Endocrine Involvement in Developmental Syndromes (Endocrine Development Vol 14)

Endocrine Involvement in Developmental Syndromes

This book has been printed with financial support from Pfizer Italia

Endocrine Development Vol. 14

Series Editor

P.-E. Mullis

Bern

Workshop, April 21–22, 2008, Rome

Endocrine Involvement in Developmental Syndromes Volume Editors

Marco Cappa Rome Mohamad Maghnie Genova Sandro Loche Cagliari Gian Franco Bottazzo Rome 26 figures, 2 in color, and 15 tables, 2009

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

Endocrine Development Founded 1999 by Martin O. Savage, London

Marco Cappa

Mohamad Maghnie

Department of Pediatrics Pediatric Hospital Bambino Gesù Rome, Italy

Department of Pediatrics IRCCS G. Gaslini University of Genova Genova, Italy

Sandro Loche

Gian Franco Bottazzo

Regional Hospital for Microcytaemia Cagliari, Italy

Department of Pediatrics Pediatric Hospital Bambino Gesù Rome, Italy

Library of Congress Cataloging-in-Publication Data Workshop on Endocrine Involvement in Developmental Syndromes (2008 : Rome, Italy) Endocrine involvement in developmental syndromes / Workshop on Endocrine Involvement in Developmental Syndromes, April 21-22, 2008, Rome ; volume editors, Marco Cappa ... [et al.]. p. ; cm. -- (Endocrine development, ISSN 1421-7082 ; v. 14) Includes bibliographical references and indexes. ISBN 978-3-8055-9041-9 (hard cover : alk. paper) 1. Pediatric endocrinology--Congresses. 2. Growth disorders--Endocrine aspects--Congresses. 3. Developmental disabilities--Endocrine aspects--Congresses. I. Cappa, Marco. II. Title. III. Series: Endocrine development ; v. 14. [DNLM: 1. Endocrine System Diseases--etiology--Congresses. 2. Congenital Abnormalities--Congresses. 3. Genetic Diseases, Inborn--complications--Congresses. W1 EN3635 v.14 2009 / WK 140 W926e 2009] RJ482.G76.W67 2009 618.92'4--dc22 2008052202 Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® Disclaimer. The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2009 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel ISSN 1421–7082 ISBN 978–3–8055–9041–9

Contents

VII Preface Cappa, M.; Bottazzo, G.F. (Rome) 1

10 20 29

38 53

61 67

83 95

Inherited and Sporadic Epimutations at the IGF2-H19 Locus in BeckwithWiedemann Syndrome and Wilms’ Tumor Riccio, A. (Caserta/Naples); Sparago, A.; Verde, G. (Naples); De Crescenzo, A. (Caserta); Citro, V.; Cubellis, M.V. (Naples); Ferrero, G.B.; Silengo, M.C. (Torino); Russo, S.; Larizza, L. (Milan); Cerrato, F. (Caserta) Epigenetic Regulation of Growth: Lessons from Silver-Russell Syndrome Eggermann, T. (Aachen) Genetic Imprinting: The Paradigm of Prader-Willi and Angelman Syndromes Gurrieri, F.; Accadia, M. (Rome) Muscle Involvement and IGF-1 Signaling in Genetic Disorders: New Therapeutic Approaches Barberi, L.; Dobrowolny, G.; Pelosi, L.; Giacinti, C.; Musarò, A. (Rome) Mitochondrial Encephalomyopathies and Related Syndromes: Brief Review Bertini, E.; D’Amico A. (Rome) Overgrowth Syndromes: A Classification Neri, G.; Moscarda, M. (Rome) C-Type Natriuretic Peptide and Overgrowth Bocciardi, R.; Ravazzolo, R. (Genova) Role of Transcription Factors in Midline Central Nervous System and Pituitary Defects Kelberman, D.; Dattani, M.T. (London) Developmental Abnormalities of the Posterior Pituitary Gland di Iorgi, N.; Secco, A.; Napoli, F.; Calandra, E.; Rossi, A.; Maghnie, M. (Genova) Hyperinsulinism in Developmental Syndromes Kapoor, R.R.; James, C.; Hussain, K. (London)

114

135 143

151 167

174

181 182

VI

Developmental Syndromes: Growth Hormone Deficiency and Treatment Mazzanti, L.; Tamburrino, F.; Bergamaschi, R.; Scarano, E.; Montanari, F.; Torella, M.; Ballarini, E.; Cicognani, A. (Bologna) Growth Hormone-Resistant Syndromes: Long-Term Follow-Up Chernausek, S.D. (Oklahoma City, Okla.) Phenotypic Aspects of Growth Hormone- and IGF-I-Resistant Syndromes Savage, M.O.; David, A.; Camacho-Hübner, C.; Metherell, L.A.; Clark, A.J.L. (London/Stockholm) Double Diabetes: A Mixture of Type 1 and Type 2 Diabetes in Youth Pozzilli, P.; Guglielmi, C. (Rome) Cryptorchidism as Part of the Testicular Dysgenesis Syndrome: The Environmental Connection Main, K.M.; Skakkebæk, N.E. (Copenhagen); Toppari, J. (Turku) Disorders of Sex Development in Developmental Syndromes Hiort, O.; Gillessen-Kaesbach, G. (Lübeck) Author Index Subject Index

Contents

Preface

Recent years have seen a significant improvement in the knowledge of genetics and developmental syndromes. In this scenario, the study of endocrinological aspects in patients with genetic syndromes acquires increasing interest and significance. The workshop on “Endocrine Involvement in Developmental Syndromes”, held in Rome on April 21–22, 2008, represented a precious opportunity to provide an updated global view of this important field of medical sciences. The scientific program of the workshop included the most recent advances in the study of developmental syndromes and epigenetics, with world-wide experts focusing their contributions on modern concepts of basic and clinical science in order to clarify genetic, clinical and biological aspects of these syndromes. This book is the result of this fruitful exchange of experience and knowledge, and it provides a thorough elucidation of endocrine involvement and epigenetic aspects of various developmental syndromes, opening new ways to manage the complexity of such a topic. We are confident that the state-of-art information provided by this book will be of great interest for endocrinologists, pediatricians, and genetists involved in the study and treatment of developmental syndromes. Marco Cappa Gian Franco Bottazzo Rome

Endocrine Involvement in Developmental Syndromes

This book has been printed with financial support from Pfizer Italia

Endocrine Development Vol. 14

Series Editor

P.-E. Mullis

Bern

Workshop, April 21–22, 2008, Rome

Endocrine Involvement in Developmental Syndromes Volume Editors

Marco Cappa Rome Mohamad Maghnie Genova Sandro Loche Cagliari Gian Franco Bottazzo Rome 26 figures, 2 in color, and 15 tables, 2009

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

Endocrine Development Founded 1999 by Martin O. Savage, London

Marco Cappa

Mohamad Maghnie

Department of Pediatrics Pediatric Hospital Bambino Gesù Rome, Italy

Department of Pediatrics IRCCS G. Gaslini University of Genova Genova, Italy

Sandro Loche

Gian Franco Bottazzo

Regional Hospital for Microcytaemia Cagliari, Italy

Department of Pediatrics Pediatric Hospital Bambino Gesù Rome, Italy

Library of Congress Cataloging-in-Publication Data Workshop on Endocrine Involvement in Developmental Syndromes (2008 : Rome, Italy) Endocrine involvement in developmental syndromes / Workshop on Endocrine Involvement in Developmental Syndromes, April 21-22, 2008, Rome ; volume editors, Marco Cappa ... [et al.]. p. ; cm. -- (Endocrine development, ISSN 1421-7082 ; v. 14) Includes bibliographical references and indexes. ISBN 978-3-8055-9041-9 (hard cover : alk. paper) 1. Pediatric endocrinology--Congresses. 2. Growth disorders--Endocrine aspects--Congresses. 3. Developmental disabilities--Endocrine aspects--Congresses. I. Cappa, Marco. II. Title. III. Series: Endocrine development ; v. 14. [DNLM: 1. Endocrine System Diseases--etiology--Congresses. 2. Congenital Abnormalities--Congresses. 3. Genetic Diseases, Inborn--complications--Congresses. W1 EN3635 v.14 2009 / WK 140 W926e 2009] RJ482.G76.W67 2009 618.92'4--dc22 2008052202 Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® Disclaimer. The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2009 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel ISSN 1421–7082 ISBN 978–3–8055–9041–9

Contents

VII Preface Cappa, M.; Bottazzo, G.F. (Rome) 1

10 20 29

38 53

61 67

83 95

Inherited and Sporadic Epimutations at the IGF2-H19 Locus in BeckwithWiedemann Syndrome and Wilms’ Tumor Riccio, A. (Caserta/Naples); Sparago, A.; Verde, G. (Naples); De Crescenzo, A. (Caserta); Citro, V.; Cubellis, M.V. (Naples); Ferrero, G.B.; Silengo, M.C. (Torino); Russo, S.; Larizza, L. (Milan); Cerrato, F. (Caserta) Epigenetic Regulation of Growth: Lessons from Silver-Russell Syndrome Eggermann, T. (Aachen) Genetic Imprinting: The Paradigm of Prader-Willi and Angelman Syndromes Gurrieri, F.; Accadia, M. (Rome) Muscle Involvement and IGF-1 Signaling in Genetic Disorders: New Therapeutic Approaches Barberi, L.; Dobrowolny, G.; Pelosi, L.; Giacinti, C.; Musarò, A. (Rome) Mitochondrial Encephalomyopathies and Related Syndromes: Brief Review Bertini, E.; D’Amico A. (Rome) Overgrowth Syndromes: A Classification Neri, G.; Moscarda, M. (Rome) C-Type Natriuretic Peptide and Overgrowth Bocciardi, R.; Ravazzolo, R. (Genova) Role of Transcription Factors in Midline Central Nervous System and Pituitary Defects Kelberman, D.; Dattani, M.T. (London) Developmental Abnormalities of the Posterior Pituitary Gland di Iorgi, N.; Secco, A.; Napoli, F.; Calandra, E.; Rossi, A.; Maghnie, M. (Genova) Hyperinsulinism in Developmental Syndromes Kapoor, R.R.; James, C.; Hussain, K. (London)

114

135 143

151 167

174

181 182

VI

Developmental Syndromes: Growth Hormone Deficiency and Treatment Mazzanti, L.; Tamburrino, F.; Bergamaschi, R.; Scarano, E.; Montanari, F.; Torella, M.; Ballarini, E.; Cicognani, A. (Bologna) Growth Hormone-Resistant Syndromes: Long-Term Follow-Up Chernausek, S.D. (Oklahoma City, Okla.) Phenotypic Aspects of Growth Hormone- and IGF-I-Resistant Syndromes Savage, M.O.; David, A.; Camacho-Hübner, C.; Metherell, L.A.; Clark, A.J.L. (London/Stockholm) Double Diabetes: A Mixture of Type 1 and Type 2 Diabetes in Youth Pozzilli, P.; Guglielmi, C. (Rome) Cryptorchidism as Part of the Testicular Dysgenesis Syndrome: The Environmental Connection Main, K.M.; Skakkebæk, N.E. (Copenhagen); Toppari, J. (Turku) Disorders of Sex Development in Developmental Syndromes Hiort, O.; Gillessen-Kaesbach, G. (Lübeck) Author Index Subject Index

Contents

Preface

Recent years have seen a significant improvement in the knowledge of genetics and developmental syndromes. In this scenario, the study of endocrinological aspects in patients with genetic syndromes acquires increasing interest and significance. The workshop on “Endocrine Involvement in Developmental Syndromes”, held in Rome on April 21–22, 2008, represented a precious opportunity to provide an updated global view of this important field of medical sciences. The scientific program of the workshop included the most recent advances in the study of developmental syndromes and epigenetics, with world-wide experts focusing their contributions on modern concepts of basic and clinical science in order to clarify genetic, clinical and biological aspects of these syndromes. This book is the result of this fruitful exchange of experience and knowledge, and it provides a thorough elucidation of endocrine involvement and epigenetic aspects of various developmental syndromes, opening new ways to manage the complexity of such a topic. We are confident that the state-of-art information provided by this book will be of great interest for endocrinologists, pediatricians, and genetists involved in the study and treatment of developmental syndromes. Marco Cappa Gian Franco Bottazzo Rome

Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 1–9

Inherited and Sporadic Epimutations at the IGF2-H19 Locus in Beckwith-Wiedemann Syndrome and Wilms’ Tumor Andrea Riccioa,b ⭈ Angela Sparagob ⭈ Gaetano Verdeb ⭈ Agostina De Crescenzoa ⭈ Valentina Citroc ⭈ Maria Vittoria Cubellisc ⭈ Giovanni Battista Ferrerod ⭈ Margherita Cirillo Silengod ⭈ Silvia Russoe ⭈ Lidia Larizzae,f ⭈ Flavia Cerratoa a

Dipartimento di Scienze Ambientali, Seconda Università di Napoli, Caserta; bIstituto di Genetica e Biofisica ‘A. Buzzati Traverso’, CNR, and cDipartimento di Biologia Strutturale e Funzionale, Università di Napoli ‘Federico II’, Naples; dDipartimento di Scienze Pediatriche e dell’Adolescenza, Università di Torino, Torino, and eLaboratorio di Citogenetica e Genetica Molecolare, Istituto Auxologico Italiano, Milano, fDivision of Medical Genetics, San Paolo School of Medicine, University of Milan, Milan, Italy

Abstract The parent-of-origin-dependent expression of IGF2 and H19 is controlled by the imprinting center 1 (IC1) consisting of a methylation-sensitive chromatin insulator. IC1 is normally methylated on the paternal chromosome and nonmethylated on the maternal chromosome. We found that 22 cases in a large cohort of patients affected by Beckwith-Wiedemann syndrome (BWS) had IC1 methylated on both parental chromosomes, resulting in biallelic activation of IGF2 and biallelic silencing of H19. These individuals had marked macrosomia and high incidence of Wilms’ tumor. A subset of these patients had 1.4- to 1.8-kb deletions with hypermethylation of the remaining IC1 region and fully penetrant BWS phenotype when transmitted maternally. Another subset of individuals with IC1 hypermethylation had a similar clinical phenotype but no mutation in the local vicinity. All these cases were sporadic and in at least two families affected and unaffected members shared the same maternal IC1 allele but not the abnormal maternal epigenotype. Similarly, no IC1 deletion was detected in 10 nonsyndromic Wilms’ tumors with IC1 hypermethylation. In conclusion, methylation defects at the IGF2-H19 locus can result from inherited mutations of the imprinting center and have high recurrence risk or arise independently from the sequence context and not transmitted to the Copyright © 2009 S. Karger AG, Basel progeny.

There is increasing evidence that aberrant chromatin states leading to aberrant gene expression patterns (epimutations) have important roles in human disease [1]. However, the causes, heritability and relationship with phenotype of many of these

lesions are still undefined. For instance, although it is generally accepted that epigenetic marks are cleared between generations, there is a number of cases in which this seems not to be the case. The human disorders caused by defects of genomic imprinting provide a paradigm for studying these issues.

Genomic Imprinting

Genomic imprinting is an epigenetic mechanism causing the expression of a minority of genes to be monoallelic and dependent on their gametic origin [2]. Correct imprinting is required for normal development, while defective imprinting is associated with human disease [3]. A 1-Mb cluster of imprinted genes is present at chromosome 11p15.5 (fig. 1). The cluster is functionally divided into two domains that are autonomously controlled by separate imprinting control regions or imprinting centers (IC1 and IC2 [4, 5]). These are CpG-rich regions that work by different mechanisms, but share as a common feature to be differentially methylated on the maternally and paternally derived chromosomes (differentially methylated regions, DMRs). Two genes, insulin-like growth factor 2 (IGF2) and H19, are located in domain 1 of the 11p15.5 imprinted gene cluster. IGF2 is a paternally expressed fetal growth factor gene with an important role in cancer development [6]. H19 is a maternally expressed noncoding RNA with possible tumor-suppressor functions [7]. The reciprocal imprinting of IGF2 and H19 is controlled by IC1 in the majority of tissues. The function of this control element has been extensively studied in the mouse. IC1 (also known as H19 DMR) is a methylation-sensitive chromatin insulator located between IGF2 and H19 [8]. Its nonmethylated maternal allele interacts with the multi-zinc finger protein CTCF. This binding is required on the maternal chromosome for maintaining the nonmethylated status of the region and preventing the activation of the IGF2 promoter by downstream enhancers that activate the H19 gene instead. On the paternal chromosome, conversely, DNA methylation prevents CTCF binding at IC1 and allows the enhancer-mediated activation of IGF2 while the H19 promoter is hypermethylated and silenced. Recent evidences indicate that the methylationsensitive binding of CTCF at IC1 mediates higher-order chromatin conformations in a parent of origin-specific manner [9]. In particular, the two parental IC1 alleles interact with two different DMRs of Igf2 on the maternal and paternal chromosomes [10]. This may partition the maternal and paternal Igf2 alleles into inactive and active chromatin domains, respectively.

