germline mutations in patients with CS has allowed the development of DNA-based predictive testing programs as part of the clinical management of individuals within these families. Given the risk of malignancy in affected individuals, early patient identification would allow increased surveillance of potentially affected organs in these patients. Genotype-Phenotype Correlations in CS In an attempt to assist in the clinical management of families with CS, genotype-phenotype correlations have been sought by different groups. In a large study of 35 CS families, the presence of a PTEN mutation seemed more likely to be associated with breast involvement [10]. However, this trend was unable to be confirmed by a similar study of a different patient group, albeit of a smaller sample size [73]. A second trend was observed between the presence of a missense mutation and the involvement of all five organ systems (i.e. breast, thyroid, central nervous system, skin and gastrointestinal tract). However, given the fact that these missense mutations occurred within the phosphatase core motif, this trend may in fact represent a positional effect [10]. Perhaps surprisingly, no useful correlations have been observed between the presence of Lhermitte-Duclos disease and PTEN mutation [10, 73, 74]. Clinically useful genotype-phenotype correlations and confirmation of putative trends will require analysis of a larger sample set. PTEN in Sporadic Component Tumours of CS PTEN has been described as ‘the most highly mutated tumour suppressor gene in the post p53 era’ [75]. In addition to being mutated in the germline of patients with CS, PTEN has also been shown to be mutated or deleted at the somatic level in a spectrum of human malignancies [4, reviewed in ref. 33]. PTEN has been shown to be the most frequently mutated gene known to be involved in endometrial carcinomas and hyperplasias, ranging from 34 to 42% in endometrial carcinomas, and is clearly a target of 10q loss in these cancers [76, 77]. PTEN is mutated in 20% of cases of endometrial hyperplasia, the likely premalignant precursor to invasive endometrial adenocarcinoma [78], and in 21% of endometrioid ovarian tumours [79]. A higher frequency of PTEN mutation is observed in microsatellite instability-positive endometrial tumours [79]. PTEN is also mutated in GBMs, with mutations occurring in between 17 and 44% of these tumours, making PTEN a likely target for the frequent chromosome 10 deletion seen in these tumours [80–86]. It would seem that PTEN mutation is largely associated with the high-grade malignancy characteristic of GBM, given that PTEN mutation is rarely found in the lower-grade gliomas such as astrocyto-

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mas. Still, the number of identified mutations is significantly lower than the LOH reported at this locus, which is in the range of 60–90%, leading to the speculation that there are most likely a number of tumour suppressor genes resident on 10q that are important in the genesis of GBM. One of these genes, DMBT1 (deleted in malignant brain tumours), mapped to 10q25.3-26.1, has been shown to have intragenic homozygous deletions in 15–38% of GBM cases [87, 88]. Despite thyroid neoplasia being a characteristic of CS, somatic PTEN mutation is rarely seen in sporadic thyroid neoplasms. In one large study of 95 sporadic thyroid tumours consisting of papillary thyroid carcinomas, follicular thyroid carcinomas (including Hürthle cell), follicular thyroid adenomas (including Hürthle cell) and anaplastic carcinomas, PTEN mutation was only seen in 3% of papillary thyroid carcinomas, and hemizygous PTEN deletion was seen in 26% of informative benign thyroid tumours and 6% of informative malignant tumours [89]. In a second study, 7% of follicular thyroid carcinomas were shown to have both PTEN mutation and loss of the wild-type allele [90]. In the same vein, somatic PTEN mutation has not been observed as a classic genetic aberration in sporadic breast carcinoma, with the mutation frequency being as low as 1.5–2% [91–93]. However, 30–40% of primary breast cancers show allelic loss at 10q23 [94–96]. Furthermore, an immunohistochemical study of breast cancer has shown a subset of samples with PTEN hemizygosity, no intragenic PTEN mutation of the remaining allele and a complete absence of immunoreactivity, suggesting that at least in breast cancer, epigenetic phenomena such as hypermethylation of the promoter region or aberrant protein turnover may be alternative mechanisms of PTEN inactivation [29]. A wide spectrum of other sporadic tumours show either PTEN mutation or LOH involving the PTEN locus. One study of sporadic prostate cancer, a tumour in which LOH at 10q23 is high, reported 43% of tumours having PTEN mutation with corresponding loss of the wild-type allele [27]. Studies of malignant melanoma cell lines have shown 17% with PTEN mutation and corresponding loss of the wild-type allele [97]. A second study of melanoma found a missense PTEN variant in 10% of the primary tumours studied [33]. Further, 3% of hepatocellular carcinoma cases show PTEN mutation [98], whilst in primary head and neck cancers, a second inactivation event at the PTEN locus was found in 8% of tumours [99]. Sixteen percent of bladder tumours with LOH at the PTEN locus were found to have a mutation in the remaining allele [100], whilst the PTEN mutation rate in colon cancer was very low at 1.4% [101]. Figure 3 provides a summary of PTEN mutations in sporadic solid tumours. Aside from the solid tumours, translocation at 10q22-24 is often seen in leukaemias and lymphomas. In one study of primary acute leukaemias and non-Hodgkin’s lymphomas, PTEN was shown to be inactivated in up to 60% of cases at either the structural, transcript or protein level [26]. Thus, it is likely that epigenetic mechanisms have a

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Fig. 3. Primary sporadic solid tumours with somatic PTEN mutation. No PTEN mutations were identified in follicular thyroid adenomas or anaplastic carcinomas. *Mutation analysis was only performed when loss of one PTEN allele was detected; i.e. this frequency represents a true ‘2-hit’ phenomenon in the samples reported.

role in PTEN inactivation and that mutation analysis alone may not fully reveal the involvement of PTEN in other tumour types. In summary, PTEN mutation may be an early event in the progression of some cancers, such as endometrial carcinoma, as is evidenced by its presence in endometrial dysplasia, and a later event in others, such as GBM.

Bannayan-Riley-Ruvalcaba Syndrome Clinical Features The classic triad of macrocephaly, lipomatosis and, in males, pigmented macules of the glans penis (‘speckled penis’) characterises the BRR syndrome [102–104]. The naming of this syndrome has been somewhat variable, given that a number of names have arisen derived from the name of the first person to

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describe different aspects of this condition, including Ruvalcaba-Myhre-Smith syndrome, Bannayan-Zonana syndrome and Riley-Smith syndrome. Most believe that there is now sufficient clinical overlap to be satisfied with one name – BRR [105]. BRR and CS show partial clinical overlap, as patients with either syndrome may develop intestinal hamartomatous polyps, macrocephaly (approaching 100% of cases in BRR) and lipomas (occurring in a minority of patients with CS). However, features such as speckled penis and a very early age of onset would appear to be specific to BRR. Other clinical manifestations of BRR include haemangiomas, mild mental retardation and developmental delay. Based on these phenotypic similarities alone and prior to knowledge of the genetics behind the hamartoma syndromes, it was postulated that BRR and CS may share a common genetic pathogenesis [106]. Whilst anecdotal cases of malignancy affecting the brain and thyroid have been reported in BRR [107, 108], to date, malignancy is not a well-described phenotype of BRR. In addition to being a multiple tumour syndrome, BRR may be considered a paediatric endocrine disorder. It is often associated with generalised overgrowth; the latter, along with macrocephaly, developmental delay and some facial features, is reminiscent of Sotos’ syndrome, and some of the first patients with BRR were diagnosed as such [109]. PTEN, the CS Susceptibility Gene, Is Also Mutated in BRR Once the CS gene had been identified as PTEN, and given the clinical overlap between the two syndromes, it was a logical step to investigate whether PTEN was also the susceptibility gene for BRR. Karyotypic analyses of a BRR patient hinted that this may be the case, as chromatin was lost between 10q23.2-10q24.1 resulting from an unbalanced translocation [105]. Earlier karyotypic studies of a patient with BRR had identified a 19;Y translocation in lymphocytes, 46,X,t(Y;19)(q11;q13), although it is likely that this may have been a coincidental finding [110]. Conclusive evidence that PTEN was also a gene for BRR was presented when one nonsense point mutation, R233X, and one missense point mutation, S170R, were identified in two unrelated BRR families [58]. Studies building on this initial report suggested that germline PTEN mutations occur in approximately 50–60% of BRR patients [10, 111, 112]. As in CS, these mutations occur scattered along the PTEN gene, although in contrast to CS, gross hemizygous deletions and also a balanced translocation likely affecting the PTEN gene have been reported [108, 111, 113]. Of interest, one of the Pten+/– mouse models supported the possibility that PTEN haploinsufficiency alone was enough of an underlying cause for characteristic developmental defects and tumour formation [42]. In this model, the wild-type allele was retained in various tumour types. However, as stated earlier, none of the Pten+/– mouse models described developed the classic

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tumours of CS, although the presence of colonic microscopic hamartomatous polyps more reminiscent of the phenotype seen in CS or BRR was reported [41–43, 49]. CS and BRR – Different Presentations of the Same Syndrome Given that identical mutations, including Q110X, R130X, R233X and R335X, have been reported in both CS and BRR, the presence of other genetic and/or epigenetic factors, such as modifier loci, must play a role in the definition of the phenotype. Not only is there overlap between a number of clinical features, but now a number of families have been reported in which both the CS and BRR phenotypes appear to be expressed [107, 111, 114]. Most often, BRR appears in the younger generation of these families, with CS being present in the older family members, suggesting some form of anticipation. Systematic studies on a larger number of families encompassing several generations are required in order to confirm this observation. At present, it would seem that some PTEN mutations are specific to either CS or BRR, such as G129E, which has been reported only in CS. It has been suggested that different PTEN germline mutants may differentially trigger various signalling pathways involving either phospholipid or proteic substrates and that this may explain some of the phenotypes [111]. Further, it is possible that this differential effect may be cell or tissue specific. For example, some mutants have been shown to have loss of phosphatase activity against in vitro protein substrates and Ptd-Ins(3,4,5)P3 [46, 53]. The CS mutation G129E, however, showed normal phosphatase activity against non-phospholipid substrates both in vitro and in cell lines but has no phosphatase activity against Ptd-Ins(3,4,5)P3 [53, 59]. The analysis of the crystal structure of PTEN suggests that mutation of this residue, located at the bottom of the active pocket near the conserved extension residues, would serve to reduce the size of this pocket, so that it could no longer accommodate the larger Ptd-Ins(3,4,5)P3 substrate, thus disrupting Ptd-Ins (3,4,5)P3 activity, with no effect on the smaller substrates of phosphotyrosine, serine or threonine [53, 57, 59]. Nonetheless, the intrafamilial variation seen in families with both BRR and CS, combined with the clinical overlap between these two hamartoma syndromes, points to the likely conclusion that BRR and CS are in fact different presentations of a single syndrome with broad clinical expression. Clinically, this underscores the importance of genetic counselling and cancer surveillance, not only for BRR patients but also for their first-degree relatives. Genotype-Phenotype Correlations in BRR Given the small number of BRR cases identified to date, detailed genotypephenotype correlations have not been able to be performed. However, in one

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study of BRR patients, the presence of a germline PTEN mutation was correlated with the presence of lipomas and also with any cancer or breast fibroadenoma [111]. The authors concluded that the presence of a PTEN mutation in either CS alone, BRR alone or CS/BRR overlap families predisposed to the presence of tumours, whether they be benign, i.e. the lipomas seen in BRR, or malignant, i.e. the breast or uterine carcinomas seen in CS or BRR/CS families.