Beckwith-Wiedemann Syndrome

The Beckwith-Wiedemann syndrome (BWS, MIM 130650) is a developmental disorder characterized by variable clinical features, including overgrowth, macroglossia,

2

Riccio · Sparago · Verde · De Crescenzo · Citro · Cubellis · Ferrero · Silengo · Russo · Larizza · Cerrato

d H19

d INS d IGF2

d TH

d ASCL2

LTRPC5 TSSC4 CD81 TSSC6 d D D D

D KCNQ1

d KCNQ1OT1/LIT1

CARS NAP1L4 TSSC3 TSSC5 CDKN1C d d d D d

Domain 2

IC1

tel IC2

cen

Domain 1

Expressed from the paternal chromosome Expressed from the maternal chromosome Biallelic expression Imprinted expression not well defined DMR methylated on the paternal chromosome DMR methylated on the maternal chromosome

Fig. 1. The 11p15.5 imprinted gene cluster.

abdominal wall defects and increased incidence of embryonal tumors that is caused by defective expression of imprinted genes located on chromosome 11p15.5 [11]. The majority of the BWS cases are sporadic. The rare familial cases show a predominantly autosomal-dominant inheritance and preferential expression following maternal transmission. Heterogeneous molecular defects are found in BWS. Only 5% of the cases (40% of the familial ones) have typical single-gene defects, consisting in lossof-function mutations of CDKN1C. About 20% of the cases have uniparental paternal disomy (UPD) of 11p15.5 loci, indicating that BWS is caused by excess of imprinted genes expressed from the paternal chromosome and/or defect of imprinted genes expressed from the maternal chromosome. The majority of the other cases show DNA methylation defects at either IC1 or IC2. Similarly to BWS, patients with nonsyndromic Wilms’ tumor also have IC1 hypermethylation, but this is restricted to cancer tissues in these cases [12]. In this chapter, we review the clinical characteristics and molecular features of the cases of BWS and Wilms’ tumor with hypermethylation at IC1.

IC1 Microdeletions

Gain of methylation at IC1 is usually found in only 5–10% of the BWS cases [11, 13]. However, the high incidence of Wilms’ tumor-associated with these molecular abnormalities makes them particularly important to study. We found 1.4- to

Epimutations at the IGF2-H19 Locus in BWS and Wilms’ Tumor

3

1.8-kb deletions of the IC1 region in 9 BWS patients with IC1 hypermethylation [14–16]. These cases were part of dominant inheritance pedigrees with maternal transmission and characterized by high recurrence risk. The deletions remove 1–2 target sequences for CTCF (CTS) resulting in hypermethylation of the residual CTSs and cosegregate with the BWS phenotype with full penetrance if maternally inherited. In 2005, Prawitt et al. [17] described a further BWS family with a 2.2-kb deletion eliminating 3 CTSs from IC1. However, maternal transmission of this deletion was not necessarily associated with the BWS phenotype. Indeed, an additional mutation consisting in duplication of the 11p15 region was present in the affected children. Interestingly, this deletion did not alter the methylation of the flanking sequences, suggesting that the hypermethylation of the imprinting center significantly contribute to the BWS pathogenesis. We also observed that the mutant alleles with gain of methylation had abnormally spaced CTSs and proposed that the 1.4- to 1.8-kb deletions resulted in lowering the affinity of IC1 for CTCF [15].

IC1 Hypermethylation without Microdeletion

Neither deletion nor any other point mutation of the IC1 region was demonstrated in 13 patients of our cohort with IC1 hypermethylation [18, and data not shown]. These were all sporadic cases and in 2 of them, the maternal IC1 allele of the index patient segregated in 1 of his healthy relatives. A detailed methylation analysis showed that the hypermethylation was extended over the entire or only 3⬘ half of the IC1 region, did not affect other imprinted loci, generally occurred in the mosaic form and was never present in the unaffected relatives. The chromosome carrying the imprinting abnormality derived from either the maternal grandfather or maternal grandmother. These results indicate that, in the absence of deletions, IC1 hypermethylation generally occurs as sporadic epimutation and is associated with low recurrence risk.

Clinical Phenotype

The clinical features of the patients carrying IC1 deletions and those with IC1 hypermethylation without accompanying deletion are very similar. However, they differ significantly from the phenotypes of the BWS patients with other molecular defects [18]. Table 1 is a summary of the characteristics of a cohort of 132 individuals with clinical diagnosis of BWS [19], subdivided into 4 classes according to the molecular defect found [20]. Features, such as pronounced macrosomia, mild or absent defects of the abdominal wall and elevated incidence of Wilms’ tumor are evident among the individuals with IC1 hypermethylation.

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Table 1. Clinical features of 132 BWS patients investigated for methylation defects at chromosome 11p15.5 Clinical features

IC1 hypermethylation IC2 11p15.5 Pat with and without hypomethylation UPD (n = 22) microdeletion (n = 43) (n = 20)

Normal methylation (n = 47)

Macrosomia (weight >90th centile) Polydramnios Macroglossia Rectum diastasis Umbilical hernia Exomphalos Inguinal hernia Hemihypertrophy Facial Asymmetry Hepato-/spleno-megaly Neonatal Hypoglycemia Ear pits/creases Facial dysmorphisms Naevus flammeous Renal abnormalities Wilms’ tumor Hepatoblastoma Other tumors Ureteral abnormalities VSD/ASD/PDA shunt Convulsions Mental retardation

16 (80)

17 (39)

8 (36)

22 (47)

5 (25) 17 (85) 12 (60) 10 (50) 0 (0) 1 (5) 7 (35) 4 (20) 12 (60) 8 (40) 7 (35) 7 (35) 5 (25) 11 (55) 5 (25) 0 (0) 0 (0) 3 (15) 1 (5) 1 (5) 1 (5)

8 (19) 42 (98) 20 (46) 17 (39) 14 (33) 4 (9) 14 (33) 8 (19) 12 (28) 18 (42) 30 (70) 20 (46) 27 (63) 7 (16) 0 (0) 0 (0) 1*(2) 2 (4) 2 (4) 5 (12) 6 (14)

6 (27) 19 (86) 9 (41) 7 (32) 1 (4) 0 (0) 16 (73) 1 (4) 9 (41) 10 (45) 7 (32) 6 (27) 8 (36) 9 (41) 1 (4) 2 (9) 0 (0) 2 (9) 0 (0) 0 (0) 3 (14)

3 (6) 38 (81) 28 (60) 17 (36) 4 (8) 3 (6) 14 (30) 6 (13) 6 (13) 8 (17) 26 (55) 18 (38) 14 (30) 10 (21) 1 (2) 0 (0) 0 (0) 2 (4) 2 (4) 2 (4) 8 (17)

Numbers in parentheses denote percent values. Percentages that significantly differ in one molecular subgroup of patients from the others are indicated in bold. * Neuroblastoma.

Epigenetic Mosaicism

We observed that all IC1 CTSs are completely and exclusively methylated on the paternal chromosome in normal leukocyte DNA while incomplete hypermethylation of the maternal allele is present in the BWS patients with the 1.4- to 1.8kb IC1 deletions and the majority of patients without deletions suggesting that this imprinting defect is generally present in the mosaic form [16, 18]. We did not observe a clear relationship between the extent of hypermethylation at the IGF2-H19 locus and the severity of the BWS phenotype. However, it is possible that

Epimutations at the IGF2-H19 Locus in BWS and Wilms’ Tumor

5

the degree of methylation of leukocyte DNA is insufficient to predict the extent of mosaicism in the whole body and tissues, which is more important as the BWS phenotype should be examined. The high incidence of hemihyperthrophy (table 1) suggests that at least some of the clinical features of the patients with IC1 deletion are influenced by mosaicism. Mosaicism can also explain the high variability of the clinical phenotype that is characteristic of this disorder. In addition, diagnostic problems may be encountered with individuals who may not have abnormal methylation in their leukocyte DNA. To avoid these problems, it may be useful in the future to analyze DNAs derived from more than one tissue (e.g. blood leukocytes and buccal mucosa).

Loss of IGF2-H19 Imprinting

It has been shown in the mouse that the CTSs within the IC1 element are required for its insulator function [21]. Furthermore, CTCF binding controls the interaction between IC1 and the Igf2 DMRs that are required to partition the Igf2 and H19 genes into transcriptionally active and inactive chromatin loops [9, 10]. We observed a gain of methylation at the IGF2 DMR0 and DMR2 in the majority of the patients with IC1 hypermethylation (with or without microdeletions) [16, 22, and data not shown]. In addition, biallelic activation of IGF2 and biallelic silencing of H19 could be demonstrated in many of these patients. It is therefore likely that the epigenetic alterations resulting from the microdeletions or sporadic epimutations of IC1 lead the maternal 11p15.5 locus to acquire high-order chromatin structures that are typical of the paternal chromosome and associated with IGF2 activation and H19 silencing (fig. 2).

Origin of the Imprinting Defects

In principle, imprinting defects at ICs can derive from failure of erasure, establishment or maintenance of the imprint [2]. The incomplete hypermethylation of the CTSs found in the BWS patients with 1.4- to 1.8-kb deletions indicates mosaicism for the imprinting defect and suggests that the methylation is acquired postzygotically and results from insufficient protection from de novo methylation of the mutated maternal IC1. In the BWS patients without microdeletions the chromosome with abnormal IC1 methylation derived from either the maternal grandfather or maternal grandmother. Since the majority of these cases also showed mosaic hypermethylation, it is likely that also in these cases the methylation defect is acquired at a postzygotic stage. However, an incomplete imprint erasure resulting in an unstable methylation cannot be excluded. Knocking-down of CTCF in mouse oocytes and in cultured cells results in gain of methylation of IC1 [23]. We ruled out the presence of a mutation of CTCF gene in

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Riccio · Sparago · Verde · De Crescenzo · Citro · Cubellis · Ferrero · Silengo · Russo · Larizza · Cerrato

IGF2

H19 Normal

IGF2

H19

BWS with IC1 microdeletions

IGF2

DMR0

H19

DMR2

IC1

BWS without IC1 microdeletions

Fig. 2. Loss of IGF2-H19 imprinting as consequence of sporadic and inherited epimutations. The diagram shows the expression of the maternal and paternal IGF2 and H19 alleles in normal individuals and BWS patients with maternally inherited IC1 microdeletions or sporadic IC1 hypermethylation. The methylated or nonmethylated status of the IGF2 DMR0, IGF2 DMR2, seven IC1 CTSs and H19 promoter is indicated by filled and open lollipops, respectively. The enhancers are indicated by ovals.

the patients without IC1 deletions. It cannot be excluded, however, that a mutation is present in other modifier genes. Considering the sporadic nature of these cases, the possibility that IC1 hypermethylation occurs as consequence of stochastic events or environmental influence should also be envisaged [24].

Absence of IC1 Microdeletions in Non-Syndromic Wilms’ Tumor

The 11p15.5 imprinted gene cluster is frequently affected in Wilms’ tumors [12]. Either maternal deletion/paternal duplication (LOH) or IC1 hypermethylation coupled to H19 silencing and IGF2 activation (LOI) can be found in a high proportion of tumors. In addition, the individuals who have somawide IC1 hypermethylation or 11p15.5 paternal UPD represent the molecular subgroup of BWS patients showing the highest risk of developing Wilms’ tumor [13]. Consistent with these observations, we found that 5/20 BWS patients with IC1 hypermethylation in our cohort had developed this neoplasm. Two of these had IC1 microdeletions [18]. However, no

Epimutations at the IGF2-H19 Locus in BWS and Wilms’ Tumor

7

IC1 deletion was found in 40 sporadic non-syndromic Wilms’ tumors. A previous American study demonstrated the absence of point mutations in the IC1 sequence of a series of sporadic Wilms’ tumors [25]. Overall, these data suggest that IC1 imprinting defects are generally not associated with a mutation in cis in non-syndromic Wilms’ tumor, as well as sporadic BWS. We recently demonstrated the presence of different methylation abnormalities at IGF2 in Wilms’ tumors and BWS, suggesting that sporadic imprinting defects arise through different mechanisms in neoplastic and non-neoplastic cells [22]

Conclusion

Although the common hallmark and probably the ultimate cause of the imprinting defects at the IGF2/H19 locus is represented by hypermethylation of IC1, our studies demonstrate that this epigenetic abnormality can result from more than one mechanism in BWS and Wilms’ tumor. In a first group of patients, we found that the epimutation is a direct consequence of a mutation in cis, consisting of a deletion of 1–2 CTSs. In these cases, the methylation defect and disease phenotype are reproduced whenever the mutation is transmitted through the maternal germline. In another group of patients, who carry no IC1 deletion, the epimutation is independent of the local DNA sequence and generally not transmitted to the progeny. Sporadic BWS and non-syndromic Wilms’ tumor belong to the second group of cases. Despite these differences, the IC1 epimutation is generally present in the patients in the mosaic form and probably acquired by postzygotic de novo methylation, providing an example of how intricate the relationship between genotype and epigenotype can be.

Acknowledgments This work was supported by grants from MIUR PRIN 2005, Istituto Superiore di Sanità, Associazione Italiana Ricerca sul Cancro, Telethon-Italia Grant No. GGP07086. F.C. was recipient of a fellowship from Società Italiana di Cancerologia and Fondazione Pezcoller.

References 1 2

3

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Feinberg AP: Phenotypic plasticity and the epigenetics of human disease. Nature 2007;447:433–440. Reik W, Walter J: Genomic imprinting: parental influence on the genome. Nat Rev Genet 2001;2:21– 32. Arnaud P, Feil R: Epigenetic deregulation of genomic imprinting in human disorders and following assisted reproduction. Birth Defects Res [C] 2005; 75:81–97.

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Ainscough JFX, Koide T, Tada M, Barton S, Surani MA: Imprinting of Igf2 and H19 from a 130 kb YAC transgene. Development 1997;124:3621–3632. Cerrato F, Sparago A, Di Matteo I, Zou X, Dean W, Sasaki H, Smith P, Genesio R, Bruggemann M, Reik W, Riccio A: The two-domain hypothesis in BeckwithWiedemann syndrome: autonomous imprinting of the telomeric domain of the distal chromosome 7 cluster. Hum Mol Genet 2005;14: 503–511.

Riccio · Sparago · Verde · De Crescenzo · Citro · Cubellis · Ferrero · Silengo · Russo · Larizza · Cerrato

6 LeRoith D, Roberts, CTJr: The insulin-like growth factor system and cancer. Cancer Lett 2003;195:127– 137. 7 Hao Y, Crenshaw T, Moulton T, Newcomb E, Tycko B: Tumour-suppressor activity of H19 RNA. Nature 1993;365:764–767. 8 Hark AT, Schoenherr CJ, Katz DJ, Ingram RS, Levorse JM, Tilghman SM: CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 2000;405:486–489. 9 Kurukuti S, Tiwari VK, Tavoosidana G, Pugacheva E, Murrell A, Zhao Z, Lobanenkov V, Reik, W, Ohlsson R: CTCF binding at the H19 imprinting control region mediates maternally inherited higherorder chromatin conformation to restrict enhancer access to Igf2. Proc Natl Acad Sci USA 2006; 103:10684–10689. 10 Murrell A, Heeson S, Reik W: Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops. Nat Genet 2004;36:889–893. 11 Weksberg R, Shuman C, Smith AC: BeckwithWiedemann syndrome. Am J Med Genet [C] 2005; 137:12–23. 12 Feinberg AP, Cui H, Ohlsson R: DNA methylation and genomic imprinting: insights from cancer into epigenetic mechanisms. Semin Cancer Biol 2002; 12:389–398. 13 Cooper WN, Luharia A, Evans GA, Raza H, Haire AC, Grundy R, Bowdin SC, Riccio A, Sebastio G, Bliek J, Schofield PN, Reik W, Macdonald F, Maher ER: Molecular subtypes and phenotypic expression of Beckwith-Wiedemann syndrome. Eur J Hum Genet 2005;13:1025–1032. 14 Sparago A, Cerrato F, Vernucci M, Ferrero GB, Cirillo Silengo M, Riccio A: Microdeletions in the human H19 DMR result in loss of IGF2 imprinting and Beckwith-Wiedemann. Nat Genet 2004;36:958– 960. 15 Cerrato F, Sparago A, Farina L, Ferrero GB, Cirillo Silengo M, Riccio A: Reply to Microdeletion and IGF2 loss of imprinting in a cascade causing Beckwith-Wiedemann syndrome with Wilm’s tumor. Nat Genet 2005;37:786–787. 16 Sparago A, Russo S, Cerrato F, Ferraiuolo S, Castorina P, Selicorni A, Schwienbacher C, Negrini M, Ferrero GB, Silengo MC, Anichini C, Larizza L, Riccio A: Mechanisms causing imprinting defects in familial Beckwith-Wiedemann syndrome with Wilms’ tumour. Hum Mol Genet 2007;16; 254–264.