Juvenile Polyposis Syndrome Clinical Features Ninety percent of all childhood and adolescent colorectal polyps are juvenile polyps [115]. Up to 2% of children and adolescents develop these polyps, but only a small proportion occur as part of JPS [116]. Juvenile polyps are usually solitary and very rarely premalignant, whilst those associated with JPS are multiple, occur throughout the gastrointestinal tract, usually in the colon but sometimes in the stomach and small bowel, and patients have an increased risk of colorectal malignancy [117–121]. In one JPS kindred, the risk of gastrointestinal malignancy exceeded 50% [122]. Histologically, these polyps have an inflammatory stroma with abundant lamina propria lacking smooth muscle, which differentiates them from Peutz-Jeghers polyps, which contain smooth muscle [122, 123]. It has been postulated that there may be a relationship between the lamina propria and the normal epithelium during the neoplastic process in JPS. Physically, the JPS polyp is a mix of largely stromal cells comprised of mesenchymal and inflammatory elements in which the epithelium is entrapped, often forming dilated cysts. The epithelial cells within and surrounding the JPS polyp are initially devoid of neoplastic features but also have an increased risk of neoplasia. Extra-colonic abnormalities in JPS are not succinctly described, although both stomach and pancreatic cancers have been reported, along with macrocephaly, cleft lip and palate, congenital heart disease and bony swellings [124]. The diagnosis of JPS is often made based on clinical exclusion of CS or BRR. Such a diagnosis by exclusion lends itself to potential misdiagnosis of this syndrome. Mapping of Putative JPS Loci – A Historical Perspective Mapping of a JPS gene has proved an interesting exercise, with the literature containing a number of conflicting reports. Initial linkage studies were able to exclude genes involved in the adenomatous polyposes (APC and DCC), as opposed to the hamartomatous polyposes [125]. In a single family with hereditary mixed polyposis syndrome, characterised by atypical juvenile polyps, colonic adenomas and carcinomas, the disease phenotype was shown to be linked to chromosome 6q [126]. A de novo interstitial deletion at 10q22.3-24.1 spanning a 25-cM

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interval including PTEN was identified in a single JPS patient with congenital abnormalities of the head, extremities and abdomen [127]. Further, juvenile polyps from JPS patients showed a high percentage of somatic deletion at 10q22 in the lamina propria, i.e. the stromal or non-epithelial component cells of these polyps [128]. However, close inspection of their shortest region of overlap revealed that this region of loss was in fact centromeric to PTEN. The authors described their interval as a putative JPS locus and called it JP1. A germline deletion of 10q23.2-q23.33 in a patient said to have JPS but with other features of a ‘Cowden-like syndrome’, including pigmented macules of the penis, has also been reported [129]. Four JPS patients have been reported with germline PTEN mutation; one of these patients was also described as having both CS and JPS [130, 131]. Some of these PTEN-linked ‘JPS patients’ may in fact be cases of CS or BRR [132]. The possibility of JPS being allelic with CS and BRR was not supported by linkage analysis in 8 JPS families that excluded linkage to PTEN and the flanking 20 cM in these families, including the putative JPS locus JP1 [133]. Further, this same study was unable to detect a germline PTEN mutation in probands from 14 JPS families and 11 sporadic cases. A second study of 11 familial cases of JPS was also unable to identify any coding mutations in PTEN [134]. Finally, a third study of a large, 5-generation family with JPS was unable to establish linkage to 10q2224 and instead established linkage to chromosome 18q21.1 [135]. SMAD4, also known as DPC4, had previously been located at this locus [136, 137]. Germline mutations predicted to truncate the SMAD4 protein were identified in 5 of 9 JPS patients [137]. It would appear that a 4-bp deletion in exon 9 of SMAD4, 1372– 1375delACAG, accounts for approximately 25% of JPS cases studied, even though a founder effect has not been demonstrated [138–140]. It is suggested that the site of this deletion is likely a mutational hot spot, possibly contributing to a high-penetrance phenotype. Certainly, this simplifies molecular genetic testing for JPS and aids in the establishment of a molecularly defined subgroup of JPS [139, 140]. Still, the issue of possible genetic heterogeneity in JPS remains, as one study was able to exclude linkage to SMAD4 in 8 JPS families and found only one SMAD4 germline mutation in a collection of 21 JPS patients [141]. Other members of the SMAD family, specifically SMAD1, SMAD2, SMAD3, SMAD5 and SMAD7, have been excluded as containing pathogenic mutations associated with JPS [140, 142]. However, it has been suggested that if an occult germline PTEN mutation is identified in a ‘JPS’ patient, the diagnosis should be reclassified as CS or BRR and the patient monitored for the breast, thyroid, skin and uterine abnormalities characteristic of these syndromes [143].

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SMAD4 (DPC4) – A Cytoplasmic Mediator in the TGF-ß Signalling Pathway SMAD4 consists of 11 exons encoding a 552-amino acid protein with a highly conserved carboxy terminus [137]. SMAD4 and other members of the SMAD gene family encode for cytoplasmic mediators in the TGF-ß signalling pathway. In the absence of functional SMAD4, colorectal cell lines have been shown to be unresponsive to TGF-ß ligands [144]. Of interest, one study has shown TGF-ß to negatively regulate PTEN, leading to increased cell migration; however, these data are yet to be reproduced by other studies (see PTEN Signalling above). The highly conserved COOH-terminus of SMAD4 (encoded by exons 8–11) is integral to the formation of SMAD4 homotrimers that complex with other SMAD proteins at the cell surface. This occurs by activation of TGF-ß or related ligands that act on TGF-ß family transmembrane serine-threonine kinase receptors, phosphorylate the SMAD proteins and form cytoplasmic complexes with SMAD4 [145]. These complexes migrate to the nucleus and act as transcriptional regulators by associating with DNA-binding proteins. Interestingly, many of the mutations reported in SMAD4 are predicted to affect the COOH-terminus, likely disrupting the ability of these molecules to complex with other SMAD proteins, therefore leading to loss of TGF-ß signalling. SMAD4 in Sporadic Tumours Somatic mutation of SMAD4 in primary tumours is seen in approximately half of pancreatic carcinoma cases, is less common in colorectal carcinoma and biliary tract carcinoma, and would appear to be only rare at the somatic level in breast, ovarian and lung carcinoma [146, 147]. Even though allelic loss is seen in many tumours at 18q, such as in GBM, mutation of SMAD4 is not seen in other cancers [146–149].

Peutz-Jeghers Syndrome Clinical Features The co-occurrence in the familial setting of gastrointestinal polyposis and benign mucocutaneous pigmentation was first observed by Peutz in 1921 [150] and later confirmed by Jeghers et al. [151] in 1949. The pigmentation of the skin and mucosa in PJS groups this disease along with BRR and CNC as one of the familial lentiginosis syndromes. Melanin spots on the lips occur in the vast majority of PJS patients, with pigmentation appearing to be age specific, first appearing in infancy or early childhood and reaching a peak at puberty. The gastrointestinal polyps found in PJS are distinct from those found in the other hamartomatous polyposis syndromes, as histologically they contain a characteristic smooth mus-

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cle component infiltrating the connective tissue core in a branching pattern [for a review, see ref. 152]. They can occur as solitary lesions or in multiple large clusters. These polyps can arise throughout the gastrointestinal tract but are most common in the jejunum. In addition to the hamartomatous polyps, adenomatous and hyperplastic polyps have also been observed and mixtures of different histologies are known to occur [152]. Gastrointestinal malignancy is a feature of PJS and a hamartoma-adenomacarcinoma sequence paralleling the adenoma-carcinoma sequence known for colorectal carcinoma has been proposed [153, 154]. Apart from gastrointestinal malignancies, PJS patients also have an increased risk of breast, ovarian, testicular, uterine and cervical cancers, as well as non-malignant lesions in these tissues [152, 154–157]. A 49-year follow-up of the ‘Harrisburg family’, the kindred originally described by Jeghers et al. [151], revealed that PJS is a premalignant condition associated with significant morbidity and increased mortality [158]. Amongst the 12 affected family members, 10 underwent 75 polypectomies and 2 developed gastric cancer and duodenal carcinoma, respectively. In another report, cancer developed in 15 of 31 patients from 13 unrelated families; the overall incidence of carcinoma in patients with PJS varies from 20 to 50%, and it appears at a relatively early age [150–157, 159, 160]. Among the nongastrointestinal neoplasms associated with PJS, endocrine tumours are the most frequent [150–157, 161]. These include thyroid nodules [162], cancer [150–157] and genital tract neoplasms. The latter include, in female patients with PJS, ovarian neoplasms from both the epithelium and stromal cells, and also adenoma malignum of the cervix and adenocarcinoma of the endometrium [163, 164]. Male patients with PJS often have Leydig cell tumours, or a Sertoli cell tumour that is uniquely found in the two lentiginoses discussed in this chapter, PJS and CNC, as well as large-cell calcifying Sertoli cell tumours (LCCSCT) [165]. LCCSCT in PJS, as in CNC, can produce estradiol, which may lead to precocious puberty or gynecomastia [161, 165]. Mapping the PJS Gene – A Historical Perspective Initial regions of interest for the PJS gene on chromosomes 1 and 6 were excluded by linkage analysis in a study of 34 PJS families [166]. A combination of comparative genomic hybridisation using DNA from PJS polyps and LOH studies was able to localise a susceptibility locus for PJS to 19p [153]. These studies suggested that it was the wild-type allele that was lost in PJS polyps at this locus, hinting that the PJS susceptibility gene was likely functioning as a tumour suppressor. It has been postulated that the telomeric position of this locus may favour complete loss of the wild-type copy through events such as telomere shortening and that it is the loss of function of this second allele that initiates the growth and formation of hamartomatous polyps. However, whilst the majority of PJS fami-