17 Prawitt D, Enklaar T, Gartner-Rupprecht B, Spangenberg C, Oswald M, Lausch E, Schmidtke P, Reutzel D, Fees S, Lucito R, Korzon M, Brozek I, Limon J, Housman DE, Pelletier J, Zabel B: Microdeletion of target sites for insulator protein CTCF in a chromosome 11p15 imprinting center in Beckwith-Wiedemann syndrome and Wilms’ tumor. Proc Natl Acad Sci USA 2005;102:4085–4090. 18 Cerrato F, Sparago A, Verde G, De Crescenzo A, Citro V, Cubellis MV, Rinaldi MM, Boccuto L, Neri G, Magnani C, D’Angelo P, Collini P, Perotti D, Sebastio G, Maher ER, Riccio A: Different mechanisms cause imprinting defects at the IGF2/H19 locus in BeckwithWiedemann syndrome and Wilms’ tumour. Hum Mol Genet 2008;17:1427–1435. 19 DeBaun MR, Tucker MA: Risk of cancer during the first four years of life in children from The BeckwithWiedemann Syndrome Registry. J Pediatr 1998;132: 398–400. 20 Priolo M, Sparago A, Mammì C, Cerrato F, Laganà C, Riccio A: MS-MLPA is a specific and sensitive technique for detecting all chromosome 11p15.5 imprinting defects of BWS and SRS in a single-tube experiment. Eur J Hum Genet 2008;16:565–571. 21 Schoenherr CJ, Levorse JM, Tilghman SM: CTCF maintains differential methylation at the Igf2/H19 locus. Nat Genet 2002;33:66–69. 22 Murrell A, Ito Y, Verde G, Huddleson J, Woodfinel K, Cirillo Silengo M, Spreafico F, Perotti D, De Crescenzo A, Sparago S, Cerrato F, Riccio A: Distinct methylation changes at the IGF2-H19 locus in congenital growth disorders and cancer. PloS ONE 2008;3:e1849. 23 Fedoriw AM, Stein P, Svoboda P, Schultz RM, Bartolomei MS: Transgenic RNAi reveals essential function for CTCF in H19 gene imprinting. Science 2004;303:238–240. 24 Jaenisch R, Bird A: Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 2003;33(suppl): 245–254. 25 Cui H, Niemitz EL, Ravenel JD, Onyango P, Brandenburg SA, Lobanenkov VV , Feinberg AP: Loss of imprinting of insulin-like growth factor-II in Wilms’ tumour commonly involves altered methylation but not mutations of CTCF or its binding site. Cancer Res 2001;61:4947–4950.

Andrea Riccio Dipartimento di Scienze Ambientali Seconda Università di Napoli, Via Vivaldi, 43 IT–81100 Caserta (Italy) Tel. +39 0823 274 599, Fax +39 082 274 605, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 10–19

Epigenetic Regulation of Growth: Lessons from Silver-Russell Syndrome Thomas Eggermann Institute of Human Genetics, University Hospital, RWTH Aachen, Germany

Abstract Human growth is a complex process and requires the appropriate interaction of many members. Central members in the growth axes are regulated epigenetically and thereby reflect the profound significance of imprinting for correct mammalian ontogenesis. A prominent imprinting disorder, Silver-Russell syndrome (SRS), is a congenital disease characterized by intrauterine and postnatal growth retardation and other features. SRS represents the first human disorder with imprinting disturbances affecting two different chromosomes: nearly one tenth of patients carry a maternal uniparental disomy of chromosome 7 and more than 38% show a hypomethylation in the imprinting control region 1 in 11p15. Interestingly, hypermethylation of the same region is associated with the overgrowth disease Beckwith-Wiedemann syndrome (BWS), thus SRS and BWS can be regarded as genetically (and clinically) opposite diseases. Because of the different imprinting regions involved, SRS is a suitable model to decipher the role of imprinting in growth and the functional interaction Copyright © 2009 S. Karger AG, Basel between imprinted genes in different genomic regions.

Growth is a complex process with both genetics and environment contributing in equally parts. Among the involved genes is a substantial number of so-called imprinted factors, e.g. these genes are expressed only from one chromosome from one parent. Generally, paternally expressed imprinted genes enhance fetal growth, whereas maternally expressed ones suppress it. Based on this observation, the genetic conflict theory has been hypothesized: it explains the evolution of imprinted genes by paternally derived genes that aim at extracting more resources from the mother whereas maternally derived genes have to balance the nutrient provision to the current fetus with that to future fetuses of the same mother [1]. With the identification of human diseases caused by epigenetic mutations, the significance of a balanced expression of imprinted genes became obvious. In addition to the profound role of imprinted genes in congenital disorders, there is evidence for epigenetic inheritance in humans which might explain the transgenerational effects of grandparental nutrition on health in next generations [2].

The close relationship between imprinting and (fetal) growth is illustrated by the recent identification of specific genetic and epigenetic alterations in Silver-Russell syndrome (SRS)(OMIM 180860). SRS is a congenital disorder mainly characterized by pre- and postnatal growth restriction. The children are relatively macrocephalic and their face is triangular-shaped with a broad forehead and a pointed, small chin. In many cases, asymmetry of limbs and body and clinodactyly of the fifth digit is present. Growth failure is often accompanied by severe failure to thrive, and feeding difficulties are frequently reported. The latter is the main clinical concern and much effort is expended in encouraging adequate feeding. For those children without catch-up growth by the age of 2, growth hormone therapy is encouraged.

Genetics of SRS

The observation of ‘classical’ genetic findings in SRS patients such as families with several affected members and cytogenetic aberrations indicated the influence of genetic factors in the etiology of SRS. Among the rare SRS families, autosomalrecessive as well as dominant inheritance have been reported. This genetic heterogeneity has also been confirmed by the observation of different chromosomal disturbances (fig. 1). Whereas in other genetic diseases these findings help to identify the specific genes, in SRS the majority of the putative genetic observations did not enlighten the molecular basis of the disorder but indeed led to more confusion. This confusion was increased by the finding that the majority of monozygotic twins are discordant (for review, see ref. [3]). In circumstances in which genetic information is incomplete, twin studies are often helpful in establishing genetic causes. In SRS, these discordant monozygotic twins were misleading for a long time but can now be explained by the data from Gicquel et al. [4] who reported on discordant monozygotic twins carrying the same 11p15 epimutation but with mosaic distribution in different tissues. Despite the heterogeneous cytogenetic findings, aberrations of four chromosomal regions have been described for several times and these regions were therefore considered to harbor SRS relevant genes.

Chromosomes 15 and 17

The identification of deletions in 15q26qter in patients with SRS features led to the assumption that an imbalanced expression of the insulin-like growth factor 1 receptor gene (IGF1R) might cause SRS. Nevertheless, pathogenic mutations in IGF1R were not found in SRS patients [5]. Based on balanced translocations in 2 patients involving 17q24-q25, a central role of this chromosomal region in SRS etiology had been long discussed. However,

(Epi)Genetics of Silver-Russell Syndrome

11

3 1

5

4

2 …

6

7

…

…

13

14

19

20

Deletion

15

…

8

9

…

11

10

16

17

21

22

UPD

12

X

18

Y

Isochromosome/marker chromosome

Duplication/trisomy Translocation breakpoint

Ring chromosome

… more than two cases

Fig. 1. Chromosomal aberrations in SRS: Review on types, frequencies and affected genomic regions. A complete list of the literature describing chromosomal aberrations in SRS is available on request.

characterization of the 17q breakpoints in both patients showed that they were not identical [6]. Furthermore, the previously reported heterozygous deletions in the growth hormone (GH) gene cluster in 17q are now regarded as apathogenic polymorphisms [7].

Chromosome 7

Cytogenetic aberrations of chromosome 7 including duplications of 7p11.2p13 and small marker chromosomes have been identified in several SRS individuals (for review, see ref. [8]) and lead to the assumption that a ‘SRS gene’ should be localized in

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the short arm of chromosome 7 (see below). However, the most spectacular finding was that of maternal uniparental disomy of chromosome 7 (UPD(7)mat) in 7–10% of SRS individuals [9]. Uniparental disomy (UPD) is the inheritance of two homologous chromosomes or chromosomal fragments (partial UPD) from only one parent in an euploid offspring. Whole chromosome UPD can be the result of meiotic as well as mitotic non-disjunction errors, but the most frequent mode of formation is via trisomic rescue: 10–15% of all recognized human conceptions are trisomic and most of them are lethal. For many chromosomes, the only way that the trisomic conception survives is that one of the three chromosomes is lost. In one third of these cases a random chromosome loss might lead to UPD. If the UPD results from a postzygotic mitotic error, it might be associated with chromosomal mosaicism. Identification of UPD is extremely helpful because it helps deciphering imprinted regions and genes. UPD has meanwhile been described for the majority of chromosomes but only chromosomes 6, 7, 11, 14 and 15 show a specific phenotype on the basis of imprinting disturbances. So far, research on chromosome 7 encoded factors has focused on two chromosomal segments. For the candidate region in 7p11.2-p13 SRS patients with duplications have been reported (for review, see ref. [10]). The region harbors an imprinted gene (growth factor receptor bound protein 10/GRB10, see below) and several factors involved in human growth and development (fig. 2). Pathogenic mutations in these genes have not yet been described in SRS (for review, see ref. [8]). The most prominent candidate in 7p is GRB10 which encodes a cytoplasmic adaptor protein and interacts with tyrosine kinase receptors. The gene shows a complex expression in mammalian tissues with various isoforms expressed either from the maternal or from the paternal copy. In mice, Grb10 is mainly expressed from the maternal allele; the loss of maternal Grb10 results in both fetal and placental overgrowth [11, 12] demonstrating its role as a growth suppressor. By contrast, Grb10 overexpression in mice causes postnatal growth retardation and insulin resistance [13]. Grb10 serves a negative regulator of insulin signaling and action in vivo [12, 13] but a role in a fetal growth pathway independent of Igf2 has also been postulated [11]. To summarize, Grb10/GRB10 plays an essential role in growth and is therefore still a good candidate for SRS. Nevertheless, neither point mutations in the coding region nor aberrant methylation of GRB10 have been detected in SRS patients despite extensive screening studies (for review, see ref. [8]). There is additional evidence that the chromosomal region 7q31 is also involved in the etiology of SRS: meanwhile four growth-restricted patients have been described with segmental maternal UPD of the long arm of chromosome 7, three of which with SRS features (fig. 2). In 7q31, three imprinted genes (MEST/PEG1; CPA4; COPG2) and two imprinted noncoding RNAs (MESTIT1, CIT1/COPG2IT1) are localized but screening studies did not detect any pathogenic variants.

(Epi)Genetics of Silver-Russell Syndrome

13

22

Candidate genes

21

p

15 14 13

Fig. 2. SRS candidate genes on chromosomes 7 and review on the extents of segmental UPD(7)mat and maternal duplications in 7p12 reported so far (* imprinted genes). IGFBP1, IGFBP3 insulin-like growth factor-binding proteins 1 and 3; PHKG1 = Phosphorylase kinase 1; EGFR = epidermal growth factor receptor; GHRHR = growth hormone-releasing hormone receptor; MEST/PEG1 = mesoderm-specific transcript; CPA4 = carboxypeptidase A4; COPG2 = coatomer protein complex subunit gamma 2; MESTIT MEST = intronic transcript; CIT1/COPG2IT1 COPG2 = intronic transcript; PAX4 = paired box gene 4.

12 11.2 11.1 11.1

Duplications Segmental UPD(7)mat

GHRHR IGFBP1 IGFBP3 GRB10*

EGFR PHKG1 c7orf10

11.2

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q

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32 33 34 35 36

MEST* MESTIT1*

CIT1* COPG2*

PAX4 CPA4

Chromosome 7

Chromosome 11

Recent exciting findings indicate an important role of the region 11p15 in the etiology of SRS (fig. 3). The first hint was the identification of maternal 11p15 duplications in growth retarded patients and more or less striking SRS features (for review: [14]). Interestingly, the opposite disturbance – duplication of paternal 11p15 – is associated with the overgrowth disease Beckwith-Wiedemann syndrome (BWS, OMIM 130650). Numerous genetic and epigenetic alterations can be detected in BWS patients (table 1; fig. 3) (for review, see ref. [15]) but in more than 50% aberrant methylation patterns in 11p15 are involved. The search for epimutations in 11p15 in SRS patients was therefore consequent and indeed, hypomethylation at the telomeric imprinting control region 1 (ICR1) in 11p15 regulating H19 and IGF2 expression could be identified in more than 38% of cases (table 1; fig. 3). A comparison of the 11p15 disturbances in BWS and SRS reveals many similarities in the molecular biology of the two disorders.

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Table 1. 11p15 (epi)mutations in SRS and BWS and their frequencies: modified from Eggermann et al. [8] Type of mutation

SRS

Frequencies

BWS

Frequencies

In ICR1

hypomethylation

>38%

hypermethylation

2–8%

In ICR2

mutations in CKDN1C

–

mutations in CDKN1C

5–10% sporadic 40% autosomal dominant traits

hypermethylation

–

hypomethylation

50–60%

maternal duplication

1 case

11p structural rearrangements

maternal duplications

4 cases

paternal duplications

1–2%

UPD of 11p15

maternal

?

paternal UPD

10–20%

Others

maternal UPD7/7p 10% duplications >40% unknown

unknown

10–20%

First of all, epimutations affect the ICR1 in both entities. In BWS, hypermethylation of the ICR1 leads to biallelic expression of IGF2, potentially doubling the IGF2 dose in specific organs. By contrast, the ICR1 hypomethylation observed in SRS should result in a suppressed IGF2 expression in the target tissues. As a result of its central role in human growth (and tumorigenesis), IGF2 is the perfect candidate for both diseases, and indeed IGF2 is highly expressed in tissues which are affected in BWS. In vitro, IGF2 expression is altered in both syndromes: in tissue samples from BWS patients IGF2 is overexpressed at the mRNA level (for review, see ref. [15]), in fibroblasts of SRS individuals IGF2 mRNA is reduced [4]. However, IGF2 serum levels in both BWS patients and SRS patients with H19 hypomethylation are normal [16, 17] but we have to consider that the liver as the major organ of postnatal IGF2 secretion expresses the factor from a nonimprinted promoter. Due to the negative findings in serum it has been concluded that ICR1 demethylation does not directly influence IGF2 secretion in SRS children but an altered IGF2 production probably leads to a diminished autoparacrine action in the fetus. The recent identification of a SRS patient with a duplication restricted to the centromeric imprinting control region 2 (ICR2) [18] which regulates the expression of CDKN1C, KCNQ1 and further genes suggests that both ICRs on 11p15 are involved in the aetiology of the disease, like in BWS where epi/mutations in the ICR2 account for more than 50% of cases (table 1). This finding and further data obtained from BWS patients and mice models suggest that ICR1 and ICR2 interact (for review, see ref. [8]).

(Epi)Genetics of Silver-Russell Syndrome

15

11pter

11cen CDKN1C

KCNQ1

IGF2

M

H19

CTCF

KCNQ1OT1 CH3

CH3

CH3 CH3

CDKN1C

KCNQ1

IGF2

H19

P KCNQ1OT1

BWS

ICR2

SRS

ICR1

Chromosomal rearrangements (duplications) CDKN1C

M

KCNQ1

IGF2

H19

IGF2

H19

IGF2

H19

M

KCNQ1OT1

CDKN1C

P

KCNQ1

M

KCNQ1OT1

CDKN1C

P

KCNQ1

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KCNQ1OT1

CDKN1C

KCNQ1

IGF2

H19

IGF2

H19

IGF2

H19

IGF2

H19

IGF2

H19

KCNQ1OT1

CDKN1C

KCNQ1 KCNQ1OT1

CDKN1C

KCNQ1 KCNQ1OT1

Uniparental disomy (UPD) CDKN1C

KCNQ1

P

IGF2

H19

IGF2

H19

KCNQ1OT1

CDKN1C

P

KCNQ1 KCNQ1OT1

Altered Methylation of ICR1

M P

CDKN1C

KCNQ1

CDKN1C

KCNQ1

IGF2

H19

IGF2

H19

M

CDKN1C

P

KCNQ1OT1

KCNQ1 KCNQ1OT1

KCNQ1OT1

CDKN1C

KCNQ1 KCNQ1OT1

Duplication of ICR2

(Epi)mutations of/in ICR2 Hypomethylation of ICR2 M

CDKN1C

P

CDKN1C

KCNQ1

IGF2

H19

IGF2

H19

KCNQ1OT1

KCNQ1

M

CDKN1C

KCNQ1

CDKN1C

KCNQ1

KCNQ1OT1

P

CDKN1C

M P

CDKN1C

KCNQ1

CDKN1C

KCNQ1

IGF2

H19

IGF2

H19

H19

KCNQ1

IGF2

H19

KCNQ1OT1

KCNQ1OT1

Pointmutations in CDKN1C

IGF2

KCNQ1OT1

Expressed

Silenced

IGF2

IGF2

CDKN1C

CDKN1C

KCNQ1OT1 KCNQ1OT1

Fig. 3. Epigenetic regulation of the two imprinting center regions (ICR) in 11p15 and the different (epi)mutations affecting 11p15 in BWS and SRS. CDKN1C = Cyclin-dependent kinase inhibitor/p57KIP2; KCNQ1 = potassium channel KQT-family member 1; KCNQ1OT1 KCNQ1 = intronic transcript 1; IGF2 = insulin-like growth factor 2.

Do Different Mutations in Functional Networks Explain the Heterogeneity of SRS?

When looking at the major chromosomal regions involved in rearrangements in SRS we see that three of them, i.e. chromosomes 7, 11 and 15, harbor members of the IGF system, a mediator of pre- and postnatal growth. SRS might therefore represent a common phenotype caused by alterations in members of this axis such as the ligand IGF2, its receptor IGF1R and the IGF signaling modulator GRB10. The effects of IGF2 on fetal growth are mainly mediated through the IGF1R. Mice knockout models indicate that loss of the paternal IGF2 causes IUGR with reduction in 40% of the body weight in newborn mice [19] whereas experimental overexpression of the same allele results in hypertrophic mice [20].

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Another axis – an ‘imprinted gene network’ – has recently been hypothesized by Arima et al. [21]. They observed that in vitro the imprinted gene PLAGL1/ZAC (pleomorphic adenoma gene-like 1) in 6q24 drives the expression of KCNQ1OT1 noncoding RNA in 11p15. PLAGL1/ZAC might affect imprinted expression along the ICR2 domain in 11p15, including CDKN1C. This would put PLAGL1/ZAC, KCNQ1OT1 and CDKN1C in a common regulatory mechanism that influences intrauterine growth. According to Arima et al. [21], the assumed regulation of CDKN1C by PLAGL1/ZAC indicates a potential role of this gene in BWS but they did not detect aberrant methylation at the PLAGL1/ZAC DMR in a small number of BWS patients. Indeed they observed changes in the methylation of the ICR2 in two patients with transient neonatal diabetes mellitus (TNDM) and 6q24 disturbances.

Genotype-Phenotype Correlation in SRS?

With the identification of the ICR1 hypomethylation in 11p15 and UPD(7)mat the molecular confirmation of the clinical diagnosis of SRS is now possible in ~50% of patients (table 1). However, we have to bear in mind that routine diagnostics is based on lymphocytes and that nearly all SRS patients with ICR1 hypomethylation are mosaics. Thus, we hypothesize that a subgroup of patients escapes molecular diagnosis because their mosaicism affects other tissues than blood cells. In these cases, the mosaic distribution of the epimutation probably influences the phenotype. This assumption is supported by the findings of several groups [4, 22, 23] who reported on a correlation between the degree of methylation at the ICR1 locus and the severity of the phenotype. The finding of different epigenetic and genetic alterations in SRS patients should enable us to delineate a genotype-phenotype correlation. Indeed, the SRS phenotype of carriers of the 11p15 epimutation is generally more severe and typical than that of UPD(7)mat carriers or those SRS without known mutation [16, 24] but exceptions exist [23]. Additionally, in single cases chromosomal anomalies might also influence the phenotype depending on the chromosomal regions involved. In total, the phenotypic transition is fluent and therefore carriers of 11p15 epimutations and UPD(7) mat can not be discriminated solely by clinical findings. We therefore suggest to test all patients with intrauterine and severe postnatal growth retardation and only slight signs reminiscent for SRS for the known mutations. Screening of larger groups of growth-retarded patients will help to establish the frequency of 11p15 epimutation and UPD(7)mat in this heterogeneous cohort. Based on the current data it can be hypothesized that the frequency of SRS is underestimated because of the difficult and often subjective clinical diagnosis which depends on the experience of the clinical investigator. As aforementioned, IGF2 serum levels are within the normal range in SRS patients possibly due to a biallelic expression in the liver whereas its predominantly paternal

(Epi)Genetics of Silver-Russell Syndrome

17

expression in the fetus makes functional consequences of an aberrant methylation possible. Recently, Binder et al. [24] demonstrated that IGF1 serum levels in 11p15 epimutation carriers are inadequately high in comparison to non-syndromic short children. Moreover, IGFB3 levels were increased in the 11p15 patients while UPD(7) mat patients showed regular IGF1 and IGFB3 serum levels. It was therefore concluded that heterogeneity of SRS does not only include the genetic and clinical level but also hormone regulation.

Conclusion

In the highly developed countries ~3% of children are born too small and 20% of them do not show catch-up growth, patients with SRS belonging to the latter group. The functional cause for the persisting growth retardation is currently unknown in the majority of cases, and the only pharmacological option available is treatment with hGH. Since the response is highly variable even in a defined cohort like SRS patients, a more specific classification of the patients based on genetic methods might help to understand this variable response and allow to specifically adapt the treatment. Thus, SRS can be regarded as a model to decipher the functional link between the different genetic and epigenetic factors identified in growth retarded individuals; future studies will further enlighten the complex interactions between growth factors and contribute to a better directed therapy.

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Moore T, Haig D: Genomic imprinting in mammalian development: a parent tug-of-war. Trends Genet 1991;7:45–49. Pembrey ME, Bygren LO, Kaati G, Edvinsson S, Northstone K, Sjöström M, Golding J, ALSPAC Study Team: Sex-specific, male-line transgenerational responses in humans. Eur J Hum Genet 2006; 14: 159–166. Bailey W, Popovich B, Jones KL: Monozygotic twins discordant for the Russell-Silver syndrome. Am J Med Genet 1995;58:101–105. Gicquel C, Rossignol S, Cabrol S, Houang M, Steunou V, Barbu V, Danton F, Thibaud N, Le Merrer M, Burglen L, Bertrand AM, Netchine I, Le Bouc Y: Epimutation of the telomeric imprinting center region on chromosome 11p15 in SilverRussell syndrome. Nat Genet 2005;37:1003–1007. Binder G, Mavridou K, Wollmann HA, Eggermann T: Screening for insulin-like growth factor-I receptor mutations in patients with Silver-Russell syndrome. J Pediatr Endocrinol Metab 2002;15; 1167–1171.

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Dörr S, Midro AT, Farber C, Giannakudis J, Hansmann I: Construction of a detailed physical and transcript map of the candidate region for Russell- Silver syndrome on chromosome 17q23q24. Genomics 2001;71:174–181. Eggermann T, Schoenherr N, Eggermann K, Wollmann HA: Hypomethylation in the 11p15 telomeric imprinting domain in a Silver-Russell patient with a CSH1 deletion (17q24) renders a functional role of this alteration unlikely. J Med Genet 2007; 44:e77. Eggermann T, Eggermann K, Schönherr N: Growth retardation versus overgrowth: Silver-Russell syndrome is genetically opposite to BeckwithWiedemann syndrome. Trends Genet 2008;24: 195–204. Kotzot D, Schmitt S, Bernasconi F, et al: Uniparental disomy 7 in Silver-Russell syndrome and primordial growth retardation. Hum Mol Genet 1995;4:583– 587.

Eggermann

10 Monk D, Bentley L, Hitchins M, Myler RA, ClaytonSmith J, Ismail S, Price SM, Preece MA, Moore GE: Chromosome 7p disruptions in Silver-Russell syndrome: delineating an imprinted candidate gene region. Hum Genet 2002;111:376–387. 11 Charalambous M, Smith FM, Bennett WR, Crewe TE, MacKenzie F, Ward A: Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism. PNAS 2003;100:8292–8297. 12 Wang L, Balas B, Christ-Roberts CY, Kim RY, Ramos FJ, Kikov CK, Li C, Deng C: Peripheral disruption of the Grb10 gene enhances insulin signaling and sensitivity in vivo. Mol Cell Biol 2007;27:6497– 6505. 13 Shiura H, Reyna S, Muri N, Dony LQ, De Fronzo RA, Liu F: Meg1/Grb10 overexpression causes postnatal growth retardation and insulin resistance via negative modulation of the IGF1R and IR cascades. BBRC 2005;329; 909–916. 14 Eggermann T, Meyer E, Obermann C, Heil I, Schuler H, Ranke MB, Eggermann K, Wollmann HA: Is maternal duplication 11p15 associated with Silver-Russell syndrome? J Med Genet 2005;42:e26. 15 Weksberg K, Smith AC, Squire J, Sadowski P: Beckwith-Wiedemann syndrome demonstrates a role for epigenetic control of normal development. Hum Mol Genet 2003;12:R61–R68. 16 Netchine I, Rossignol S, Dufourg MN, Azzi S, Rousseau A, Perin L, Houang M, Seunou V, Esteva B, Thibaud N, Demay M-CR, Danton F, Petricko E, Bertrand A-M, Heirnichs C, Carel J-C, Loeuille G-A, Pinto G, Jaquemont M-L, Gicquel C, Cabrol S, Le Bouc Y: 11p15 ICR1 loss of methylation is a common and specific cause of typical Russell-Silver syndrome: clinical scoring system and epigeneticphenotypic correlations. J Clin Endocrinol Metabol 2007;92: 3148–4154.

17 Schneid H, Vazquez MP, Vachev P, Gournelen M, Cabrol S, Le Bouc Y: The Beckwith-Wiedemann syndrome phenotype and the risk of cancer. Med Pediatr Oncol 1997;28; 411–415. 18 Schönherr N, Meyer E, Schmidt A, Wollmann HA, Eggermann T: The centromeric 11p15 imprinting center is also involved in Silver-Russell syndrome. J Med Genet 2007;44:59–63. 19 DeChiara TM, Efstratiadis A, Robertson EJ: A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 1990;345:78–80. 20 Sun F-L, Dean WC, Kelsey G, Allen ND, Reik W: Transactivation of Igf2 in a mouse model of Beckwith-Wiedemann syndrome. Nature 1997;389: 809–815. 21 Arima T, Kamikara T, Hayashita T, Kato K, Inoue T, Shirayashi Y, Oskimura M, Soejima H, Makai T, Wake N: ZAC, Lit1 (KCNQ1OT1) and p57KIP2 (CKDN1C) are in an imprinted gene network that might play a role in Beckwith-Wiedemann syndrome. Nucleic Acids Res 2005;33:2650–2660. 22 Bliek J, Terhal P, van den Bogaard M-J, Maas S, Hamel B, Salieb-Beugelaar G, Simon M, Letteboer T, van der Smagt J, Kroes H, Mannens M: Hypomethylation of the H19 gene causes not only Silver-Russell syndrome (SRS) but also isolated asymmetry or an SRS-like phenotype. Am J Hum Genet 2006;78:604–614. 23 Zeschnigk M, Albrecht B, Buiting K, Kanber D, Eggermann T, Binder G, Gromoll J, Prott E-C, Seland S, Horsthemke B: IGF2/H19 hypomethylation in Silver-Russell syndrome and isolated hemihypoplasia. Eur J Hum Genet 2008;16:328–334. 24 Binder G, Seidel A-K, Martin DD, Schweizer R, Schwarze P, Wollmann HA, Eggermann T, Ranke MB: The endocrine phenotype in Silver-Russell syndrome is defined by the underlying epigenetic alteration. J Clin Endocrinol Metab 2008;93:1402– 1407.

Thomas Eggermann, PhD Institute of Human Genetics University Hospital, RWTH Aachen Pauwelsstrasse 30, DE–52074 Aachen (Germany) Tel. +49 241 808 8008, Fax +49 241 808 2394, E-Mail [email protected]

(Epi)Genetics of Silver-Russell Syndrome

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Genetic Imprinting: The Paradigm of Prader-Willi and Angelman Syndromes Fiorella Gurrieri ⭈ Maria Accadia Institute of Medical Genetics, Catholic University, Rome, Italy

Abstract Imprinted genes are expressed from only one of the two parental alleles. A consequence of genomic imprinting is that viable embryos must receive two haploid genome complements from parents of opposite sex. The parental-specific expression is obtained through epigenetic modifications (DNA methylation, histone tail modifications) which alter the conformation of chromatin fiber and therefore regulate the expression of the underlying genes. Deletions, duplication, mutations or alterations of imprinting of the only active allele, as well as uniparental disomy or loss of imprinting of the inactive allele lead to an unbalance (loss of function or gain of function) in the dosage of the gene product and may have phenotypic consequences. Two such examples in human pathology are represented by the Prader-Willi and Angelman syndromes, whose phenotypes result from loss of paternal or maternal contribution of the 15q11-q13 genomic region, respectively. Prader-Willi syndrome is characterized by pre- and postnatal hypotonia, feeding difficulties in early life and subsequent hyperphagia with obsessive/compulsive food searching, obesity, short stature, hypogonadism and acromicria. Psychomotor development is mildly affected and behavioral problems are more relevant. Patients with Angelman syndrome show a completely different phenotype characterized by severe mental retardation, absent speech, autistic-like behavior, severe epilepsy and postnatal Copyright © 2009 S. Karger AG, Basel microcephaly.

The term ‘genomic imprinting’ refers to the different expression of an allele depending on its parent-specific origin, so that only the paternal or the maternal allele is expressed in certain tissues or developmental stages. A direct consequence of this process is a physiologic functional haploidy of imprinted genes, which makes them more likely to be associated with disease. Normal development in mammals requires genes to be inherited from both parents. The functional differences between the maternal and paternal genomes are clearly recognized in two human tumors: the ovarian teratoma, arising from ovarian germ cells, and the hydatidiform mole, arising from cytotrophoblast. The ovarian teratoma has a gynogenetic origin and contains elements derived from all three germinal layers (ectoderm, mesoderm, endoderm)

[1], whereas the hydatidiform mole has an androgenetic origin and is composed by extra-embryonic trophoblast elements [2]. Nuclear transplantation experiments in mouse provided further evidence of the different developmental program of the maternal and paternal genome in embryo. Mouse eggs manipulated to contain two maternal pronuclei (gynogenotes) showed better formation of the embryo, but very poor development of extraembryonic tissues. In contrast, in mouse eggs with two paternal pronuclei (androgenotes), an exuberant trophoblast growth and failure of embryonic development was noted. These experiments pointed out that the maternal and paternal contributions to the embryonic genome in mammals were not equivalent and the correct development of the embryo required both of them [3]. It was also noted that this parental effect did not involve the whole genome, but was limited to specific chromosomal regions containing clusters of imprinted genes, which were differentially marked in the maternal and paternal germ lines [4].

Meaning of Epigenetic Reprogramming

Genomic imprinting is an epigenetic process. But what does ‘epigenetic’ mean? Actually, this term has been reinvented twice in the history of biology and development. The term epigenetic was first introduced in 1942 by Conrad Hal Waddington to describe biological differences between tissues that result from the process of development. He used the metaphor ‘genetic landscape’, suggesting that cell fates were established in development in a way similar to a marble rolling down to the lowest point of a ground in which ridges and furrows delineate the path and make it irreversible. Therefore, such a path depends not only on the intrinsic nature of the marble, but also on the conditioning through external forces [5]. Today, the term epigenetic refers to the modification of a given DNA trait, not changing its sequence, but rather its function through DNA methylation and histone tail modifications. Such modifications alter the conformation of the chromatin fiber, interfering with the transcriptional machinery and with DNA-binding proteins thus regulating the expression of the underlying genes [6]. This process is very important during the development of a multicellular organism, in which different cells and tissues acquire different programs of gene expression. From this point of view, genomic imprinting is a particular type of epigenetic regulation in which the activity of a gene is modified depending on the sex of the transmitting parent. Imprinting requires three main steps: gamete DNA marking, maintenance of the marking in embryo and adult somatic tissues, and resetting of marking at the beginning of gametogenesis [7]. In mammalian embryos there are two major cycles of epigenetic reprogramming of the genome: during preimplantation and during germ cell differentiation. These events have been widely studied in mouse embryos. After fertilization, the paternal and maternal genomes undergo a genomewide demethylation, followed by de novo methylation around the time of implantation. Imprinted alleles are protected from this wave of demethylation and

Prader-Willi and Angelman Syndromes

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remethylation to maintain their proper dosage effects. The second step in epigenetic reprogramming is necessary to reset imprinting in germ cells. This process takes place in primordial germ cells and consists of DNA demethylation, involving also imprinted genes, and subsequent remethylation according to the sex, so that in ovocytes all imprinted genes will have a maternal mark and in sperm cells a paternal one [8]. Occasionally, epigenetic information appears to be inherited through the germ line, and this is likely to be a result of incomplete erasure in the germ line (or after fertilization) [9].

Paradigm of Prader-Willi and Angelman Syndromes: Imprinting Regulation and Evolutionary Aspects

Deletions, duplication, mutations or alterations of imprinting of the only active allele, as well as uniparental disomy or loss of imprinting of the inactive allele lead to an unbalance (loss of function or gain of function) in the dosage of the gene product and may have phenotypic consequences. The first clinical syndromes recognized in humans as a result of anomalies at imprinted loci were Prader-Willi (PWS) and Angelman syndromes (AS) in 1989 [10]. PWS and AS result from loss of paternal or maternal contribution of the same genetic region at 15q11.q13 (PWS-AS imprinting domain). The PWS-AS imprinting domain has a bipartite structure, revealed by the smallest region of overlap in AS and PWS patients due to an imprinting center deletion. The PWS imprinting center (PWS-IC) is located in exon 1 of SNRPN and is defined by a cluster of CpG sites that are not methylated on the paternal allele and methylated on the maternal allele. The AS imprinting center (AS-IC) resides 35–40 kb upstream of SNRPN exon 1 and its proposed function is to promote the methylation of the adjacent PWS-IC in the maternal germline. This IC regulates the expression of several paternally expressed genes (among those SNRPN, MAGEL2, MKRN3, NDN and more than seventy snoRNAs) and two maternally expressed genes (UBE3A and ATP10C) (fig. 1) [11]. Proximal to this region the non-imprinted P gene is also mapped, encoding a tyrosine transporter whose deficiency contributes to the skin and ocular hypopigmentation that occurs in PWS and AS patients with deletion [12].The AS phenotype is caused by several genetic mechanisms leading to loss of function of the UBE3A gene, which encodes for an ubiquitin protein ligase, E6-AP, and is expressed only by the maternal allele in certain brain regions [13]. For PWS, instead, the phenotype seems to result from loss of function of a number of paternally expressed genes on chromosome 15q11-q13 [14]. In an evolutionary perspective, the PWS-AS domain was constructed relatively recently, between 180 and 105 million years ago. In marsupials, in which the first evidence of imprinting was found, the PWS-AS domain is absent, SNRPN and UBE3A are not on the same chromosome and a completely different gene, CNGA3 (now located on human chromosome 2) is situated in place of SNRPN, ~60 kb distant from UBE3A. The construction of the

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P1 0 AT

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Fig. 1. The PWS-AS imprinting domain. The imprinting center (IC) has bipartite structure: PWS-IC and AS-IC. On the maternal allele AS-IC confers methylation (M) of PWS-IC. As a result, all paternally expressed genes are silenced (empty boxes) and UBE3A and ATP10 are active (full boxes, arrows indicate transcription sense). On the paternal allele, PWS-IC remains unmethylated and activates all paternally expressed genes (e.g. MAGEL2, NDN, SNRPN).