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lies are linked to 19p13.3 [167], a second minor locus has been identified at 19q13.4 [168]. The PJS gene at 19p13.3 was mapped in 1998 by two independent groups – one naming the locus STK11 (for serine threonine kinase 11) and the other keeping an earlier name given to the then unlocalised and uncharacterised gene LKB1 [169, 170]. Together, these two initial reports presented 17 PJS families, 16 of which (95%) had germline mutations in STK11, the majority of which were speculated to lead to the truncation of proteins with the effect of generating proteins with incomplete catalytic domains. Later reports of mutation screening in PJS families have yielded substantially lower frequencies, i.e. 10–63%, with mutations occurring in all 9 exons and mutational hot spots at codons 51–84 of exon 1 as well as in the relatively short exon 7 [171–176]. The large difference in the mutation frequencies in these cohorts studied, from as low as 10% [174] to as high as between 50 and 100% [175, 176], could reflect the ascertainment criteria, the mutation detection screening methods used, a geographical bias or genetic heterogeneity. Whilst oncogenes functioning as protein kinases are responsible for cancer susceptibility in a number of inherited cancer syndromes, including multiple endocrine neoplasia type 2 (RET), familial renal papillary cancer (MET) and familial melanoma (CDK4), this is the first example of a gene predisposing to a cancer syndrome by inactivation of its encoded kinase activity [169]. STK11 (LKB1) – A Serine-Threonine Kinase STK11/LKB1 is a novel serine-threonine kinase containing 9 exons transcribed in a telomeric to centromeric direction. Its transcripts are 3.0 and 3.3 kb and would appear to be ubiquitously expressed [169, 170]. Its 433-amino acid residues show high homology (almost 84%) with XEEK1 (named for Xenopus egg and embryo kinase 1), a Xenopus cytosolic serine-threonine protein kinase [177]. A mouse homologue sharing approximately 93% overall identity with human STK11 has been identified, mapping to mouse chromosome 10 [178]. The core kinase domain is highly conserved (approaching 98%) between mice and humans. A nuclear localisation signal has been identified within the protein sequence, which is highly suggestive that mouse Lkb1 is a nuclear protein. Interestingly, wild-type STK11 shows both nuclear and cytoplasmic localisation. The effect of a number of mutations identified in the germline of PJS patients has been explored in vitro. As predicted, the majority of mutations would appear to abrogate kinase function; however, one mutation, a small in-frame deletion, caused accumulation of STK11 in the nucleus, thus altering its subcellular distribution [179, 180]. Residues 43–88 are believed to be critical for nuclear targeting [180]. Residues 1–346 are required for the kinase function of STK11, with a deletion mutant of 1–310 losing kinase activity [180]. Growth suppression

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of STK11 requires a functional kinase domain [181]. Thus, it would appear in most cases that elimination of the kinase activity of STK11 provides a background against which the PJS phenotype forms. Further, loss of the ability to be retained in the cytoplasm would appear to be pathogenic. It is postulated that cytoplasmic retention might be caused by the interaction of STK11 with its cytoplasmic substrate protein and that mutant STK11 may lose its ability to interact with and phosphorylate its substrate protein. A specific growth-inhibitory activity has been identified for STK11 that seems to be caused by a G1 cell cycle block [181] and does not involve increased apoptosis. Thus, STK11 likely functions at the level of a G0/G1 checkpoint rather than as a regulator of cell death [181]. STK11 in Sporadic Component Tumours of PJS Unlike PTEN, STK11/LKB1 has not been shown to have a significant role in any malignancy outside of PJS. Whilst LOH at the STK11 locus has been observed in between 19 and 53% of colorectal lesions, only a subset with a predominance of high-grade dysplasia adenomas and invasive carcinomas have somatic STK11 mutation [173, 182–187]. Thus, it is possible that STK11 has a role in tumour progression as opposed to tumour initiation in colorectal neoplasia [182]. Somatic STK11 mutation is only rarely found in pancreatic and biliary adenocarcinomas, sporadic testicular cancer and melanoma [175, 183, 185, 186]. Whilst the risk of breast cancer is elevated in PJS patients, no somatic mutations in STK11 have been identified, even though a small subset of primary breast cancers have LOH at 19p [187]. Fifty percent of ovarian cancers show allelic loss at 19p13.3; however, STK11 is rarely mutated in these tumours outside of PJS [184]. It is likely that allelic loss on 19p13.3, especially in the case of ovarian tumours, targets a gene other than STK11 [184]. It is also possible that gene clusters, rather than single genes, are targeted by these LOH events. Alternatively, STK11 may be regulated by a mechanism other than somatic mutation in sporadic cancers, such as hypermethylation of promoter sequences. Low levels of STK11 mRNA detected in a subset of cervical adenocarcinomas and melanoma may be evidence for alternative regulation of this gene in some sporadic tumours [181].

Carney Complex Clinical Features In 1985, 39 patients with the complex of spotty skin pigmentation, myxomas and endocrine overactivity were reported. In 1984, some of these patients had been found to have a pituitary-independent Cushing syndrome and an unusual adrenal pathology characterised by multiple, small, pigmented, adrenocortical

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Table 4. Clinical and pathological components of CNC Spotty skin pigmentation Myxomas Endocrine tumors

Schwannoma Other tumors

hypermelanosis, lentigines, blue and combined nevi heart, skin, breast, tongue, palate PPNAD testicular LCCSCT pituitary adenoma other: thyroid follicular adenoma, ovarian cysts sympathetic nerve chain, upper gastrointestinal tract breast ductal adenoma, predisposition to various cancers (?)

nodules and internodular cortical atrophy [161, 188–190]. This condition, which proved to be primary and bilateral, is now commonly referred to as primary pigmented nodular adrenal disease (PPNAD). It had been previously reported to be inherited in at least two families, one from Cuba and another from Switzerland [188–191]. Shortly afterward, this syndrome, apparently transmitted in a manner consistent with dominant inheritance [192, 193], was identified in three generations of a family. Soon, it was realised that ‘Carney complex’ described a clinical syndrome that included a number of associations reported earlier. The characteristic pathology of PPNAD had been described in children and young adults with Cushing syndrome as early as 1949 and in a number of case reports thereafter [161, 193]. Accordingly, several familial cases of cutaneous and cardiac myxomas associated with lentigines and blue nevi of the skin and mucosae had been described under the acronyms NAME (for nevi, atrial myxoma, myxoid neurofibromata and ephelides) and LAMB (for lentigines, atrial myxoma, mucocutaneous myxoma and blue nevi) [194, 195]. When compared to their sporadic counterparts, virtually all of the individual components of CNC are unusual (table 4). For example, the usual cardiac myxoma is a single tumour in the left atrium of an older, usually female, patient; in CNC, the tumour is often multiple, affects any or all cardiac chambers, occurs at a relatively young age and is equally distributed between the sexes [196]. The cutaneous myxomas have a predilection for the eyelids and external ear canals, although they may affect any part of the skin [197, 198]. Mammary myxoid fibroadenoma is usually an isolated finding in an otherwise normal breast [199]. In the complex, the tumour is often multiple and bilateral, and the non-tumourous breast commonly shows microscopic foci of non-neoplastic myxomatous change between the masses [199, 200]. The classic facies of CNC are characterised by relative hypertelorism and centrofacial spotty pigmentation that involves the vermilion border of the lips and the conjunctiva. The pigmented spots may be either (1) tan, irregularly

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shaped and poorly outlined, several millimetres in diameter and freckle-like, or (2) small, sharply delineated and dark brown to black. The conjunctival pigmentation typically affects the lacrimal caruncle and the conjunctival semilunar fold and may involve the sclera. Commonly, one or more eyelid masses (myxomas) are present. The combination of facial, labial and conjunctival spotty pigmentation and eyelid tumours presents a diagnostic picture that should precipitate investigation for cardiac myxoma or myxomas. Indeed, 5–10% of patients with CNC have one or more intraoral pigmented spots, and the female external genitalia are commonly heavily pigmented. Skin pigmentation in CNC is of multiple pathologic types (table 4). Most of the lesions are lentigines or represent other examples of hypermelanosis (increased melanin in basal cells and throughout the thickness of the epidermis). Blue nevi (the usual type, as well as the exceptionally rare epithelioid type), combined and common junctional, dermal and compound nevi, and café-au-lait spots (CALS) also occur in the syndrome [161, 201, 202]. Cushing syndrome, which appears to be the most common endocrine manifestation of CNC and affects about one third of patients, is caused by a distinct adrenal lesion, PPNAD, which is uniquely associated with the complex and only rarely occurs as an isolated, sporadic lesion. In this disorder, the glands are most commonly normal in size or small and peppered with black or brown nodules set in a cortex that is usually atrophic. This atrophy is pathognomonic and reflects the autonomous function of these nodules and the suppressed levels of pituitary ACTH. Despite their small size (less than 6 mm), the nodules are visible with computed tomography or magnetic resonance imaging of the adrenal glands, most likely because of the surrounding atrophy [203]. The combination of atrophy and nodularity gives the glands an irregular contour, which is distinctly abnormal and diagnostic, especially in younger patients with Cushing syndrome. Occasionally, one or both of the glands may be larger and harbour adenomas with a calcified centre, while macronodules larger than 10 mm may be present in older patients. Patients with CNC often present with a variant Cushing syndrome called ‘atypical’ Cushing syndrome (ACS) [204], which is characterised by an asthenic, rather than obese, body habitus caused by severe osteoporosis, short stature and muscle and skin wasting. ACS was recognised as early as 1956 and has since been described in several cases of patients with Cushing syndrome [205–207]; only recently, however, was this condition associated with CNC [208, 209]. A recent review of the literature indicated that almost all the reported cases of ACS could be attributed to PPNAD, including that of a patient who presented 27 years after unilateral adrenalectomy [208]. Patients with ACS tend to have normal or nearnormal 24-hour cortisol production, but this is characterised by the absence of the normal circadian rhythmicity of this hormone [204–210]. Occasionally, normal cortisol production is interrupted by days or weeks of hypercortisolism, which