PWS-AS domain was reached in eutherian through translocation of SNRPN beside UBE3A and insertion of miRNAs, snoRNAs and retroposed genes from all over the genome. Imprinting arose during or after this complex rearrangement (fig. 2) [15].

Prader-Willi Syndrome

This syndrome was clinically described by Prader and colleagues in 1956, but only in 1989 were the molecular bases of PWS and AS unraveled and related to an imprinting disorder [10]. A recent epidemiological study in the United Kingdom estimates an incidence of ~1 in 25,000 births and a prevalence of ~1 in 50,000 [16]. PWS is characterized by pre- and postnatal hypotonia, with a history of decreased fetal movements, poor suck and early failure to thrive. The fetus is usually podalic and delivery is commonly after term and requires cesarean section. Polyhydramnios is highly frequent. The hypotonia is most severe in early infancy, but improves somewhat over time. In the neonatal period feeble reflexes, lethargy and weak cry are often present. Feeding is usually an issue in the first few months of life and gavage is frequently required. Minor anomalies are often described and the typical facial appearance shows almond-shaped palpebral fissures, narrow bifrontal diameter, narrow nasal

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Eutheria 180 MYA

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210 MYA

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105 MYA Lactation

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Fig. 2. Evolution of imprinting in mammals. Imprinting is not present in non-mammals nor in monotremes, the most primitive mammals, which are capable of lactation but are still oviparous. The first evidence of imprinting has been found in marsupials. Modified from Hore et al. [15].

bridge and downturned mouth with thin upper lip, but not everyone has these features. Most patients have strabismus early in life. Frequently noted is also acromicria of the hands and feet with tapering fingers. Obesity is a hallmark of this syndrome, it occurs between the ages of 1 and 6 years and is due to hyperphagia. It is the major cause of morbidity and mortality in PWS and medical problems related to obesity are cardiopulmonary issues, increased risk for type II diabetes, obstructive sleep apnea and gastrointestinal complications [17, 18]. Hypoplastic genitalia, with cryptorchidism, scrotal hypoplasia and small penis in males and hypoplastic labia minora and clitoris in females are related to hypothalamic hypogonadism. An incomplete pubertal development is usually present, menarche may occur late and some female may have oligoamenorrhea or amenorrhea. Short stature is common and growth hormone (GH) deficiency has been demonstrated in most patients. Therapy with GH results in significant improvement of height and body composition (decreased fat mass and increased lean body mass) [17]. However, a significant number of deaths in children treated with GH has been reported, raising some concern about the safety of GH treatment in this population. A recent study shows that the major cause of death in children with PWS, who received GH treatment or did not, is a respiratory disease and no difference is found between the two groups. Nevertheless, since most deaths

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Clinical features and genetic mechanisms AS Loss of maternal contribution

PWS Loss of paternal contribution

• Mild- to moderate mental retardation • Hypotonia • Obesity • Hypogonadism • Short stature

• Severe mental retardation • Seizures • Ataxic movements • Absent speech • Inappropriate laughter

70%

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Fig. 3. Clinical features and genetic mechanisms of PWS and AS. Modified from Cassidy et al. [14]. Pictures of PWS and AS patients: courtesy of Prof. Giovanni Neri.

occurred in the first 9 months of therapy, a possible aggravating role of GH at the start of treatment cannot be excluded [18]. The incidence of spinal deformity in PWS is high: scoliosis can occur at any age and kyphosis usually develops in early adulthood. Psychomotor development is mildly affected: walking is achieved at around 24 months and language development at 3–4 years. Mental retardation is in the mildto-moderate range and most patients have an IQ ranging from 60 to 70. Many show an unusual skill in doing jigsaw puzzles, suggesting a particular attention to visual details. Behavioral problems are frequent and include not only food-related problems (excessive appetite, absent sense of satisfaction, obsession with eating), but also temper tantrums, impulsivity, sleep disturbance, skin picking, obsessive-compulsive symptoms and anxiety [14, 17]. The genetic abnormality of PWS is the lack of expression of paternally inherited genes in chromosome 15q11-q13. Approximately 70% of affected individuals have a cytogenetically small deletion of chromosome segment 15q11-q13. Maternal uniparental disomy (UPD) accounts for 25%, imprinting defect (small deletion or mutation) for 2–5% and the remaining Cys at codon 120 (R120C). J Clin Endocrinol Metab 1998;83:3727–3734. 71 Vallette-Kasic S, Barlier A, Teinturier C et al: PROP1 gene screening in patients with multiple pituitary hormone deficiency reveals two sites of hypermutability and a high incidence of corticotroph deficiency. J Clin Endocrinol Metab 2001;86: 4529– 4535. 72 Mendonca BB, Osorio MG, Latronico AC, Estefan V, Lo LS, Arnhold IJ: Longitudinal hormonal and pituitary imaging changes in two females with combined pituitary hormone deficiency due to deletion of A301,G302 in the PROP1 gene. J Clin Endocrinol Metab 1999;84:942–945. 73 Asteria C, Oliveira JH, Abucham J, Beck-Peccoz P: Central hypocortisolism as part of combined pituitary hormone deficiency due to mutations of PROP-1 gene. Eur J Endocrinol 2000;143:347–352. 74 Pernasetti F, Toledo SP, Vasilyev VV, Hayashida CY, Cogan JD, Ferrari C, Lourenco DM, Mellon PL: Impaired adrenocorticotropin-adrenal axis in combined pituitary hormone deficiency caused by a two-base pair deletion (301–302delAG) in the prophet of Pit-1 gene. J Clin Endocrinol Metab 2000;85:390–397. 75 Riepe FG, Partsch CJ, Blankenstein O, Monig H, Pfaffle RW, Sippell WG: Longitudinal imaging reveals pituitary enlargement preceding hypoplasia in two brothers with combined pituitary hormone deficiency attributable to PROP1 mutation. J Clin Endocrinol Metab 2001;86:4353–4357. 76 Bottner A, Keller E, Kratzsch J, Stobbe H, Weigel JF, Keller A, Hirsch W, Kiess W, Blum WF, Pfaffle RW: PROP1 mutations cause progressive deterioration of anterior pituitary function including adrenal insufficiency: a longitudinal analysis. J Clin Endocrinol Metab 2004;89:5256–5265.

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77 Agarwal G, Bhatia V, Cook S, Thomas PQ: Adrenocorticotropin deficiency in combined pituitary hormone deficiency patients homozygous for a novel PROP1 deletion. J Clin Endocrinol Metab 2000;85:4556–4561. 78 Fofanova O, Takamura N, Kinoshita E, Vorontsov A, Vladimirova V, Dedov I, Peterkova V, Yamashita S: MR imaging of the pituitary gland in children and young adults with congenital combined pituitary hormone deficiency associated with PROP1 mutations. AJR Am J Roentgenol 2000;174:555– 559. 79 Voutetakis A, Argyropoulou M, Sertedaki A, Livadas S, Xekouki P, Maniati-Christidi M, Bossis I, Thalassinos N, Patronas N, Dacou-Voutetakis C: Pituitary magnetic resonance imaging in 15 patients with Prop1 gene mutations: pituitary enlargement may originate from the intermediate lobe. J Clin Endocrinol Metab 2004;89:2200–2206. 80 Parks JS, Brown MR, Baumbach L, et al: Natural history and molecular mechanisms of hypopituitarism with large sella turcica. Am Endocrine Soc Meet, New Orleans, 1998, abstr P3–409, p 470. 81 Bodner M, Castrillo JL, Theill LE, Deerinck T, Ellisman M, Karin M: The pituitary-specific transcription factor GHF-1 is a homeobox-containing protein. Cell 1988;55:505–518. 82 Andersen B, Rosenfeld MG: POU domain factors in the neuroendocrine system: lessons from developmental biology provide insights into human disease. Endocr Rev 2001;22:2–35. 83 Rhodes SJ, DiMattia GE, Rosenfeld MG: Transcriptional mechanisms in anterior pituitary cell differentiation. Curr Opin Genet Dev 1994;4:709–717. 84 Li S, Crenshaw EB III, Rawson EJ, Simmons DM, Swanson LW, Rosenfeld MG: Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 1990; 347:528–533. 85 Andersen B, Rosenfeld MG: Pit-1 determines cell types during development of the anterior pituitary gland: a model for transcriptional regulation of cell phenotypes in mammalian organogenesis. J Biol Chem 1994;269:29335–29338.

86 Rhodes SJ, Chen R, DiMattia GE, Scully KM, Kalla KA, Lin SC, Yu VC, Rosenfeld MG: A tissue-specific enhancer confers Pit-1-dependent morphogen inducibility and autoregulation on the pit-1 gene. Genes Dev 1993;7:913–932. 87 Tatsumi K, Miyai K, Notomi T, Kaibe K, Amino N, Mizuno Y, Kohno H: Cretinism with combined hormone deficiency caused by a mutation in the PIT1 gene. Nat Genet 1992;1:56–58. 88 Cohen LE, Wondisford FE, Salvatoni A, Maghnie M, Brucker-Davis F, Weintraub BD, Radovick S: A ‘hot spot’ in the Pit-1 gene responsible for combined pituitary hormone deficiency: clinical and molecular correlates. J Clin Endocrinol Metab 1995;80:679– 684. 89 Radovick S, Nations M, Du Y, Berg LA, Weintraub BD, Wondisford FE: A mutation in the POUhomeodomain of Pit-1 responsible for combined pituitary hormone deficiency. Science 1992;257: 1115–1118. 90 Kishimoto M, Okimura Y, Fumoto M, Iguchi G, Iida K, Kaji H, Chihara K: The R271W mutant form of Pit-1 does not act as a dominant inhibitor of Pit-1 action to activate the promoters of GH and prolactin genes. Eur J Endocrinol 2003;148:619–625. 91 Pfaffle RW, DiMattia GE, Parks JS, Brown MR, Wit JM, Jansen M, Van-der-Nat H, Van-den-Brande JL, Rosenfeld MG, Ingraham HA: Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia. Science 1992;257:1118– 1121. 92 Pfaffle RW, Martinez R, Kim C, Frisch H, Lebl J, Otten B, Heimann,G: GH and TSH deficiency. Exp Clin Endocrinol Diabetes 1997;105(suppl 4):1–5. 93 Turton JPG, Reynaud R, Mehta A et al: Novel mutations within the POU1F1 gene associated with variable combined pituitary hormone deficiency. J Clin Endocrinol Metab 2005;90:4762–4770. 94 Cohen LE, Radovick S: Molecular basis of combined pituitary hormone deficiencies. Endocr Rev 2002; 23:431–442.

Mehul Tulsidas Dattani, MD Developmental Endocrine Research Group Institute of Child Health, 30 Guilford Street London WC1N 1EH (UK) Tel. +44 0207 905 2657, Fax +44 0207 404 6191, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 83–94

Developmental Abnormalities of the Posterior Pituitary Gland Natascia di Iorgi ⭈ Andrea Secco ⭈ Flavia Napoli ⭈ Erika Calandra ⭈ Andrea Rossi ⭈ Mohamad Maghnie Departments of Pediatrics and Neuroradiology (A.R.), IRCCS G, Gaslini Institute, University of Genova, Genova, Italy

Abstract While the molecular mechanisms of anterior pituitary development are now better understood than in the past, both in animals and in humans, little is known about the mechanisms regulating posterior pituitary development. The posterior pituitary gland is formed by the evagination of neural tissue from the floor of the third ventricle. It consists of the distal axons of the hypothalamic magnocellular neurones that shape the neurohypophysis. After its downward migration, it is encapsulated together with the ascending ectodermal cells of Rathke’s pouch which form the anterior pituitary. By the end of the first trimester, this development is completed and vasopressin and oxytocin can be detected in neurohypophyseal tissue. Abnormal posterior pituitary migration such as the ectopic posterior pituitary lobe appearing at the level of median eminence or along the pituitary stalk have been reported in idiopathic GH deficiency or in subjects with HESX1, LHX4 and SOX3 gene mutations. Another intriguing feature of abnormal posterior pituitary development involves genetic forms of posterior pituitary neurodegeneration that have been reported in autosomal-dominant central diabetes insipidus and Wolfram disease. Defining the phenotype of the posterior pituitary gland can have significant clinical implications for management and counseling, as well as providing considerable insight into normal and abnormal mechanisms of posterior pituitary development in Copyright © 2009 S. Karger AG, Basel humans.

The molecular mechanisms and roles of transcription factors in anterior pituitary development are now much better understood than in the past, both in animals and in humans, but little is known about the mechanisms that regulate posterior pituitary development. The anterior, intermediate and posterior lobes of the pituitary gland develop from separate embryonic cell lineages: the oral ectoderm and neural ectoderm, respectively. Pituitary organogenesis begins when an area of oral ectoderm at the roof of the presumptive oral cavity invaginates upwards to form the Rathke’s pouch, which

Timeline Prenatal time scale (months)

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Differentiation of neurones involves cascades of transcription factors which progressively define their phenotype

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9 Birth

Optic chiasm

Anterior pituitary

Mamillary body Posterior pituitary

Completion of the formation of the posterior pituitary involves the development of axonal projections from the PVN and SON through the neural stalk, terminating in the posterior pituitary lobe

Fig. 1. Timeline of the development of the posterior pituitary neurone. SIM1, ARNT2, OTP and BRN2 are genes involved in the cascade of transcription factors. PVN = Paraventricular nuclei; SON = supraoptic nuclei: SS = synthesizing somatostatin; TRH = thyrotropin-releasing hormone; CRH = corticotrophin-releasing hormone; AVP = arginine vasopressin; OT = oxytocin. With permission.

eventually becomes the anterior and intermediate lobes of the gland. The posterior pituitary evolves from an infundibulum that develops by downward evagination of the dorsal presumptive diencephalon soon after Rathke’s pouch begins to extend upward. The two structures maintain close contact while cells migrate from the mesoderm and neural crest into the space between the presumptive brain and oral cavities. The ventral wall of Rathke’s pouch becomes the anterior lobe whereas the posterior wall of the pouch develops into the pars intermedia [1]. The following sections of this article will focus on the most important aspects of diseases involving abnormal posterior pituitary gland development.

Abnormal Development of the Posterior Pituitary Lobe

The hypothalamo-neurohypophysis consists of magnocellular neurons that produce the peptide hormones vasopressin and oxytocin. The cell bodies are located in the

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paraventricular nucleus (PVN) and supraoptic nucleus (SON) in the hypothalamus, and axons project to the neurohypophysis where the hormones are secreted into the bloodstream. During embryogenesis, neuroepithelial cells from the lining of the third ventricle migrate to the walls of the third ventricle where they mature into PVN. Some cells continue to migrate laterally towards and above the optic chiasm to form the SON. Their unmyelinated axons traverse the basal hypothalamus, form the neural stalk and terminate at the floor of the third ventricle and in the median eminence. The early differentiation of these cell lineages has recently been made clearer through an understanding of the role of transcription factors in hypothalamic development [2]. The SIm1, ARNt2, OTP, and BRN2 genes appear to be involved in the cascade of transcription factors implicated in the development of the neuroendocrine hypothalamus leading to the completion of posterior pituitary development by the end of the first trimester (fig. 1), when vasopressin and oxytocin can be detected in neurohypophyseal tissue [3]. Null mutation of UNcx4.1 showed a phenotype consisting of an ectopic localization that extends vasopressinergic axons from PVN and SON; these axons do not halt at the proper position in the neurohypophysis, but instead grow into the anterior pituitary lobe [4]. In humans, a similar picture has been reported in association with cerebral malformations [5]. In a recent study, a Hes1-null pituitary gland was revealed to be reduced in size but was otherwise morphologically normal compared with the control. Indeed, in Hes1-Hes5 double-mutant mice, the evagination of the infundibulum was affected and the neurohypophysis was lost compared to both the wild-type and Hes1-null mice, suggesting that both Hes genes are essential for the formation of the neurohypophysis [6]. In the nervous system, both Hes1 and Hes5 are essential for the regulation of neural stem cells, while in the endocrine system, Hes1 controls pancreatic cell differentiation: indeed, an analysis of mice deficient for Prop1 has indirectly implicated involvement of the Notch pathway in pituitary development [7].

Relationship between Posterior Pituitary Development and Pituitary Diseases

Abnormal posterior pituitary development can be subdivided into two main categories (fig. 3). The first is associated with a migration defect and the second with neurodegeneration of the hypothalamic PVN and SON nuclei. Developmental abnormality of the posterior pituitary will lead to an ectopic posterior pituitary (EPP) at the median eminence or along the pituitary stalk with partial or complete pituitary stalk agenesis with or without additional central nervous system malformations where the genetic and the idiopathic forms are closely linked and might have a common origin [8]. On the other hand, the neurodegeneration of the hypothalamic nuclei will lead to posterior pituitary dysfunction and central diabetes

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Posterior pituitary pathologies

Developmental abnormality–migration ectopic posterior pituitary

Neurodegenerative neurones

Wolfram syndrome Autosomal-dominant FNDI

Partial or complete agenesis pituitary stalk CNS malformations

Partial or complete agenesis pituitary stalk

Astrocytoma Choristoma (granular cell tumor)

Genetic syndromes idiopathic

Neurohypophyseal germinoma

Posterior pituitary function

Fig. 2. Tree diagram showing the two main categories of developmental anomalies of the posterior pituitary gland.

insipidus. Whether posterior pituitary function is affected in patients with developmental abnormalities of the posterior pituitary remains uncertain.