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gives rise to yet another variant called ‘periodic Cushing syndrome’. Periodic Cushing syndrome is frequently found in children with the complex [209, 210]. All patients with PPNAD and classic CS, ACS or periodic Cushing syndrome respond to dexamethasone with a paradoxical rise in cortisol production [208, 210]. In a recent study of the largest series reported to date, all patients with PPNAD responded to the graded administration of dexamethasone during classic Liddle’s test with a rise in both urinary free cortisol and 17-hydroxy-corticosteroid production [210]. The test may be used diagnostically for the identification of PPNAD, even in patients who have normal baseline cortisol levels and do not have clinical stigmata of CS. About 10% of patients with CNC have a growth hormone (GH)-secreting pituitary adenoma that results in acromegaly [193]. Although most of the known patients with this condition had macroadenomas, a number of recently investigated cases showed that abnormal 24-hour GH and prolactin secretion can precede the development of a pituitary tumour in CNC [211, 212]. The disorder, therefore, provides an unusual opportunity for prospective screening of affected patients without clinical acromegaly. In one such case, serial measurements of GH or insulin-like growth factor 1 (IGF1), or both, became progressively abnormal over several years, but a possible pituitary mass was identified on computed tomography examination only recently; partial hypophysectomy revealed minute foci of a GH-producing adenoma and pituitary hyperplasia. Hyperplasia appears to have been present in the pituitary gland of all patients with CNC, acromegaly and a GH-producing microadenoma operated to date [213]. Endocrine involvement in CNC also includes three types of testicular tumours: LCCSCT (among the rarest of testicular neoplasms), adrenocortical rests and Leydig cell tumour [161, 193, 214]. About one third of affected male patients have these masses. The LCCSCT, a bilateral, multicentric and benign neoplasm, may secrete estrogens and cause precocious puberty, gynecomastia, or both [214]. LCCSCTs are frequent in PJS, too [165]. However, in PJS, the ovaries are also frequently affected [163, 164]; in CNC, the ovaries may be affected by cystic formation and occasionally epithelial cancer, but not by germ cell tumours [215]. Since 1985, the number of identified patients with CNC has more than quadrupled [161]. Information derived from these cases has resulted in a reordering of the frequency of occurrence of the components of CNC, with spotty skin pigmentation currently the most frequent component. In addition, three new components of the syndrome have been identified: psammomatous melanotic schwannoma, epithelioid blue nevus and ductal adenoma of the breast [216–218]. Because thyroid follicular neoplasms, both benign and malignant, have been found in a number of patients, it is possible that thyroid involvement will prove to be a component of the syndrome [219].

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Identification of the CNC Genetic Loci Two genetic loci have been determined for CNC by linkage analysis of polymorphic markers from likely areas of the genome [220, 221]. Initially, positive lod scores were obtained for nine markers on the short arm of chromosome 2, identifying an approximately 4-cM-long area in the cytogenetic band 2p16 (CNC locus), which was likely to contain the gene(s) responsible for CNC [212]. This region includes the D2S123 locus, where another genetic syndrome, hereditary non-polyposis colorectal cancer, had been mapped [220, 222]. The gene for hereditary nonpolyposis colorectal cancer (hMSH2) codes for a protein that plays a direct role in DNA mismatch repair, increasing microsatellite stability and enhancing mutation predisposition in human cells; this gene was excluded from being a candidate for the complex [222]. Another recently identified gene, hMSH6, also involved in DNA stability, is adjacent to hMSH2, but outside the defined region on 2p16 [222]. Although chromosomal instability may be a feature of several tumour cell lines established from patients with the lentiginosis syndromes, so far none of the genes involved in DNA repair or preservation of chromosomal integrity have been implicated in the pathogenesis of the lentiginoses. Indeed, the formation of telomeric associations and dicentric chromosomes is a frequent feature of fibroblasts derived from the myxoid tumours excised from patients with CNC [223–226]. Also, a recent study found similar features in in vitro cultured adrenocortical cells derived from PPNAD nodules [226]. Earlier investigations had found chromosomal instability in cultured skin fibroblasts, peripheral blood lymphocytes and adenomatous polyp cells established from patients with familial polyposis coli and PJS [227– 229]. In these studies, no specific chromosomal breaks or exchange points were revealed, although several sites were involved in three or more rearrangements. One of the first genes that was screened for mutations in patients with CNC was the gsp proto-oncogene (GNAS1) [230]; the locations of several other genes that code for components of the guanine nucleotide-binding proteins (G-proteins) were excluded by linkage analysis [220]. It was the substantial clinical overlap between McCune-Albright syndrome and CNC that made it likely that the gene(s) responsible for CNC participate in G-protein-controlled or -related signalling systems [161, 213]. The identification of the 2p16 locus for CNC [230] was followed by the description of a family that did not map to chromosome 2 [231]. This finding was then confirmed in another large kindred that had a number of recombinant genotypes with the first locus [232]. Genetic heterogeneity was confirmed in this syndrome when a second locus on 17q22-24 was identified [221]. This was followed by the recent identification of the PRKAR1A gene coding for the type I· regulatory subunit (RI·) of protein kinase A (PKA) as the gene responsible for CNC in most families that mapped to chromosome 17 and some sporadic cases of the disease (see below) [233].

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Fig. 4. Summary of the PRKAR1A gene and its mutations identified in families with CNC and sporadic cases. The DNA sequences are numbered according to the NCBI’s reference sequence for PRKAR1A, GB accession No. NM-002734.

Germline PRKAR1A Mutations in CNC Several ESTs from the PRKAR1A gene had been mapped to 17q22-24 [234]. LOH analysis using polymorphic markers from this region revealed consistent changes in tumours from patients with CNC, including those from one family previously mapped to 17q22-24 [221, 233]. Four more families were identified with CNC that was also mapped to this region [233]. Investigation of a polymorphic site within the 5) region of the PRKAR1A gene showed segregation with the disease in both families and retention of the allele bearing the disease gene in their tumours [233]. Heteroduplex analysis of each exon of this gene and sequencing demonstrated that three of the families had the same mutation in the PRKAR1A gene, a 2-bp deletion in exon 4B resulting in a frame shift predicted to lead to premature truncation of the PRKAR1A protein (578delTG). A founder effect was excluded in these families by haplotype analysis. Furthermore, the same mutation was found to have occurred de novo in an apparently sporadic case of CNC. Another germline mutation in exon 8 of PRKAR1A was found in an additional sporadic case with CNC (GG873CT), whereas no mutations were found in all but one of the kindreds that had been mapped to 2p16 (in this one family, one person was a silent carrier of the mutation and was previously thought to be unaffected) (fig. 4). Both 578delTG and GG873CT are predicted to lead to truncation of the RI· protein. The LOH data suggest that the PRKAR1A gene may function as a tumour

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suppressor gene in the tissues affected by CNC. Indeed, a defective cyclic nucleotide-dependent pathway has long been considered a candidate mechanism for the various manifestations of CNC [161, 230], including tumours similar to those of McCune-Albright syndrome (MAS) [212, 213] and paradoxical responses to hormonal stimuli [210]. However, measurements of baseline and post-stimulation intracellular cAMP levels in cultured tumours from patients with CNC [Stratakis, C.A., unpubl. observations] and mutation analysis of the GNAS1 gene [230] gave negative results. Thus, the defect in CNC was placed downstream from cAMP activation, and the PKA complex, a critical step in cAMP-dependent signalling, was seen as a likely candidate for the identification of mutations in patients with CNC. However, a recent study identified LOH for the PRKAR1A gene in CNC tumours, suggesting a tumour suppression function for normal RI·. Might haploinsufficiency for one of the subunits of PKA, an enzyme that has been linked to cancer through its overexpression [234–237], lead to an increase in cAMP signalling and explain the multiple tumour formation and tendency for carcinogenesis that is seen in patients with CNC? One possibility is that truncated or absent RI· may inhibit the normal formation of the multimeric PKA enzymatic complex, leading to dysregulated PKA activity [237]. Alternatively, the truncated RI· may interfere with the function of the normal components of the PKA complex in a dominant-negative manner, or, perhaps RI· deficiency leads to overexpression of the genes coding for the other regulatory subunits of the PKA complex, as has been demonstrated in other contexts (e.g. animal models in which one of the subunits has been knocked out) [237]. Other possible mechanisms include an uninhibited catalytic subunit of the PKA complex, which by itself when mutated may lead to unregulated PKA activity, or a reduced turnover of the cAMP molecule, because both 578delGT and GG873CT mutations interrupt the cAMP-binding domains of RI· [238]. Genetic Heterogeneity in CNC The identification of the gene causing CNC on chromosome 17 left a group of families that appear to map collectively to chromosome 2 (although none of them had a lod score over 3) for which the syndrome has not been molecularly elucidated. There are also families that seem to map neither to chromosome 2 nor to chromosome 17, allowing for a third possible locus harbouring gene(s) responsible for the complex. In tumours from patients with CNC, genetic changes in the chromosome 2p16 locus, including both copy number gain and loss, have been identified [215, 239]. Interestingly, these changes are shared by both chromosome 2- and chromosome 17-mapping families, indicating, perhaps, a common molecular mechanism [215, 239].

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Given the substantial clinical overlap between syndromes like BRR, PJS and CNC, it is not unlikely that some patients, especially sporadic cases of CNC, will end up being diagnosed with one of these overlapping conditions. It is characteristic of the potential diagnostic errors in patients with one of the lentiginoses that one of the first families identified with CNC and the first one that was found to harbour PRKAR1A mutations was at first misdiagnosed with PJS [240]. It remains to be seen whether functional characterisation of the responsible genes for all these disorders will provide a clearer molecular basis for the observed clinical overlaps.

The Related Syndromes – LEOPARD Syndrome, Proteus Syndrome and Cronkhite-Canada Syndrome A number of other rare syndromes show clinical overlap with CS, BRR, JPS, PJS and CNC, in addition to having other endocrine manifestations. LEOPARD syndrome is also known as ‘multiple lentigines syndrome’. First suggested by Gorlin et al. [241] in 1969, the acronym describes the association of lentigines (multiple, darkly pigmented and present on the lips, but absent from other mucosal sites), electrocardiographic abnormalities, ocular hypertelorism (with other dysmorphic features), pulmonary stenosis, abnormalities of the genitalia (hypogonadism), (mental) retardation and deafness (sensorineural) [241]. This condition was also referred to as ‘cardiocutaneous syndrome’ in older reports which had not recognised the pleomorphism of the phenotype [242, 243]. Watson syndrome, characterised by lentigines, CALS, pulmonic stenosis and mental retardation, as well as the association of heart defects, pigmented lesions and deafness in other patients, could represent variant forms of the LEOPARD syndrome [244]. LEOPARD syndrome and its variants are inherited in an autosomal dominant manner. Variable expression within the same family, a feature of all the lentiginosis syndromes, is frequently seen. This is best exemplified by a kindred where the propositus, an 11-year-old boy, had many lentigines and severe heart problems. His father and 5 out of 6 siblings with generalised lentiginosis had no other abnormalities, and 9 other relatives from three generations who had lentigines were otherwise healthy [245]. Almost all the patients had low intelligence and other delays, as well as various skeletal abnormalities [245]. In addition, neural tube defects have been described in association with LEOPARD syndrome, and the facies of many patients are reminiscent of other conditions that include heart or skeletal defects, or both, such as Noonan syndrome [244–246]. The patients can also have other pigmented lesions, including dark CALS, junctional nevi and abnormal pigmentation of the iris and retina [247, 248]. Neoplasms, although uncommon, can also be present in LEOPARD syndrome and include rhabdomyosarcoma and granular cell myoblastoma [249]. Kindreds with LEO-

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PARD syndrome and those with Noonan syndrome and pigmented lesions have been tested for linkage to the neurofibromatosis type 1 (NF-1) locus on chromosome 17q11.2 [250, 251]; indeed, a patient with NF-1 and lentigines had mutations in the NF-1 gene [252]. The overgrowth syndrome PS, named after the Greek god Proteus (‘the polymorphous’), who could change his shape at will to avoid capture, was likely the disease suffered by the elephant man [253]. Patients with PS display multiple hamartomas including lipomas, macrocephaly (14% of patients) and pigmented skin lesions, leading the authors of one report to note a significant difficulty in differentiating between BRR and PS [254]. To date, an underlying genetic defect in PS has not been identified, although the vast majority of cases would appear to be sporadic. One sporadic case was found to have a de novo mosaic chromosomal abnormality likely to be a direct duplication of a segment of chromosome 1, 1q11→q25; however, it cannot be excluded that this may be a coincidental finding [255]. Given the sporadic nature of PS, it has been postulated that this condition is the result of a post-zygotic event leading to a mosaic effect that may be lethal if it were to occur in the non-mosaic state [256]. CC syndrome also shows phenotypic overlap with the classic hamartoma syndromes, the gastrointestinal polyps found in the stomach, small bowel and colon histologically resembling the appearance of juvenile polyps. However, the pigmentation observed in CC is diffuse rather than spotty, as is seen in PJS [257]. In CC, all cases appear to be sporadic; however, unlike PS, all patients have presented as adults [258]. The aetiology of CC syndrome is unknown.

Summary The classic hamartoma syndromes and the related conditions discussed in this chapter show varying degrees of phenotypic and genetic overlap. Knowledge of the susceptibility genes underlying their phenotypes has provided additional information for the classification of these syndromes. Germline PTEN mutations appear to cause both CS and BRR. These two syndromes are therefore likely to be different manifestations of a single disease with variable expression. It has been suggested that PTEN mutation-positive CS and BRR should be grouped as a single entity for clinical purposes and classified as the ‘PTEN hamartoma-tumour syndrome’ [111]. Germline PTEN mutations are unlikely to cause JPS. However, germline SMAD4 mutation, especially a well-described 4-bp deletion, can be used to confirm a clinical diagnosis of JPS. Like the hamartoma syndromes, the lentiginoses also show substantial clinical overlap; it remains to be seen whether this is reflected in the molecular pathways that are involved in the pathogenesis of these syndromes.

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Given that the degree and type of cancer susceptibility between the hamartoma and lentiginosis syndromes is different, clarification of these syndromes at the molecular level is predicted to allow directed cancer surveillance. The molecular story is still evolving with regards to aspects of genetic heterogeneity, signalling pathways and the manner in which these hamartoma genes function in the development of their respective syndromes. It is likely that in all of these syndromes, tumours develop against a background created by loss of the growth-suppressive function of their susceptibility gene via mechanisms including disruption of the cell cycle and the activation of anti-apoptotic pathways.

Acknowledgements The authors would like to acknowledge works in the field not cited due to space restrictions. D.J.M. would like to thank Oliver Gimm and Roberto Zori for their critical reading of parts of this chapter and many helpful comments. C.A.S. would like to thank Dr. J.A. Carney (Mayo Clinic) for his continuing friendship and mentorship, and his help in the preparation of sections of this text.

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169 Hemminki A, Markie D, Tomlinson I, Avizienyte E, Roth S, Loukola A, Bignell G, Warren W, Aminoff M, Höglund P, Järvinen H, Kristo P, Pelin K, Ridanpää M, Salovaara R, Toro T, Bodmer W, Olschwang S, Olsen AS, Stratton MR, de la Chapelle A, Aaltonen LA: A serine/threonine kinase gene defective in Peutz-Jeghers syndrome. Nature 1998;391:184–187. 170 Jenne DE, Reimann H, Nezu J, Friedel W, Loff S, Jeschke R, Müller O, Back W, Zimmer M: Peutz-Jeghers syndrome is caused mutations in a novel serine threonine kinase. Nat Genet 1998;18: 38–43. 171 Westerman AM, Entius MM, Boor PPC, Koole R, de Baar E, Offerhaus GJA, Lubinski J, Lindhout D, Halley DJJ, de Rooij FWM, Wilson JHP: Novel mutations in the LKB1/STK11 gene in Dutch Peutz-Jeghers families. Hum Mutat 1999;13:476–481. 172 Wang Z-J, Churchman M, Avizienyte E, McKeown C, Davies S, Evans DGR, Ferguson A, Ellis I, Xu W-H, Yan Z-Y, Aaltonen LA, Tomlinson IPM: Germline mutations of the LKB1 (STK11) gene in Peutz-Jeghers patients. J Med Genet 1999;36:365–368. 173 Resta N, Simone C, Mareni C, Montera M, Gentile M, Susca F, Gristina R, Pozzi S, Bertario L, Bufo P, Carlomagno N, Ingrosso M, Rossini FP, Tenconi R, Guanti G: STK11 mutations in PeutzJeghers syndrome and sporadic colon cancer. Cancer Res 1998;58:4799–4801. 174 Jiang C-Y, Esufali S, Berk T, Gallinger S, Cohen Z, Tobi M, Redston M, Bapat B: STK11/LKB1 germline mutations are not identified in most Peutz-Jeghers syndrome patients. Clin Genet 1999; 56:136–141. 175 Ylikorkala A, Avizienyte E, Tomlinson IPM, Tiainen M, Roth S, Loukola A, Hemminki A, Johansson M, Sistonen P, Markie D, Neale K, Phillips R, Zauber P, Twama T, Sampson J, Järvinen H, Mäkelä TP, Aaltonen LA: Mutations and impaired function of LKB1 in familial and non-familial Peutz-Jeghers syndrome and a sporadic testicular cancer. Hum Mol Genet 1999;8:45–51. 176 Boardman LA, Couch FJ, Burgart LJ, Schwartz D, Berry R, McDonnell SK, Schaid DJ, Hartmann LC, Schroeder JJ, Stratakis CA, Thibodeau SN: Genetic heterogeneity in Peutz-Jeghers syndrome. Hum Mutat 2000;16:23–30. 177 Su J-Y, Erikson E, Maller JL: Cloning and characterization of a novel serine/threonine protein kinase expressed in early Xenopus embryos. J Biol Chem 1996;271:14430–14437. 178 Smith DP, Spicer J, Smith A, Swift S, Ashworth A: The mouse Peutz-Jeghers syndrome gene Lkb1 encodes a nuclear protein kinase. Hum Mol Genet 1999;8:1479–1485. 179 Mehenni H, Gehrig C, Nezu J, Oku A, Shimane M, Rossier C, Guex N, Blouin J-L, Scott HS, Antonarakis SE: Loss of LKB1 kinase activity in Peutz-Jeghers syndrome, and evidence for allelic and locus heterogeneity. Am J Hum Genet 1998;63:1641–1650. 180 Nezu J, Oku A, Shimane M: Loss of cytoplasmic retention ability of mutant LKB1 found in PeutzJeghers syndrome patients. Biochem Biophys Res Commun 1999;261:750–755. 181 Tiainen M, Ylikorkala A, Mäkelä TP: Growth suppression by Lkb1 is mediated by a G1 cell cycle arrest. Proc Natl Acad Sci USA 1999;96:9248–9251. 182 Dong SM, Kim KM, Kim SY, Shin MS, Na EY, Lee SH, Park WS, Yoo NJ, Jang JJ, Yoon CY, Kim JW, Kim SY, Yang YM, Kim SH, Kim CS, Lee JY: Frequent somatic mutations in serine/threonine kinase 11/Peutz-Jeghers syndrome gene in left-sided colon cancer. Cancer Res 1998;58:3787– 3790. 183 Avizienyte E, Roth S, Loukola A, Hemminki A, Lothe RA, Stenwig AE, Fossa SD, Salovaara R, Aaltonen LA: Somatic mutations in LKB1 are rare in sporadic colorectal and testicular tumors. Cancer Res 1998;58:2087–2090. 184 Wang Z-J, Churchman M, Campbell IG, Xu W-H, Yan Z-Y, McCluggage WG, Foulkes WD, Tomlinson IPM: Allele loss and mutation screen at the Peutz-Jeghers (LKB1) locus (19p13.3) in sporadic ovarian tumours. Br J Cancer 1999;80:70–72. 185 Su GH, Hruban RH, Bansal RK, Bova GS, Tang DJ, Shekher MC, Westerman AM, Entius MM, Goggins M, Yeo CJ, Kern SE: Germline and somatic mutations of the STK11/LKB1 Peutz-Jeghers gene in pancreatic and biliary cancers. Am J Pathol 1999;154:1835–1840. 186 Guldberg P, thor Straten P, Ahrenkiel V, Seremet T, Kirkin AF, Zeuthen J: Somatic mutation of the Peutz-Jeghers syndrome gene, LKB1/STK11, in malignant melanoma. Oncogene 1999;18:1777– 1780.