Abnormal Posterior Pituitary Development and Phenotype Craniofacial dysmorphisms and eye abnormalities are often manifestations of major developmental syndromes and can give useful clues about possible defects of the brain and/or hypothalamus and pituitary gland [9–10]. Some facial malformation syndromes including septo-optic dysplasia (SOD) and Pallister-Hall are easy to recognize by visual and/or physical examination. Indeed, the diagnosis of hypopituitarism in childhood can sometimes be straightforward when short stature and persistent growth failure are associated with frontal bossing, mid-facial hypoplasia and truncal adiposity; however, this presentation tends to be the exception rather than the rule and thus the clinical phenotype may not be particularly impressive. Recent study shows, however, that patients with an EPP at magnetic resonance imaging (MRI) had significantly higher canthal index CI values (the relative distance between the eyes; hypertelorism is generally defined as CI >42, and hypotelorism as CI 6 years and 29 patients >8 years; 24 patients developed spontaneous menarche. FH were 148.6 ± 5.4 cm in patients treated for a period 4 years; 154.2 + 4.6 cm >8 years of GH therapy (fig. 1). At multiple regression analysis, height velocity in the 1st year of therapy, pretherapy height, GH duration (p = 0.00001), age at the start of GH therapy (p = 0.008) and target height (TH) (p = 0.009) are the most important factors influencing FH. Spontaneous menarche seems to have a negative influence on FH (T = –1.9, p = 0.06). Our results confirm the importance of dosage, age at the start and duration of GH therapy, in particular a minimum of 4 years of therapy, to obtain a good result in FH in subjects with TS.

Noonan Syndrome

Noonan syndrome (NS) is a quite frequent genetic condition (1/1,000–2,500 live births).

Etiology An autosomal-dominant inheritance has been suggested. The PTPN11 gene has been identified as the disease gene, located on chromosome 12 (12q24.1), in about 50% of the NS patients. This gene encodes the protein tyrosine phosphatase SHP2, that is involved in postreceptor signaling of developmental processes in the RAS-MAPK (mitogen-activated protein kinase) pathway; the mutation produces a gain of function that impairs the GH post-receptorial signal. Recently, other genes have been identified in this pathway (KRAS, SOS1, RAF1).

Clinical Data The variable phenotype accounts for the difficulty in diagnosis, that remains mainly clinical, therefore many scoring systems have been prepared and the most practical was prepared by van der Burgt. The characteristic pattern of dysmorphic features includes the face, trunk and extremities, congenital heart defects such as pulmonary valvular stenosis and other cardiac abnormalities, cryptorchidism, bleeding diathesis.

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Growth Growth retardation is a consistent feature and most affected patients have a height below the 3rd centile with a FH of 162 cm for males and 153 cm for females.

Growth Hormone Secretion and Therapy GH secretion has been shown to be frequently abnormal, but classical GHD has not been found; however, it has been shown that some patients show alterations of the GH-IGF-1 axis (neurosecretory dysfunction). No correlation was found between GHD and growth response to therapy. In the literature, short-term studies on few patients showed that GH therapy was effective in increasing height velocity. Recently, in a Swedish study [7] on 25 prepubertal children treated with GH, 18 of them reached the FH, very close to the TH, with the gain of 1.7 SD on pretreatment height. In the study of Noordam and Otten [8], 38 patients treated with GH reached FH after a mean of 7.1 years of GH treatment. The positive response in the 1st year of treatment (about 1 SD) was maintained during the following years and even up to FH. Some recent studies have shown that the presence of PTPN11 mutation may influence height velocity and FH. In the study by Binder et al. [9], GH secretion and IGF-1/ IGFBP3 levels were compared in PTPN11-positive with PTPN11-negative patients; the levels of IGF-1 (–2.0 vs. –1.1 SD) and IGFBP3 (–0.9 vs. 0.4 SD) were found to be lower in the PTPN11+ group with GH levels higher during spontaneous secretion at night and on arginine stimulation. The authors suggest that PTPN11 mutations may cause a mild GH resistance by a postreceptor signaling defect, which may contribute

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to growth failure and the relatively poor response to GH in NS. Patients with NS with heart disease may be submitted to GH therapy and only the subjects with hypertrophic cardiomyopathy (HCM) have been excluded. In all NS subjects, before GH therapy and yearly during treatment, echocardiography should be recommended to exclude and monitor the development of HCM [10].

Personal Data In our group of 38 NS patients, studied for PTPN11, 16 children were treated with GH in a 10-year follow-up. 6 patients had the PTPN11 mutation. After the 1st year of GH therapy, there were no significant height differences between PTPN11– and PTPN11+ patients (–2.7 vs. –2.4 SDS ). After 5 years of therapy PTPN11– patients achieved a stature of –2.1 SDS and PTPN11+ remained at –2.3 SDS. After 10 years of therapy PTPN11– achieved a stature of -1.5 SDS and PTPN11+ –2.2 SDS (fig. 2). In conclusion, after 10 years of therapy, PTPN11– had a height gain vs. pretherapy height of +1.7 SD in comparison with +0.1 SD of PTPN11+ patients. Our data seem to confirm the hypothesis of a mild GH resistance in NS subjects caused by the SHP2 mutation.

Prader-Willi Syndrome

Clinical Signs The characteristic signs are short stature, hypotonia, hypogonadism, hyperphagia with progressive obesity, developmental delay (DD), behavior abnormalities, sleep disturbances and dysmorphic features. The clinical diagnosis is made according to scoring systems, recently revised. Other typical signs are short stature, respiratory problems and abnormalities in the autonomous regulation.

Etiology A deletion of a segment of the paternally derived chromosome 15q11-q13 is found in 70–75% of patients, 20–25% have a maternal disomy of the same region, 2–5% have imprinting defect mutations, and about 1% of patients a balanced translocation.

Growth Short stature is frequently observed in Prader-Willi syndrome (PWS). During the 1st year, growth is below the 3rd percentile, but later remains at the 10th percentile

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or below until 10 or 12 years of age. Thereafter, height velocity often declines due to a lack of growth spurt and a mean adult height of 155 cm for males and 148 cm for females have been reported. Specific growth charts for PWS have been prepared.

GH Secretion and Therapy In these patients, a dysregulation of the GH/IGF axis has been reported. In fact, a reduced spontaneous GH secretion to stimulation tests, and low serum IGF-1 and IGF-BP3 levels have been reported [11]. The reduced GH secretion together with the hypogonadotropic hypogonadism (retarded and incomplete sexual development), the hyperphagia and the high pain threshold suggest a hypothalamic-pituitary dysfunction for PWS patients. In 2000, the FDA approved the use of GH in these subjects. In several studies, the efficacy of GH treatment has been demonstrated. The initial positive effects on height velocity appear to be sustained throughout treatment. Moreover, many studies report that in patients treated for many years, growth continues to improve and TH-SDS can be reached. Long-term GH therapy shows a reduction in fat mass, an increase in muscle mass, and improved motor development. This was also documented in control studies in particular on younger patients [12]. The adverse effects reported for PWS patients are similar to those observed in GHD patients. The rapid growth associated with GH therapy may aggravate scoliosis. PWS is a condition with high risk factors for sudden death such as: upper airway obstruction and autonomic impairment of ventilatory control. It has been suggested that GH therapy may increase lymphoid and soft tissue inducing upper apnea events and may compromise pre-existing altered gas exchange with the augmented volume load. Furthermore, during GH therapy, hypopnea and central apnea events decreased in number and duration, as recorded by polysomnography. A recent review by Tauber et al. [13] showed that most deaths in PWS were due to respiratory infections and the causes of deaths were the same in treated and untreated PWS patients. Nevertheless, most cases of sudden death, in patients who received GH, occurred in the first months of treatment. Even though a causal relationship between GH therapy and sudden death has not been demonstrated, an accurate clinical protocol of management and follow-up of PWS patients at the start of therapy is mandatory.

Floating-Harbor Syndrome (Boston Floating Hospital and Harbor General Hospital [Pelletier et al., 1973])

Clinical Signs Floating-Harbor syndrome has as main features: short stature, developmental delay (DD), in particular speech, typical face: triangular with bulbous nose, prominent

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nasal bridge and broad columella, large nares and hypoplastic alae, deep-set eyes, wide mouth with thin lips. Normal occipitofrontal circumference (OFC) for height age.

Etiology Gene unknown.

Growth Small birth-length, during childhood very short stature (–4 to –6 SD), delayed bone age, normal puberty.

GH Secretion and Therapy Some cases of patients with GHD are reported [14, 15] with an increase in height velocity obtained with GH therapy. Nevertheless, there are no conclusive data as to the effect of GH therapy in this condition.

Personal Data We report a patient, 3.5 years of age, with severe short stature (-6.3 SD), typical face, bone age delay, GH deficit, who gained +2 SD with GH therapy over 5 years.

Kabuki Syndrome

Clinical Signs Minimal diagnostic criteria are DD, typical face: long palpebral fissures with eversion of the lateral part of the lower lids, epicanthal folds, short columella with depressed nasal tip, prominent or cupped ears, prominent filtrum, associated congenital anomalies: cleft palate, skeletal abnormalities, CHD (aortic coarctation, VSD, ASD, tetralogy of Fallot), cataract, optic nerve hypoplasia, hearing loss. Immune deficiency, autoimmune disorders.

Etiology Mostly sporadic. Various chromosomal abnormalities have been seen (interstitial duplication of 1p, del 6p, dup 12q).

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Growth At birth generally normal, postnatal short stature (< –2 SD) in 80% of cases.

GH Secretion and Therapy Some cases of GHD or neurosecretory dysfunction are reported [16]. Some cases have been treated with GH therapy with long-term good or moderate response [17]. The efficacy of GH therapy has not been proved in this syndrome. Isolated premature telarche and precocious puberty have been reported.

Personal Data Two patients (male ad female) showed GH deficiency and submitted to therapy with moderate results. The male suspended GH therapy due to autoimmune hemolytic anemia. The female showed early puberty treated with GnRH agonist.

Pallister-Hall Syndrome

Clinical Signs The clinical diagnostic criteria for PHS requires the presence of insertional polydactyly and hypothalamic hamartoma or isolated hamartoma or polydactyly in a relative of the proband. Other signs are bifid epiglottis, imperforate anus and other anomalies. This condition has variable clinical manifestations.

Etiology An autosomal dominant inheritance has been suggested. Isolated cases also occur. PHS is caused by mutations in the GLI3 gene located on chromosome 7 at 7p13. This gene codes a zinger finger domain DNA-binding protein expressed during embryogenesis. The mutations reported for PHS predict truncation of the protein C-terminal of the DNA-binding domain [18].

Growth Data of growth rate and final height are not available.

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GH Secretion and Therapy Different studies report a deficit in GH secretion in PHS patients [19, 20]. In particular, low mean and peak night-time GH and low levels of IGF-1 (neurosecretory dysfunction) have been reported. These findings suggest GH deficiency should be considered one of the causes of short stature in these patients, but the mechanism of GH deficiency associated with PHS is not clear. It has been hypothesized that the hypothalamic hamartoma determines hypothalamic dysfunction associated with low GH levels. Some adult patients presented normal stature and normal growth rate during childhood, so it suggests that in some cases GH decreases after childhood years or that other factors stimulate a normal growth.

Personal Data A female presented hypothalamic hamartoblastoma, postaxial polydactyly, genital anomalies, bifid epiglottis, neurosensorial deafness, and ASD. At 7.9 years she was 110 cm (50th centile; she showed GH deficiency at stimulation tests and low nocturnal GH concentration and still undergoes GH therapy. Her FH was 158.6 cm (25th centile for the GP). She presented normal growth spurt at puberty [18, 19] (fig. 3).

Silver-Russel Syndrome

Clinical Signs The main features are severe intrauterine and postnatal growth retardation, a typical facial appearance, limb asymmetry, fifth finger clinodactily and hemihypertrophy.

Etiology Most cases are sporadic (prevalence 1–30/100,000); genetic mutations have been identified as causes of Silver-Russel syndrome (SRS): chromosome 11p15 (epi)genetic mutations (35% of cases) and maternal uniparental disomy of chromosome 7 (10% of cases).

Growth SRS has been considered as a model of the role of imprinting in growth, human chromosome 11p15 contains a cluster of genes (IGF2 and CDKN1C) important for the

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control of fetal and early childhood growth. Height SDS of children with UPD-7 decreased significantly during early childhood, and children with 11p15 epimutation are shorter and leaner than children with no detected genetic alteration. Mean birth length is –3 SDS, by the age of 4 years height SDS is of –3.5 to –4.4 and height is below and parallel to the 3rd centile and FH is –4.2 SDS (151.2 cm in males and 19.9 cm in females).

GH Secretion and Therapy Some studies reported that SRS patients have an impaired spontaneous secretion with reduced pulse frequency during the night [21]. Until now the contribution of GH/ IGF-1 axis defects on severe growth deficiency in SRS has not been determined and the genetic defects seem to play a more important role [22]. Very few data have been published on long-term therapy in SRS. There is an initial height gain comparable with small for gestational age (SGA).

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Personal Data In our study case, 7 of 9 SRS patients showed GH deficiency (4 partial and 3 severe) and were submitted to GH therapy. At the start of therapy, IGF-1 levels were below 1 SDS. Before puberty all the patients showed a good result in catch-up growth in particular an earlier beginning gave a better result. Nevertheless, the 3 subjects (2 males and 1 female) that reached the FH seldom arrived over the 3rd centile. The height gain obtained with GH was partially wasted at puberty for bone age advancement. The girls treated with GnRH agonist therapy for early puberty increased her height of 1 SD on pre-treatment height.

Skeletal Dysplasia

Skeletal dysplasias are genetic disorders that compromise linear growth and body proportion. It is a large group that includes more than 200 entities clinically distinct and genetically heterogeneous, in fact the genetic defect involves the collagen, matrix proteins, ion transporters and growth factor receptors. Most of the conditions are autosomal dominant; however, some are autosomal recessive. Their clinical diversity often makes these disorders difficult to diagnose and many attempts have been made to give a nosological classification to facilitate diagnosis. The most prevalent group is the achondroplasia group with achondroplasia (ACH) and hypochondroplasia (HCH) where the limbs are more affected than the trunk. We will consider also other skeletal dysplasias such as dyschondrosteosis (Leri-Weill syndrome), spondiloepiphyseal dysplasia (SED), pseudoachondroplasia (PSACH), Ellis-van-Creveld syndrome (EVC). The clinical evidence suggest that GH secretion or action is not responsible for growth deficiency in skeletal dysplasias, but some effects on growth acceleration have been reported. The deformities interfere with the efficacy of GH therapy. In skeletal dysplasias, differently than in GHD, height gain in the 1st year of therapy does not correlate with long-term efficacy. Moreover, GH given at an appropriated time can maintain height velocity within the normal range, even when the natural history of the condition suggests a decrease of height velocity. GH therapy associated to leg lengthening in some cases (ACH and HCH) may provide a better FH.

Personal Data In our series, 22 of 60 patients with skeletal dysplasia showed GH deficiency at pharmacological tests and were submitted to GH therapy: 6 ACH patients, 5 HCH, 7 SED, 3 PSACH, 1 Leri-Weill dyschondrosteosis and 1 EVC patient. Six patients reached the FH (2 ACH patients, 2 PSACH, 1 HCH and 1 EVC). In ACH subjects, H-SDS gain on baseline was

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+1.6SD on ACH growth charts, PSACH patients gained +1.4 SD on PSACH charts. In all the patients sitting height/height ratio showed no modification during therapy.

Achondroplasia

Clinical Signs Severe disproportional short stature with rizomelic shortening of legs and arms, macrocephaly with a prominent forehead, midface hypoplasia.

Etiology It is caused by mutations in the transmembrane region of the fibroblast growth factor receptor 3 (FGFR3). These mutations cause an increased FGFR3 activity that interferes with osteogenesis.

Growth Mean adult height in achondroplasia (ACH) is about 125 cm for women and 132 cm for men (–7SD below the average). Fetal growth is almost normal with birth length about –1.6 SD below the mean. Linear growth is fairly normal for the first postnatal months and later shows a profound decrease with a compromission of body proportions.

Growth Hormone Secretion Although clinical evidence suggests that patients with ACH have normal GH, some studies have reported a hypothalamic-pituitary dysfunction, including abnormal GH secretion with blunted response on different pharmacological tests and low IGF-1 levels.

Growth Hormone Therapy Given the skeletal abnormality, a reduced sensitivity to the action of GH and IGF-1 would be expected. Hence, supraphysiological GH doses have been used. There are several reports on GH therapy in ACH, although most involved few patients with a short-term follow-up. There are no definitive data available in these patients for FH and development in body proportions, in these patients. A number of short-term trials have reported that GH administration significantly increases the rate of growth.

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First year response is typically a 2- to 3-cm increase in growth velocity in prepubertal children (+0.5 SD height gain); GH treatment for up to 5 years can produce a total height gain of about +1 SD. In the subsequent years, a decrease in growth velocity was observed, but still greater than before therapy in most patients [23, 24]. This rapid desensitization to therapy may be due to abnormal functions of chondrocytes such as cell survival. Further studies are therefore essential to establish the duration of administration, dose and timing for the start of therapy. Sitting height SDS improved significantly during therapy, but body proportions did not show any significant change and this is a reassuring finding. In some studies, no bone age acceleration was observed under GH treatment; in contrast, other studies have reported a significant bone age acceleration. It has been found that OGTT was normal both before and after 12 months of GH treatment.