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187 Bignell GR, Barfoot R, Seal S, Collins N, Warren W, Stratton MR: Low frequency of somatic mutations in the LKB1/Peutz-Jeghers syndrome gene in sporadic breast cancer. Cancer Res 1998; 58:1384–1386. 188 Carney JA, Gordon H, Carpenter PC, Shenoy BV, Go VLW: The complex of myxomas, spotty pigmentation, and endocrine overactivity. Medicine (Baltimore) 1985;64:270–283. 189 Salomon F, Froesch ER, Hedinger CE: Familial Cushing syndrome (Carney complex) (letter). N Engl J Med 1990;322:1470. 190 Shenoy BV, Carpenter PC, Carney JA: Bilateral primary pigmented nodular adrenocortical disease. Rare cause of the Cushing syndrome. Am J Surg Pathol 1984;8:335–344. 191 Schweizer-Cagianut M, Froesch ER, Hedinger CE: Familial Cushing’s syndrome with primary adrenocortical microadenomatosis (primary adrenocortical nodular dysplasia). Acta Endocrinol (Copenh) 1980;94:529–535. 192 Carney JA, Hruska LS, Beauchamp GD, Gordon H: Dominant inheritance of the complex of myxomas, spotty pigmentation and endocrine overactivity. Mayo Clin Proc 1986;61:165–172. 193 Carney JA, Young WF: Primary pigmented nodular adrenocortical disease and its associated conditions. Endocrinologist 1992;2:6–21. 194 Atherton DJ, Pitcher DW, Wells RS, Macdonald DM: A syndrome of various cutaneous pigmented lesions, myxoid neurofibromata and atrial myxoma: The NAME syndrome. Br J Dermatol 1980; 103:421–429. 195 Rhodes AR, Silverman RA, Harrist TJ, Perez-Atayde AR: Mucocutaneous lentigines, cardiomucocutaneous myxomas, and multiple blue nevi: The ‘LAMB’ syndrome. J Am Acad Dermatol 1984; 10:72–82. 196 Carney JA: Differences between nonfamilial and familial cardiac myxoma. Am J Surg Pathol 1985; 9:53–55. 197 Kennedy RH, Flanagan JC, Eagle RC Jr, Carney JA: The Carney complex with ocular signs suggestive of cardiac myxoma. Am J Ophthalmol 1991;111:699–702. 198 Ferreiro JA, Carney JA: Myxomas of the external ear and their significance. Am J Surg Pathol 1994; 18:274–280. 199 Carney JA, Toorkey BC: Myxoid fibroadenoma and allied conditions (myxomatosis) of the breast. A heritable disorder with special associations including cardiac and cutaneous myxomas. Am J Surg Pathol 1991;15:713–721. 200 Courcoutsakis NA, Chow CK, Shawker T, Carney JA, Stratakis CA: Breast imaging findings in the complex of myxomas, spotty pigmentation, endocrine overactivity, and schwannomas (Carney complex). Radiology 1997;205:221–227. 201 Carney JA, Ferreiro JA: The epithelioid blue nevus. A multicentric familial tumor with important associations, including cardiac myxoma and psammomatous melanotic schwannoma. Am J Surg Pathol 1996;20:259–272. 202 Carney JA: Carney complex: The complex of myxomas, spotty pigmentation, endocrine overactivity and schwannomas. Semin Dermatol 1995;14:90–98. 203 Doppman JL, Travis WD, Nieman L, Miller DL, Chrousos GP, Gomez TM, Loriaux DL: Cushing syndrome due to primary pigmented nodular adrenocortical disease: Findings at CT and MR imaging. Radiology 1989;172:415–420. 204 Mellinger RC, Smith RW: Studies of the adrenal hyperfunction in 2 patients with atypical Cushing’s syndrome. J Clin Endocrinol Metab 1955;16:350–366. 205 Kracht J, Tamm J: Bilaterale kleinknotige Adenomatose der Nebennierenrinde bei Cushing-Syndrom. Virchows Arch Pathol Anat 1960;333:1–9. 206 Levin ME: The development of bilateral adenomatous adrenal hyperplasia in a case of Cushing’s syndrome of eighteen years’ duration. Am J Med 1966;40:318–324. 207 De Moor P, Roels H, Delaere K, Crabbe J: Unusual case of adrenocortical hyperfunction. J Clin Endocrinol Metab 1965;25:612–620. 208 Sarlis NJ, Chrousos GP, Doppman JL, Carney JA, Stratakis CA: Primary pigmented nodular adrenocortical disease: Reevaluation of a patient with Carney complex 27 years after unilateral adrenalectomy. J Clin Endocrinol Metab 1997;82:2037–2043. 209 Gomez-Muguruza MT, Chrousos GP: Periodic Cushing’s syndrome in a short boy: Usefulness of the ovine corticotropin releasing hormone test. J Pediatr 1989;115:270–273.

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210 Stratakis CA, Sarlis NJ, Kirschner LS, Carney JA, Doppman JL, Chrousos GP, Papanicolaou DA: Paradoxical response to dexamethasone assists with the diagnosis of primary pigmented nodular adrenocortical disease. Ann Intern Med 1999;131:585–591. 211 Watson JC, Stratakis CA, Bryant-Greenwood PK, Koch CA, Kirschner LS, Nguyen T, Carney JA, Oldfield EH: Neurosurgical implications of Carney complex. J Neurosurg 2000;92:413–418. 212 Raff SB, Carney JA, Krugman D, Doppman JL, Stratakis CA: Prolactin secretion abnormalities in patients with the ‘syndrome of spotty skin pigmentation, myxomas, endocrine overactivity and schwannomas’ (Carney complex). J Pediatr Endocrinol Metab 2000;13:373–379. 213 Pack S, Kirschner LS, Pak E, Carney JA, Zhuang Z, Stratakis CA: Pituitary tumors in patients with the ‘complex of spotty skin pigmentation, myxomas, endocrine overactivity and schwannomas’ (Carney complex): Evidence for progression from somatomammotroph hyperplasia to adenoma. J Clin Endocrinol Metab 2000;85:3860–3865. 214 Premkumar A, Stratakis CA, Shawker TH, Papanicolaou DA, Chrousos GP: Testicular ultrasound in Carney complex. J Clin Ultrasound 1997;25:211–214. 215 Stratakis CA, Papageorgiou T, Premkumar A, Kirschner LS, Taymans SE, Pack S, Zhuang Z, Oelkers WH, Carney JA: Ovarian cysts in patients with Carney complex: Clinical and genetic studies and evidence for predisposition to cancer. J Clin Endocrinol Metab 2000;85:4359–4366. 216 Carney JA: Psammomatous melanotic schwannoma. A distinctive, heritable tumor with special associations, including cardiac myxoma and the Cushing syndrome. Am J Surg Pathol 1990;14: 206–222. 217 Carney JA, Toorkey BC: Ductal adenoma of the breast with tubular features. A probable component of the complex of myxomas, spotty pigmentation, endocrine overactivity, and schwannomas. Am J Surg Pathol 1991;15:722–731. 218 Carney JA, Stratakis CA: Ductal adenoma of the breast (letter). Am J Surg Pathol 1996;20:1154– 1155. 219 Stratakis CA, Courcoutsakis N, Abati A, Filie A, Doppman JL, Carney JA, Shawker TH: Thyroid gland abnormalities in patients with the ‘syndrome of spotty skin pigmentation, myxomas, and endocrine overactivity’ (Carney complex). J Clin Endocrinol Metab 1997;82:2037–2043. 220 Stratakis CA, Carney JA, Lin J-P, Papanicolaou DA, Karl M, Kastner DL, Pras E, Chrousos GP: Carney complex, a familial multiple neoplasia and lentiginosis syndrome. Analysis of 11 kindreds and linkage to the short arm of chromosome 2. J Clin Invest 1996;97:699–705. 221 Casey M, Mah C, Merliss AD, Kirschner LS, Taymans SE, Denio AE, Korf B, Irvine AD, Hughes A, Carney JA, Stratakis CA, Basson CT: Identification of a novel genetic locus for familial cardiac myxomas and Carney complex. Circulation 1998;98:2560–2566. 222 Stratakis CA, Pras E, Lin J-P, Kastner DL, Carney JA, Chrousos GP: Carney complex, a multiple endocrine neoplasia and familial lentiginosis syndrome: Clinical analysis and linkage to the D2S123 locus (chromosome 2p16). Am J Hum Genet 1995;57:A54. 223 Dewald GW, Dahl RJ, Spurbeck JL, Carney JA, Gordon H: Chromosomally abnormal clones and nonrandom telomeric translocations in cardiac myxomas. Mayo Clin Proc 1987;62:558–567. 224 Dijkhuizen T, van der Derg E, Molenaar WM, Meuzelaar JJ, de Jong B: Cytogenetics of a case of cardiac myxoma. Cancer Genet Cytogenet 1992;63:73–75. 225 Richkind KE, Wason D, Vidaillet HJ: Cardiac myxoma characterized by clonal telomeric association. Genes Chromosomes Cancer 1994;9:68–71. 226 Stratakis CA, Jenkins RB, Pras E, Mitsiades CS, Raff SB, Stalboerger P, Tsigos C, Carney JA, Chrousos GP: Cytogenetic and microsatellite alterations in tumors from patients with the syndrome of myxomas, spotty skin pigmentation, and endocrine overactivity (Carney complex). J Clin Endocrinol Metab 1996;81:3607–3614. 227 Takai S, Iwama T, Tonomura A: Chromosome instability in cultured skin fibroblasts from patients with familial polyposis coli and Peutz-Jeghers syndrome. Jpn J Cancer Res 1986;77:759–766. 228 Griffin CA, Lazar S, Hamilton SR, Giardello FM, Long P, Krush AJ: Cytogenetic analysis of intestinal polyps in polyposis syndromes: Comparison with sporadic colorectal adenomas. Cancer Genet Cytogenet 1993;67:14–20. 229 Richard F, Muleris M, Dutrillaux B: Chromosome instability in lymphocytes from patients affected by or genetically predisposed to colorectal cancer. Cancer Genet Cytogenet 1994;73:23–32.

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230 DeMarco L, Stratakis CA, Boson WL, Yakbovitz O, Carson E, Adrade LM, Chrousos GP, Nordenskjold M, Friedman E: Sporadic cardiac myxomas and tumors from patients with Carney complex are not associated with activating mutations of the Gs· gene. Hum Genet 1996;98:185–188. 231 Basson CT, MacRae CA, Korf B, Merliss A: Genetic heterogeneity of familial atrial myxoma syndromes (Carney Complex). Am J Cardiol 1997;79:994–995. 232 Taymans SE, Macrae CA, Casey M, Merliss A, Lin J-P, Rocchi M, Kirschner LS, Basson CT, Stratakis CA: A refined genetic, radiation hybrid, and physical map of the Carney complex (CNC) locus on chromosome 2p16; evidence for genetic heterogeneity in the syndrome. Am J Hum Genet 1997; 61(suppl): A84. 233 Kirschner LS, Carney JA, Pack SD, Taymans SE, Giatzakis C, Cho YS, Cho-Chung YS, Stratakis CA: Mutations of the gene encoding the protein kinase A type I· regulatory subunit in patients with Carney complex. Nat Genet 2000;26:89–92. 234 Boshart M, Weih F, Nichols M, Schutz G: The tissue-specific extinguisher locus TSE1 encodes a regulatory subunit of c-AMP dependent protein kinase. Cell 1991;66:849–859. 235 Esapa CT, Harris PE: Mutation analysis of protein kinase A catalytic subunit in thyroid adenomas and pituitary adenomas. Eur J Endocrinol 1999;141:409–412. 236 Orellana SA, McKnight GS: Mutations in the catalytic subunit of cAMP-dependent protein kinase result in unregulated biological activity. Proc Natl Acad Sci USA 1992;89:4726–4730. 237 Scott JD: Cyclic nucleotide-dependent protein kinases. Pharmacol Ther 1991;50:123–145. 238 Beebe SJ, Holloway R, Rannels SR, Corbin JD: Two classes of cAMP analogs which are selective for the two different cAMP-binding sites of type II protein kinase demonstrate synergism when added together to intact adipocytes. J Biol Chem 1984;259:3539–3547. 239 Taymans SE, Kirschner LS, Pack S, Ping Z, Stratakis CA: YAC-BAC contig of the Carney complex (CNC) critical region on 2p16 and copy number gain of 2p16 in CNC tumors: Evidence for a novel oncogene? Am J Hum Genet 1999;65(suppl):A326. 240 Stratakis CA, Kirschner LS, Taymans SE, Tomlinson IPM, Marsh DJ, Torpy DJ, Giatzakis C, Eccles DM, Theaker J, Houlston RS, Blouin J-L, Antonarakis SE, Basson CT, Eng C, Carney JA: Carney complex, Peutz-Jeghers syndrome, Cowden disease, and Bannayan-Zonana syndrome share cutaneous and endocrine manifestations, but not genetic loci. J Clin Endocrinol Metab 1998;83: 2972–2976. 241 Gorlin RJ, Anderson RC, Blaw M: Multiple lentigenes syndrome. Am J Dis Child 1969;117:652– 662. 242 Polani PE, Moynahan EJ: Progressive cardiomyopathic lentiginosis. Q J Med 1972;41:205–225. 243 Senanez H, Mane-Garzon F, Kolski R: Cardio-cutaneous syndrome (the ‘LEOPARD’ syndrome). Review of the literature and a new family. Clin Genet 1976;9:266–276. 244 Watson GH: Pulmonary stenosis, cafe-au-lait spots, and dull intelligence. Arch Dis Child 1967;42: 303–307. 245 Gorlin RJ, Anderson RC, Moller JH: The Leopard (multiple lentigines) syndrome revisited. Birth Defects Orig Artic Ser 1971;7:100–115. 246 Agha A, Hashimoto K: Multiple lentigenes (Leopard) syndrome with Chiari I malformation. J Dermatol 1995;22:520–523. 247 Arnsmeiler SL, Paller AS: Pigmentary anomalies in the multiple lentigines syndrome: Is it distinct from the LEOPARD syndrome? Pediatr Dermatol 1996;13:100–104. 248 Smith RF, Puliciccio LU, Holmes AV: Generalized lentigo, electrocardiographic abnormalities, conduction disorders and arrhythmia in three cases. Am J Cardiol 1970;25:501–506. 249 Heney D, Lockwood L, Allibone EB, Bailey CC: Nasopharyngeal rhabdomyosarcoma and multiple lentigines syndrome: A case report. Med Pediatr Oncol 1992;20:227–228. 250 Bahuau M, Flintoff W, Assouline B, Lyonnet S, Le Merrer M, Prieur M, Guilloud-Bataille M, Feingold N, Munnich A, Vidaud M, Vidaud D: Exclusion of allelism of Noonan syndrome and neurofibromatosis type 1 in a large family with Noonan syndrome-neurofibromatosis association. Am J Med Genet 1996;66:347–355. 251 Ahlbom EB, Dahl N, Zetterqvist P, Anneren G: Noonan syndrome with cafe-au-lait spots and multiple lentigines syndrome are not linked to the neurofibromatosis type 1 locus. Clin Genet 1995; 48:85–89.

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252 Wu R, Legius E, Robberecht W, Dumoulin M, Cassiman JJ, Fryns JP: Neurofibromatosis type 1 gene mutation in a patient with features of LEOPARD syndrome. Hum Mutat 1996;8:51–56. 253 Tibbles JAR, Cohen MM: The Proteus syndrome: The Elephant man diagnosed. Br Med J 1983; 293:683–685. 254 Bialer MG, Riedy MJ, Wilson WG: Proteus syndrome versus Bannayan-Zonana syndrome: A problem in differential diagnosis. Eur J Pediatr 1988;148:122–125. 255 Say B, Carpenter NJ: Report of a case resembling the Proteus syndrome with a chromosome abnormality. Am J Med Genet 1988;31:987–989. 256 Biesecker LG, Happle R, Mulliken JB, Weksberg R, Graham JM, Viljoen DL, Cohen MM Jr: Proteus Syndrome: Diagnostic criteria, differential diagnosis, and patient evaluation. Am J Med Genet 1999;84:389–395. 257 Harned RK, Buck JL, Sobin LH: The hamartomatous polyposis syndromes: Clinical and radiologic features. Am J Roentgenol 1995;164:565–571. 258 Cronkhite LW, Canada WJ: Generalized gastrointestinal polyposis: An unusual syndrome of polyposis, pigmentation, alopecia, and onychotrophia. N Engl J Med 1955;252:1011–1015. 259 Iida S, Tanaka Y, Fujii H, Hayashi S, Kimura M, Nagareda T, Moriwaki K: A heterozygous frameshift mutation of the PTEN/MMAC1 gene in a patient with Lhermitte-Duclos disease – Only the mutated allele was expressed in the cerebellar tumor. Int J Mol Med 1998;1:925–929. 260 Iida S, Nakamura Y, Fujii H, Kimura M, Moriwaki K: A heterozygous germline mutation of the PTEN/MMAC1 gene in a patient with Cowden disease. Int J Mol Med 1998;1:565–568. 261 Kurose K, Araki T, Matsunaka T, Takada Y, Emi M: Variant manifestations of Cowden disease in Japan: Hamartomatous polyposis of the digestive tract with mutation of the PTEN gene. Am J Hum Genet 1999;64:308–310. 262 Raizis AM, Ferguson MM, Robinson BA, Atkinson CH, George PK: Identification of a novel PTEN mutation (L139X) in a patient with Cowden disease and Sjögren’s syndrome. Mol Pathol 1998;51:339–341.