Hypochondroplasia

This is a genetic form of rhizomelic short stature with a wide variation in clinical expression.

Clinical Signs The patients do not have the typical macrocephaly seen in patients with ACH, and the incidence is difficult to determine since many mild forms remain undiagnosed. For the diagnosis of hypochondroplasia (HCH), the invariable radiological finding is the failure of increase in the vertebral interpedicular distance in the lumbar spine from L1 to L5.

Etiology The molecular defect is the same FGFR3 mutation seen in ACH and it was found in about 50% of HCH patients. The most prevalent mutation is the Asn540Lys. FGFR3 mutations activate the signaling of the receptor in the absence of ligand. The severity of the condition depends on the degree of the activation of the signaling pathway. Forms with a mild phenotype are often FGFR3 mutation negative.

Growth Mean adult height in HCH is 133–151 cm for women and 145–164 cm for men. The typical feature of many patients is the lack or the attenuation of growth spurt during puberty.

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Growth Hormone Secretion and Therapy Some authors reported a defect of IGF-1 in HCH patients. There are few studies on GH treatment in HCH patients. The height gain SDS was found to be of 1 DS after 2–3 years of treatment, but in some patients a gain of more than 3 SD has been reported. Some studies have shown that GH used earlier in childhood is not very effective, but others have shown better results if treatment is started at an early age. Moreover, it seems that the results are better when GH is administrated at puberty, when GH is able to regain the missing growth spurt [25]. In fact, the best responses were seen in patients treated when their puberty spurt failed. It is important to observe that pre-treatment height velocity in FGFR3 mutation-positive and mutation-negative patients is similar, but the response to GH treatment over 4 years is greater in the mutation-negative patients; moreover, in patients with the FGFR3 mutation a significant increase in sitting height has been reported which accentuated the disproportion. In many studies, no bone age acceleration was observed. GH treatment seems to be effective in patients with HCH even in severe forms more than in ACH patients. The difference is given by the difference in chondrocytes response to GH. In any case, FH data in these patients are necessary to confirm the effect of GH therapy.

Pseudoachondroplasia

Clinical Signs This is characterized by disproportionate very short stature, deformity of the legs, short fingers, ligamentous laxity, a waddling gait and early onset of osteoarthritis. Head size and facial features are normal.

Etiology A mutation in the cartilage oligomeric matrix protein (COMP) is found in 40% of the cases; the mutation interferes with the normal folding of the protein and causes an accumulation of the protein in the endoplasmic reticulum leading to the death of the cell and disrupts the formation of a normal extracellular matrix. This effect causes a decrease in cells in the bone matrix and a reduction in bone size. It is dominantly inherited.

Growth Final height is around 80–130 cm.

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Growth Hormone Secretion and Therapy Treatment of PSACH with GH has been reported in a small number of patients. In some cases, an empirical approach was used for individual patients. In this condition, the response to treatment was generally poor; in particular, in cases with deformities of the lower extremities GH therapy may worsen the deformities [25].

Ellis-van Creveld Syndrome

Clinical Signs ECV is a skeletal and ectodermal dysplasia characterized by short ribs, limbs, postaxial polydactyly, teeth and nails dysplasia and congenital heart defects (atrial septal defects). Etiology It is a rare autosomal-recessive disease with genetic heterogeneity. Mutations of the ECV1 and ECV2 genes (chr 4p16) have been identified as causative in this syndrome. Growth Short-limb dwarfism present at birth. Mean FH 109–152 cm. The association EVC and GH deficiency has been reported and GH therapy showed a satisfying effect on growth [26, 27]. Personal Data Our EVC patient with severe GH deficiency at pharmacological tests and spontaneous secretion showed a good growth gain with GH therapy during prepubertal age [27], but FH-SDS was just above the pretherapy height.

Dyschondrosteosis or Leri-Weill Syndrome

Clinical Signs Leri-Weill syndrome is a osteochondrodysplasia with mesomelic short stature and Madelung deformity of the wrist. Frequent signs are cubitus valgus, high-arched palate, and scoliosis.

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Etiology This skeletal disorder is caused by mutation or deletion of SHOX gene, a homeobox gene located at the pseudoautosomal region of the X and Y chromosome (SHOX haploinsufficiency or deficiency). Several mutations and deletions have been described. The phenotype of SHOX haploinsufficiency is highly variable ranging from short stature without dysmorphic signs to mesomelic skeletal dysplasia: Leri-Weill syndrome; the rare homozygous form referred to as Langer mesomelic dysplasia. SHOX regulates linear growth and may in part be involved in regulation of IGF-I gene expression or metabolism.

Growth The adult height in patients with Leri-Weill syndrome is variable but in most cases reduced, whereas the height reduction seems to be gender specific with a greater loss of height in females.

Growth Hormone Secretion and Therapy There are different studies on GH treatment in patients with Leri-Weill syndrome [28]. In these patients, an increase in height SDS and height velocity SDS over 24 months of therapy have been reported.

Discussion

Many developmental syndromes have short stature as a characteristic clinical feature. In recent years, the scientific community has shown an increased interest in these conditions. Advances in molecular genetics have contributed to the identification of the genetic etiology of a growing number of dysmorphic syndromes. The larger availability of recombinant GH has expanded the interest towards GH secretion and therapy to a wide variety of these conditions. In genetic syndromes growth defect is generally related to a deficit in cellular growth, caused by the underlying genetic defect and few of them are characterized by a severe disorder in GH secretion as a symptom of the disease. Nevertheless, in dysmorphic syndromes, some unknown abnormality in GH/ IGF-1 action may be hypothesized that contributes to the growth abnormality. For this reason, it is important to assess physiological growth hormone secretion, IGF-1 and IGFBP3 levels and therefore the detection of more subtle defects in GH secretion.

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Genetic syndromes, in particular when growth defect is prenatal, represent a natural model for further investigations in some unknown pathway of GH mechanism of action. On the other hand, also in physiological states the mechanisms that determine the achievement of an appropriate stature remain largely unknown. In dysmorphic syndromes with growth defect, the finding of a dysfunction in the GH/IGF-1 axis would permit a therapeutic attempt to obtain a higher FH than the expected height according to the natural history of the condition. The recent molecular studies on the etiology of Noonan syndrome have highlighted the link between the genetic defect of NS and the mechanisms of action of GH (JAK-STAT pathway). In NS, a partial GH insensitivity is due to the molecular defect found in more than 65% of the patients with this condition [9]. In the syndromes like PHS and PWS, where a defect in GH/IGF-1 axis is part of the syndrome, the benefit of GH-therapy is demonstrated on short-term-growth and also on FH [10]. In PWS the effect of GH extends to the general well-being: improvement of body composition, energy, muscle mass, cognitive functioning, decrease in hypotonia [12, 19, 20]. SRS represents a syndrome of IUGR associated with various dysmorphic features; in the mild forms the diagnosis may not be easy. In this condition, a defect in GH secretion, in particular in the nocturnal physiological profiles, are very common. GH therapy produces an increase in growth rate and does not worsen body asymmetry. Some authors suggest [21], although in a few patients, that this increase continues also when therapy had been stopped with an increase in FH as well. Differently from other authors, we found that height gain obtained with GH was partially wasted at puberty for bone age advancement, partially controlled by GnRH agonists therapy. Skeletal dysplasia is a large category of disorders that involve long bone and spine to a different extent, leading to disproportionate shortening. The molecular basis has been only recently identified in many of them and GH deficiency is not generally involved in the growth defect. Nevertheless, the experience of the beneficial effect of GH therapy in other conditions such as TS [5, 6, our data], without GH insufficiency, has led to clinical trials with supraphysiological doses. Some studies have reported an increase in growth velocity without conclusive data on FH [25]. This category of disorders includes so many conditions with different molecular defects, the number of treated patients is too little and FH data are indispensable for each group of skeletal dysplasia to obtain more conclusive results. Our experience of GH therapy in skeletal dysplasia is limited to the subjects that showed a GHD and could be treated according to the Italian healthcare laws. We found in ACH subjects who reached FH, a H-SDS gain on baseline of +1.6 SD on ACH growth charts and PSACH patients +1.4 SD on PSACH charts. In all the patients sitting height/height ratio showed no modification during therapy.

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Recognized Benefits of Growth Hormone Therapy

Therapeutic correction of partial GH deficiency and action in particular in some conditions such as PWS, SRS and NS. Improvement in growth velocity in prepuberty to reduce the difference with the age-matched children during schoolage. Gain of only 8 or 10 cm in height is of benefit to a child if it takes him into the bottom of the normal range and if his FH is moved from 120 to 130 cm. Benefit on the general well-being of the patients, e.g. in particular, modification of body composition, increase of energy, muscle mass, cognitive functioning, decrease in hypotonia.

Unsolved Problems

Systematic and accurate studies of the GH/IGF-1 axis are not available in most dysmorphic syndromes to detect subtle defects in GH secretion or a neuro-secretory dysfunction. The efficacious dosage of GH therapy has not been established for many conditions. Large databases, NCGS and KIGS, collected a high number of patients with dysmorphic syndromes, but FH data are available only for some less-rare conditions such as TS and PWS. Most of the trials are not randomized and in consideration of the small number of patients for each condition there are no defined homogeneous groups. Usual prediction methods are flawed in their application to children with syndromes: bone age estimation is often difficult and frequently pubertal growth spurt is blunted.

Complications and Warning

For some children with MR, GH therapy reduces the potential benefit even though there is an increase in FH. There is a cost for health care, for the family and child (daily injections, repeated visits to the hospital). In many rare diseases, the safety of GH therapy at supraphysiological doses has not been proven; in fact, cancer risk linked to the genetic defect and the natural history of many syndromes is not well known. Thus, care is needed with monitoring of IGF-1 and IGFBP-3.

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Conclusions

• Genetic syndromes may represent a natural model for further investigations in some unknown pathways of the GH mechanism of action. • A systematic and accurate study of the GH/IGF-1 axis may be important to detect subtle defects in GH secretion or a neurosecretory dysfunction. Some unknown abnormality in GH/IGF-1 action may be hypothesized contributing to the growth abnormality in dysmorphic syndromes. • Study of the GH/IGF-1 axis could help the definition of diagnostic criteria for more subtle forms of GHD that remain a dilemma also in nondysmorphic subjects. • To avoid bias in this difficult field, the use of specific growth charts to evaluate the effect of the therapy is advisable. • Long-term international multicenter studies with FH data will be mandatory to collect statistically significant groups of subjects for each syndromic condition. • In the future, the interest towards genetic syndromes and the skill in detecting very mild dysmorphic signs will permit a correct diagnosis in the large and sometimes not-well-defined group of idiopathic short stature.

References 1

2

3

4

Mazzanti L, Nizzoli G, Tassinari D, Bergamaschi R, Magnani C, Chiumello G, Cacciari E: Spontaneous growth and pubertal development in Turner’s syndrome with different karyotypes. Acta Paediatr 1994;83:299–304. Pirazzoli P, Mazzanti L, Bergamaschi R, Perri A, Scarano E, Nanni S, Zucchini S, Gualandi S, Cicognani A, Cacciari E: Reduced spontaneous growth hormone secretion in patients with Turner’s syndrome. Acta Paediatr 1999;88:610–613. Wasniewska M, De Luca F, Bergamaschi R, Guarneri MP, Mazzanti L, Matarazzo P, Petri A, Crisafulli G, Salzano G, Lombardo F: Early treatment with GH alone in Turner syndrome: prepubertal catch-up growth and waning effect. Eur J Endocrinol 2004; 151:567–572. Davenport ML, Crowe BJ, Travers SH, Rubin K, Ross JL, Fechner PY, Gunther DF, Liu C, Geffner ME, Thrailkill K, Huseman C, Zagar AJ, Quigley CA: Growth hormone treatment of early growth failure in toddlers with Turner syndrome: a randomized, controlled, multicentric trial. J Clin Endocrinol Metab 2007;92:3406–3416.

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van Parenen YK, de Muinck Keizer-Schrama SMPF, Stijnen T, Sas TCJ, Jansen M, Otten BJ, HoorwedNijman JJG, Vulsma T, Stokvis-Brantsma WH, Rouwe CW, Reeser HM, Gerver WJ, Gosen JJ, Rongen-Westerlaken C, Drop SLS: Final height in girl with Turner syndrome after long-term growth hormone treatment in three dosages and low estrogens. J Clin Endocrinol Metab 2003;88:1119–1125. The Canadian Growth Hormone Advisory Committee: Impact of growth hormone supplementation on adult height in Turner syndrome: results of the Canadian Randomized Controlled Trial. J Clin Endocrinol Metab 2005;90:3360–3366. Osio D, Dahlgren J, Wikland KA, Westphal O: Improved final height with long-term growth hormone treatment in Noonan syndrome. Acta Paediatr 2005;94:1232–1237. Otten J, Noordam K: Short Stature in Noonan Syndrome: Results of Growth Hormone Treatments. Growth Hormone in Pediatrics – 20 years of KIGS. Basel, Karger, 2007, pp 347–355. Binder G, Neuer K, Ranke MB, Wittekindt NE: PTPN11 mutations are associated with mild growth Hormone resistance in individuals with Noonan Syndrome. J Clin Endocrinol Metab 2005;90:5377– 5381.

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10 Brown DC, Macfarlane CE, McKenna WJ, Patton MA, Dunger DB, Savage MO, Kelnar CJ: Growth hormone therapy in Noonan’s syndrome: non-cardiomyopathic congenital heart disease does not adversely affect growth improvement. J Pediatr Endocrinol Metab 2002;15:851–852. 11 Lee PDK, Allen DB, Angulo MA, Cappa M, Carrel AL, Castro-Magana M, Chiumello G: Consensus statement – Prader-Willi syndrome: growth hormone (GH)/Insulin-like growth factor axis deficiency and GH treatment. Endocrinologist 2000;10: 71S–73S. 12 Grugni G, Crinò A, Bosio L, et al: Genetic Obesity Study Group of Italian Society of Pediatric Endocrinology and Diabetology (ISPED). The Italian National Survey for Prader-Willi syndrome: an epidemiologic study. Am J Med Genet [A] 2008; 146:861–872. 13 Tauber M, Diene G, Molinas C, Hebert M: Review of 54 cases of death in children with Prader Willi syndrome. Am J Med Genet 2008;146A:881–887. 14 Wieczorek D, Wusthof A, Harms E, Meinecke P: Floating-harbor syndrome in two unrelated girls: mild short stature in one patient and effective growth hormone therapy in the other. Am J Med Genet 2001;104:47–52. 15 Cannavo S, Bartolone L, Lapa D, Venturino M, Almoto B, Violi A, Trimarchi F: Abnormalities of GH secretion in a young girl with Floating-Harbor syndrome. Ital Endocrinol Invest 2002;25:58–64. 16 Gabrielli O, Bruni S, Bruschi B, Carloni I, Coppa GV: Kabuki syndrome and growth hormone deficiency: description of a case treated by long-term hormone replacement. Clin Dysmorphol 2002;11: 71–72. 17 Tawa R, Kaino Y, Ito T, Goto Y, Kida K, Matsuda H: A case of Kabuki make-up syndrome with central diabetes insipidus and growth hormone neurosecretory dysfunction. Acta Paediatr Jpn 1994;36:412– 415. 18 Johnston JJ, Olivos-Glander I, Killoran C, et al: Molecular and clinical analyses of Greig cephalopolysyndactyly and Pallister-Hall syndromes: robust phenotype prediction from the type and position of GLI3 mutation. Am J Hum Genet 2005;76:609–622. 19 Zucchini S, Mazzanti L, Ambrosetto P, Salardi S, Cacciari E: Usual magnetic resonance imaging findings of the sella region in subjects with hypopituitarism: report of 4 cases. J Pediatr Endocrinol Metab 1998;11:35–44.

20 Galasso C, Scirè G, Fabbri F, Spadoni GL, Killoran CE, Biesecker LG, Boscherini B: Long-term treatment with growth hormone improves final height in a patient with Pallister-Hall syndrome. Am J Med Geent 2001;99:128–131. 21 Stanhope R, Albanese A, Azcona C: Growth hormone treatment of Russell-Silver syndrome. Horm Res 1998;49:37–40. 22 Binder G, Seidel AK, Martin D, Schweizer R, Schwarze C, Wollmann H, Heggerman T, Ranke M: The endocrine phenotype in Silver-Russell syndrome is defined by the underlying epigenetic alteration? J Clin Endocrinol Metab 2008;93:1402–1407. 23 Hertel NT, Eklöf O, Ivarsson S, Aronson S, Westphal O, Sipilä I, Kaitila I, Bland J, Veimo D, Müller J, Mohnike K, Neumeyer L, Ritzen M, Hagenäs L: Growth hormone treatment in 35 prepubertal children with achondroplasia: a five-year dose-response trial. Acta Paediatr 2005;94:1402–1410. 24 Tanaka H, Kubo T, Yamate T, Ono T, Kanzaki S, Seino Y: Effect of growth hormone therapy in children with achondroplasia: growth pattern, hypothalamic-pituitary function, and genotype. Eur J Endocrinol 1998;138:275–280. 25 Hertel T: Growth hormone treatment in skeletal dysplasia: the KIGS experience; in Ranke MB, Price DA, Reiter EO (eds): Growth Hormone Therapy in Pediatrics – 20 Years of KIGS. Basel, Karger, 2007, pp 356–368. 26 Pirazzoli P, Mazzanti L, Mandini M, Cau M, Ravagli L, Cacciari E: GH-deficiency in Ellis-vanCreveld Sindrome: Response to Replacement Therapy; in Bierich JR, Cacciari E, Raiti S (eds): Growth Abnormalities. Serono Symposia. New York, Raven Press, 1989, vol 56, pp 391–394. 27 Versteegh FG, Buma SA, Costin G, de Jong WC, Hennekam RC: Growth hormone analysis and treatment in Ellis-van Creveld syndrome, EvC Working Party. Am J Med Genet [A] 2007;143:2113–2121. 28 Blum WF, Crowe BJ, Quigley CA, Jung H, Cao D, Ross JL, Braun L, Rappold G: SHOX Study Group. Growth hormone is effective in treatment of short stature associated with short stature homeoboxcontaining gene deficiency: two-year results of a randomized, controlled, multicenter trial. J Clin Endocrinol Metab 2007;92:219–228.