Deborah J. Marsh, Cancer Genetics, Kolling Institute of Medical Research Royal North Shore Hospital, St Leonards, Sydney, NSW 2065 (Australia) Tel. +61 2 9926 7176, Fax +61 2 9926 8484, E-Mail [email protected] Dr. Constantine Stratakis, NIH, NICHD, DEB, Building 10, Rm 10N262 Bethesda, MD 20892-1862 (USA) Tel. +1 301 4964 686/402-1998, Fax +1 301 4020 574, E-Mail [email protected]

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Subject Index

Adnexal papillary cystadenomas, von Hippel-Lindau syndrome 137 Anticipation, multiple endocrine neoplasia type 1 55 Bannayan-Riley-Ruvalcaba syndrome clinical features 183, 184 hamartoma syndrome 168 PTEN gene mutations Cowden syndrome comparison 185 genotype-phenotype correlations 185, 186 germline mutations 184, 185 overview 4, 5, 169 Calcitonin, medullary thyroid carcinoma diagnosis 109, 110, 112, 117 Candidate gene, cloning of disease genes 27, 28 Carbonic anhydrase, VHL knockout and overexpression 153 Carney complex clinical features acromegaly 194 Cushing syndrome 193, 194 overview 192 skin spots 192, 193 testicular tumors 194 genes GNAS1 proto-oncogene 195 heterogeneity 197, 198 mapping 195 mutations overview 5 PRKARIA germline mutations 196, 197 historical perspective of study 191, 192

Chromosomal translocation candidate gene approach 29–31 fluorescence in situ hybridization 30 multiple endocrine neoplasia type 2 31, 32 PAX8-PPARÁ1 32, 33 rapid amplification of cDNA ends 30, 31 Comparative genomic hybridization gene amplifications and deletions 33 sporadic cancer analysis 26 Cowden syndrome clinical features 170 hamartoma syndrome 168 PTEN gene mapping 170, 171 mutations overview 4, 5, 169 genotype-phenotype correlations 181 germline mutations 179–181 sporadic tumors 181–183 CpG islands, see DNA methylation Cronkhite-Canada syndrome, features 199 Cullin-2, VHL interactions 144, 145, 147, 150 Denaturing gradient gel electrophoresis, gene mutation screening 40, 41 Differential display pituitary tumor-transforming gene cloning 35 principle 35 DNA methylation CpG islands 11 methyltransferases 11 tumorigenesis role 11, 12

214

DNA microarray applications 37 assay principles 36, 37 E3 ubiquitin ligase complex hypoxia-inducible factor 1· as substrate 148, 149 protein interactions 145, 148, 149 Elongins C/B, VHL interactions 144, 147, 148, 150 Ethics, genetic privacy 16 Exon trapping, cloning of disease genes 27 Expressed sequence tag, gene sequence 28, 29 Fibronectin, extracellular matrix abnormalities with VHL knockout 151–153 Fluorescence in situ hybridization chromosomal translocations 30 sporadic cancer analysis 26 Focal adhesion kinase, PTEN interactions 176, 177 Founder effect, definition 25 Genetic counseling, overview 15, 16 Genomic imprinting Beckwith-Wiedemann syndrome role 12, 13 clustering of genes 12 tumorigenesis role 12, 13 Glial-derived neurotrophic factor, RET signaling role 83, 84 Hirschsprung disease, RET mutations 91, 92, 107 Hypoxia-inducible factor 1· E3 ubiquitin ligase complex substrate 148, 149 VHL knockout effects on levels 150, 151, 155 JunD, MENIN interactions 67, 68 Juvenile polyposis syndrome clinical features 186 gene linkage analysis 187 mapping 186, 187 mutations heterogeneity 187

Subject Index

overview 169 SMAD4 187, 188 hamartoma syndrome 168 LEOPARD syndrome clinical features 198, 199 gene mutations 198, 199 Linkage analysis familial cancer syndromes 23–25 genetic heterogeneity 25 Loss of heterozygosity, sporadic cancer analysis 26, 27 Medullary thyroid carcinoma C cells 107, 108 calcitonin levels in serum 109, 110, 112 CEA detection 110, 111, 117 familial medullary thyroid carcinoma gene mutations 84, 85, 87, 105, 106, 123 fine-needle aspiration 109 imaging 110, 111 incidence 107 index patient diagnosis 108–110 lymph node metastasis 109 sporadic cases 108 treatment chemotherapy 115, 116 follow-up 116, 117 lymphadenectomy 113 MEN 2B 115 metastasis 115 octreotide 116 prophylactic thyroidectomy 113, 114 radiation therapy 115 radioiodine therapy 115 thyroidectomy 113 MEN1 anticipation 55 cloning strategies 25, 60 locus 60 MENIN product, see MENIN mutation genetic testing 58 germline mutations 62, 63 multiple mutations 61 screening techniques 39, 40 sporadic mutations 63 types 63, 64 organization 64

215

MEN1 (continued) ortholog studies advantages 68, 69 mouse 69 rat 69 sequence homology 70–72 zebrafish 69, 70 phenocopies 55 transcript distribution 65 MENIN cell cycle expression 66 function 67, 68, 72 gene, see MEN1 JunD interactions 67, 68 nuclear localization signal 66, 67 tumor suppression activity 68 Western blot analysis 65 Multiple endocrine neoplasia type 1 age at diagnosis 53 chromosome instability 72 epidemiology 51 family case management 58–60 gene mutations, see MEN1 genotype-phenotype correlation 54, 55 mitogenic factor in plasma 72 modifier genes 55, 56 prospects for research 73 tumors adrenals 53 nonendocrine tumors 53 pancreas 52, 57 parathyroid 52 pituitary 53 thyroid 53 treatment of tumors pancreas 57 parathyroid 60 pituitary 57, 58 surgery 57 surveillance 60 types 51, 52 variants 56 Multiple endocrine neoplasia type 2 gene mutation, see RET subtypes 104 genotype-phenotype correlation 86, 87, 104–107 hyperparathyroidism in MEN 2A diagnosis 121, 122 treatment 122

Subject Index

medullary thyroid carcinoma, see Medullary thyroid carcinoma MEN 2B-specific disorders 123 pheochromocytoma, see Pheochromocytoma prospects for research 124 RET mutation-negative families 124 skin amyloidosis in MEN 2A 122 p53, gene mutation screening 42 PAX8-PPARÁ1, chromosomal translocation 32, 33 Peutz-Jeghers syndrome clinical features 188, 189 hamartoma syndrome 168 STK11 gene mapping 189, 190 mutations overview 169 sporadic tumors 191 protein structure and function 190, 191 Pheochromocytoma associated syndromes 117, 131 diagnosis 118 imaging 118 multiple endocrine neoplasia type 2 risks 117 sporadic disease and gene mutations 120, 121 treatment follow-up 120 pharmacotherapy 119 radiation therapy 120 surgery 119 von Hippel-Lindau syndrome classification of patients 134, 135 diagnosis 136 VHL mutations 120, 121, 135 Pituitary tumor-transforming gene differential display cloning 35 protein binding factor 38 Positional cloning familial cancer syndromes 23–25 linkage analysis 23, 24 principles 22, 23 PRKARIA Carney complex germline mutations 196, 197 cloning strategies 25 locus 195

216

mutation screening techniques 39, 40 tumor suppression function 197 Protein kinase C, VHL interactions 149 Proteomics disciplines 15 mass spectrometry 38, 39 protein chips 38, 39 Proteus syndrome, features 199 Proto-oncogene, definition 10 PTEN Bannayan-Riley-Ruvalcaba syndrome mutations Cowden syndrome comparison 185 genotype-phenotype correlations 185, 186 germline mutations 184, 185 overview 4, 5, 169 cloning strategies 25, 42 Cowden syndrome gene mapping 170, 171 mutations genotype-phenotype correlations 181 germline mutations 179–181 overview 4, 5, 169 sporadic tumors 181–183 homologs and function in other species 173 knockout mouse phenotypes 174, 175 locus 170, 171 methylation 12 mutation screening techniques 39–41 protein C2 domain and recruitment 177 cell cycle regulation 178 focal adhesion kinase interactions 176, 177 functional overview 178, 179 PDZ-binding domain 172, 177 phosphatase activity 176, 178 phosphatidylinositol 3-kinase signaling 175, 177, 178 phosphorylation 172, 173 structure 171, 172 subcellular localization 172 pseudogene 173 structure 171 transforming growth factor-ß downregulation 177

Subject Index

Rapid amplification of cDNA ends, chromosomal translocations 30, 31 Representational difference analysis, principle 35, 36 Restriction fragment length polymorphism, familial cancer syndromes 24 RET chromosomal translocation 31, 32 cloning strategies 25 developmental regulation 82 locus 104 mutations by disease type familial medullary thyroid carcinoma 84, 85, 87, 105, 106 genotype-phenotype correlation 86, 87, 104–107 Hirschsprung disease 91, 92, 107 MEN 2A 84, 105 MEN 2B 85, 86, 104, 106, 107 papillary thyroid carcinoma 92, 93 pheochromocytoma 120, 121 sporadic tumors 87, 88 receptor tyrosine kinase product ligand/co-receptor complexes 83, 84 mutation and function 86–88, 90 signaling 88–91 structure 82, 83 therapeutic targeting 93 screening for mutation accuracy 2 prophylactic thyroidectomy indications 113, 114 recommendations 111, 112 techniques 39–41 splice variants 91 SCF complexes, VHL interactions 145, 147, 148 Serial analysis of gene expression, principle 36 Single nucleotide polymorphism distribution in human genome 14 genotype-phenotype correlation 13, 14 Single-strand conformation polymorphism, gene mutation screening 40, 41 SMAD4 function 188 juvenile polyposis syndrome mutations 187, 188

217

STK11 mapping 189, 190 Peutz-Jeghers syndrome mutations overview 169 sporadic tumors 191 protein structure and function 190, 191 Subtractive hybridization, cloning of disease genes 27 Transforming growth factor-ß, PTEN downregulation 177 Tumor suppressor gene, see also specific genes definition 10, 11 Knudson two-hit theory of cancer 10, 26, 27, 61 VHL cloning strategies 25 homologs 138 knockout effects carbonic anhydrase overexpression 153 cell phenotype dedifferentiation 153, 154 fibronectin extracellular matrix abnormalities 151–153 hypoxia-inducible protein overexpression 150, 151, 155 locus 137 methylation 12 mutation in tumors familial vs. sporadic tumors 139 genotype-phenotype correlation 139, 140 loss of heterozygosity 138, 140 pheochromocytoma mutations 120, 121, 135, 138, 139 renal cell carcinomas 138 mutation screening techniques 39, 40

Subject Index

protein product E3 ubiquitin ligase complex hypoxia-inducible factor 1· as substrate 148, 149 protein interactions 145, 148, 149 interactions Cullin-2 144, 145, 147, 150 Elongins C/B 144, 147, 148, 150 protein kinase C 149 SCF complexes 145, 147, 148 isoforms 140, 142 structure 140 subcellular localization 142 target genes 151, 152 tumor suppression activity 142, 143 structure 137 transcription and tissue distribution 137, 138 von Hippel-Lindau syndrome adnexal papillary cystadenomas 137 associated tumors 131, 132 endolymphatic sac tumors 137 gene, see VHL hemangioblastomas histology 132 locations 133 morphology 133 treatment 133, 134 historical perspective of research 132 natural history 4 pancreatic lesions 136, 137 pheochromocytoma classification of patients 134, 135 diagnosis 136 VHL mutations 120, 121, 135, 138, 139 renal cell carcinoma 134 therapeutic prospects 154, 155 Yeast two-hybrid system, protein interactions 38

218