Prof. Laura Mazzanti Department of Pediatrics, Rare Disease, Syndromology and Auxology Unit S. Orsola-Malpighi Hospital, University of Bologna Via Massarenti, 11, IT–40138 Bologna (Italy) Tel. +39 051 636 3723, Fax +39 051 636 3722, E-Mail [email protected]

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Growth Hormone-Resistant Syndromes: Long-Term Follow-Up Steven D. Chernausek Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, Okla., USA

Abstract Recombinant human IGF-I (rhIGF-I) has been approved as a therapeutic agent for short stature due to primary IGF-I deficiency following clinical trials that lasted more than 10 years. The first children treated with rhIGF-I were those with short stature resulting from defects in growth hormone (GH) action, either due to GH receptor abnormalities or the development of GH-neutralizing antibodies that arose following GH exposure. The administration of rhIGF-I to such children results in improvements in growth rate that are sustained over many years. This appears to improve adult height, but so far the effects are not as robust as when GH-deficient children are treated with human GH. Side effects are common but manageable and seldom necessitate discontinuation of therapy. Copyright © 2009 S. Karger AG, Basel

Children with growth hormone (GH) insensitivity were among the first to be treated with recombinant human IGF-I (rhIGF-I). The results of therapeutic trials in shortterm and long-term resulted in approval of rhIGF-I (Increlex®) for treatment of ‘primary IGF-I deficiency’ initially in the United States and later in Europe and other regions. Severe primary IGF-I deficiency is defined as a circulating concentration of IGF-I more than 3 SD below the mean for age and sex and height below –3 SD for age and sex in the face of normal nutrition and secretion of GH. It is also approved for treatment of short children with GH deficiency who develop high titers of GH- neutralizing antibodies following exposure to GH. IGF-I is established as an effective treatment for short stature due to GH insensitivity. It seemed logical to have some of the first trials in children be in those individuals with GH receptor deficiency or severe GH insensitivity. This was because there was no effective therapy for them and because, if the somatomedin hypothesis were correct, replacing IGF-I should effectively restore growth. However, it is useful to consider the questions faced by investigators at the onset of clinical studies. Would IGF-I, in fact, be anabolic and growth promoting over the long term? (If the direct

GH action was needed, rhIGF-I might be ineffectual in complete GH insensitivity.) What sort of dose and regimen would be best? Would the insulin-like effects of IGF-I prove problematic and limit its use? What unanticipated adverse effects would occur? Many of these questions have been answered by studies of several cohorts of children with GH insensitivity who received rhIGF-I therapy for various periods. Much of the data come from the largest and longest treated cohort of patients studied by the GH Insensitivity Syndrome (GHIS) Collaborative Group and it is that which is discussed here. Several other studies have been conducted with concordant results and these are cited as well.

Treatment Rationale

Growth hormone (GH) resistance is seen in its most robust form in complete GH receptor deficiency or Laron syndrome [1] These individuals are physiologically ‘blind’ to GH and suffer from extreme short stature, repetitive bouts of hypoglycemia, abnormalities of muscle and fat composition, and other metabolic disturbances. This occurs because production of IGF-I is substantially reduced and because direct actions of GH are not present. Individuals with GH insensitivity (defined as complete lack of GH effects as seen in homozygous null GH receptor states) are severely affected and potentially would benefit from therapies to overcome the GH resistance. Though GH insensitivity is rare, several studies show that there are intermediate forms of GH resistance. Thus, in a broad sense, methods to overcome GH resistance are needed.

Treatment Strategies

The most logical and time-tested therapeutic approach would be to restore normal concentrations of missing elements with appropriate distribution within the body. Individuals with GH insensitivity/resistance lack the direct actions of GH upon the skeleton and other tissues and the GH inducible factors IGF-I, IGF binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS). For subjects in whom the GH receptor itself is missing or functionally abnormal, the most logical step would be to bypass the GH receptor, but activate the immediate downstream intracellular components (the jak-stat pathway). In theory this would be curative, but has the problem of potential lack of specificity since jak-stat pathways are used by other stimuli and, to date, there are no pharmaceuticals that might yield this effect. Because human GH (hGH) has been used for a long time and has shown a very good safety profile, supraphysiologic doses of hGH might be considered in certain forms of incomplete or partial GH resistance. In fact, the somewhat high doses of GH used to treat Turner syndrome, short children born SGA, and some cases of idiopathic short stature may reflect a degree of underlying GH resistance in these children. However, rhGH will not work in the

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severe forms of GH insensitivity and it is not necessarily true that extremely supraphysiologic doses of rhGH is the best approach in these individuals. Administration of IGF-I either alone or in combination with other circulating factors is also a logical approach to GH resistance because IGF-I is the principal mediator of the growth-promoting actions of GH and, theoretically, should promote growth in the absence of GH. IGF circulates in the blood as a ternary complex with IGFBP-3 and ALS. Since all are lowered in GH insensitivity, one might consider giving the entire complex back as the optimal therapy. Though there have been no attempts to use ternary complex treatment in humans, clinical trials have been conducted with an IGF-I /IGFBP-3 complex [2]. The potential advantages are a relatively prolonged half-life since ‘free’ IGF is more rapidly cleared from the circulation. Other advantages are the potential direct effects of IGFBP-3 and the possibility that residual ALS could complex with the IGFBP-3 and IGF-I, yielding the normal circulating form. However, because IGFBP-3 is a relatively large molecule, a substantial amount of protein needs to be given to deliver the quantity of IGF desired and the initial preparations required somewhat complex steps for the patient. Moreover, it is unclear whether the complex is absorbed intact (as complex) or whether IGF-I and IGFBP-3 separate and are absorbed at different rates following subcutaneous administration. Clinical trials using IGF-I/IGFBP-3 complex resulted in approval for use in severe primary IGF deficiency in the US, like rhIGF-I. However, litigious events resulted in its withdrawal from the US market. This leaves rhIGF-I as the only currently available therapy for GH resistance. The advantages of using rhIGF-I are that it is an active ligand for the type I IGF receptor (the receptor that mediates the growth promoting and metabolic actions of IGFI). Administration is simple with relatively rapid and complete absorption following subcutaneous injection. However, in the absence of IGFBP-3, the serum half-life of IGF-I is relatively short. Thus, twice daily injections are required in complete GH insensitivity.

Clinical Studies in Children with GH Insensitivity

Linear Growth Most patients with GH insensitivity have basal growth rates in the 2- to 3-cm/year range depending on their age. When rhIGF-I is given, growth promptly increases (see table) with the best growth occurring during the first year. In the large collaborative study when subjects were given at least 100 μg/kg b.i.d. for 2 years (n = 19) the growth velocity in the first year was 8.7 cm/year and in the second 6.1 cm/year [3]. This is equivalent to that reported in other studies [4–6]. Because the first-year growth rate is approximately 2 cm/year above the mean for age, there is some catch up in height. Thereafter, growth rates are typically normal for age, resulting in maintenance of height percentiles, but not much additional catch up growth.

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Table 1. Summary of published rhIGF-I clinical trials Trial

Ranke et al. [6] Klinger and Laron [5] Guevara-Aguirre et al. [4] Chernausek et al. [3]

Subjects

17 9 7 15 76

Height at start (SDS)

–6.6 –5.6 –8.0 –8.5 –6.5

rhIGF-I dose μg/kg

40–120 b.i.d. 175–200 daily 80 b.i.d. 120 b.i.d. 80–120 b.i.d.

Growth velocity cm/year baseline year 1

year 2

3.9 4.7 3.0 3.4 2.7

6.4 6.0 5.6 6.4 5.8

8.8 8.2 9.1 8.8 8.0

The effect of rhIGF-I therapy on final height is a natural question, but difficult to judge since no randomized controlled trial to final height has been conducted. Typical heights for adults with GH insensitivity syndrome are very low, and thus any meaningful increase, would be of benefit. One can estimate height gained in the small number of patients reported thus far by comparing their final stature with heights expected based on the growth charts developed by Laron et al. [7]. Final height data on 6 individuals were reported by the GHIS collaborative group, 3 of whom had received GnRH agonist treatment as well [3]. The duration of treatment was between 5.5 and 10 years. Using this relatively crude estimate, 5 of the 6 individuals gained more than 10 cm in height. The greatest apparent gain was seen in a male, who received therapy for nearly 8 years and had an estimated improvement in height of 23 cm without GnRH agonist treatment. Despite these encouraging results, most of these individuals did not achieve an adult height within the normal range. The reasons for this are multifactorial. First of all, nearly all the patients were far behind in height at the initiation of therapy. Indeed, all but one was below –4 SDs for height at the start. Secondly, patients with GH insensitivity syndrome, when given IGF appear not to grow as fast as severely GH deficient patients given GH [8]. The reasons for this are not completely clear, but may be attributed to relatively short half-life in the absence of IGFBP-3, restricted access of IGF-I to the growth plate, and the lack of GH direct effect.

Organ Growth Data from transgenic animals overexpressing IGF-I suggest that the kidney and spleen are particularly responsive to IGF-I. In patients with GH insensitivity, kidneys are typically small for age, even when accounting for height [3]. When rhIGF-I treatment is given, kidney length increases usually into the normal range. In a small number of patients, kidney size exceeds the norm but without any apparent adverse effect on

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250

Pretreatment On treatment

225 200

Glucose (mg/dL)

175 150 125 100 75 50 25 0 ⫺5 ⫺4 ⫺3 ⫺2 ⫺1

0

1

2

3

4

5

6

7

8

9

Day from rhIGF-1 initiation

Fig. 1. Blood glucose measures at treatment initiation, taken before lunch. Open circles represent before represent measures before treatment, and closed circles during rhIGF-I therapy. Dosing started at 40–60 μg/kg/dose b.i.d. and advanced to 120 μg/kg/dose. From Chernausek et al. [3], with permission.

kidney function. Similarly, spleen length is small for age, even when accounting for height, and usually normalizes in response to rhIGF-I.

Metabolic Effects There has been substantial concern that the hypoglycemia that occurs in patients with GH resistance might be aggravated with rhIGF-I treatment due to the insulinlike effects. Indeed, when rhIGF-I is given subcutaneously, circulating concentrations increase rapidly over 2 h and circulating blood glucose concentrations will usually fall unless carbohydrate is provided. Therefore, blood glucose concentrations were monitored frequently during the first clinical studies. This included in-hospital monitoring as well as home blood glucose measures. Figure 1 shows blood glucose measures from several patients before and during rhIGF-I initiation. Both hyper- and hypoglycemic episodes are evident prior to during rhIGF-I therapy. These results reflect the known underlying abnormalities of carbohydrate homeostasis seen in GH insensitivity [9,

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Table 2. Adverse events associated with rhIGF-I therapy Adverse event

Approximate frequency Management %

Comment

Hypoglycemia

50

administer rhIGF-I 30 min before meal

most hypoglycemia is due to underlying GH insensitivity; rhIGF-I induced hypoglycemia nearly always avoided when adequate carbohydrate intake follows injection

Lymphoid tissue growth resulting in hypoacusis, snoring, sleep apnea

20–25

tonsillectomyadenoidectomy; tympanostomy tube placement

may be more common with long-term exposure; hypoplasia of midface structures in GHIS may contribute

Hypertrophy at injection site

30

rotation of injection sites

Intracranial hypertension

4

temporarily discontinue rhIGF-I, restart at lower dose when ICH resolves

Coarsening of facial features

sporadic

improves with most often seen at end cessation of therapy at of therapy in final height conjunction with pubertal changes

also seen with hGH treatment.

Taken from Chernausek et al. [3].

10]. Though there have been clearly hypoglycemic events resulting from the administration of IGF-I, rhIGF-I injections, when given in conjunction with adequate meals, have little impact on glucose excursions. Plasma cholesterol is in the normal range for the majority and appeared to increase modestly over time. Similarly, there was a modest increase in triglycerides.

Effect on Body Composition Patients with GH insensitivity may have increased body fat compared to others and IGF-I may stimulate adipogenesis. Therefore, there has been some concern that rhIGFI may increase body fat. Studies by the collaborative group revealed modest changes in

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BMI over the course of treatment. Percent of body fat estimated by DEXA in 22 subjects showed body fat at 22% at baseline, a modest increase at 2 years, and then a return to the previous levels. Other studies have shown higher levels of body fat [11].

Adverse Events (table 2) The GHIS collaborative study found hypoglycemia to be the most common adverse event. However, this was usually related to the underlying GH insensitivity and not to the administration of rhIGF-I. Certain other adverse events appear due to growth of lymphoid tissue stimulated by IGF-I. These result from tonsillar and adenoidal hypertrophy, which led to hearing deficits in some and tympanostomy tube placement in 22% of the subjects. One third of the patients had lipohypertrophy at injection sites, but this was largely avoided with attention to site rotation. Intracranial hypertension, a known complication of rhGH therapy, also occurred with rhIGF-I therapy. The occurrence of this in three of the 76 subjects reported by the collaborative group suggests the incidence may be higher than with rhGH but such conclusions are limited by the small numbers. Another concern has been putative coarsening of facial features with rhIGF-I [12]. These changes were initially thought be similar to those seen in acromegaly, but subsequent studies involving measures of cephalometry indicate that this is not the case, but rather, result from soft tissue changes and increased soft tissue growth of the nose, lips, and other aspects of the face. This occurs somewhat sporadically and is difficult to quantify. There is improvement in this following withdrawal of rhIGF-I. Since patients with severe GH insensitivity have unusual facies, it is difficult to know to what to degree, if any, rhIGF-I altered there adult facial appearance.

Conclusion and Future Directions

Important areas for future research include efforts to optimization of therapy via dose changes, alternative regimens or different forms rhIGF-I, testing of rhIGF-I in less severe forms of GH resistance, and using GH and rhIGF-I in combination.

References 1

2

Rosenfeld RG, Rosenbloom AL, Guevara-Aguirre J: Growth hormone (GH) insensitivity due to primary GH receptor deficiency. Endocr Rev 1994;15:369–390. Camacho-Hubner C, Underwood LE, Yordam N, Yuksel B, Smith AV, Attie KM, Savage MO: Once daily rh IGF-1/rhIGFBP-3 treatment improves growth in children with severe primary IGF-I deficiency: results of a multicenter clinical trial. Proc Endocrine Soc 88th Ann Meet, Boston, 2006, abstr 40–1, p 132.

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Chernausek SD, Backeljauw PF, Frane J, Kuntze J, Underwood LE: Long-term treatment with recombinant insulin-like growth factor (IGF)-I in children with severe IGF-I deficiency due to growth hormone insensitivity. J Clin Endocrinol Metab 2007; 92:902–910.

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Guevara-Aguirre J, Rosenbloom AL, Vasconez O, Martinez V, Gargosky SE, Allen L, Rosenfeld RG: Two-year treatment of growth hormone (GH) receptor deficiency with recombinant insulin-like growth factor I in 22 children: comparison of two dosage levels and to GH-treated GH deficiency. J Clin Endocrinol Metab 1997;82:629–633. Klinger B, Laron Z: Three year IGF-I treatment of children with Laron syndrome. J Pediatr Endocrinol Metab 1995;8:149–158. Ranke MB, Savage MO, Chatelain PG, Preece MA, Rosenfeld RG, Wilton P: Long-term treatment of growth hormone insensitivity syndrome with IGFI: results of the european multicentre study. The working group on growth hormone insensitivity syndromes. Horm Res 1999;51:128–134. Laron Z, Lilos P, Klinger B: Growth curves for Laron syndrome. Arch Dis Child 1993;68:768–770. Blethen SL, Compton P, Lippe BM, Rosenfeld RG, August, GP, Johanson A: Factors predicting the response to growth hormone (GH) therapy in prepubertal children with gh deficiency. J Clin Endocrinol Metab 1993;76:574–579.

9 Brain CE, Hubbard M, Preece MA, Savage MO, Aynsley-Green A: Metabolic status of children with growth hormone insensitivity syndrome and responses to treatment with iIGF-I. Horm Res 1998;50:61–70. 10 Laron Z, Avitzur Y, Klinger B: Carbohydrate metabolism in primary growth hormone resistance (Laron syndrome) before and during insulin-like growth factor-I treatment. Metabolism 1995;44:113–118. 11 Laron Z, Ginsberg S, Lilos P, Arbiv M, Vaisman N: Body composition in untreated adult patients with Laron syndrome (primary GH insensitivity). Clin Endocrinol (Oxf) 2006;65:114–117. 12 Backeljauw PF, Underwood LE: Therapy for 6.5–7.5 years with recombinant insulin-like growth factor I in children with growth hormone insensitivity syndrome: a clinical research center study. J Clin Endocrinol Metab 2001;86:1504–1510.

Steven D. Chernausek Department of Pediatrics University of Oklahoma Health Sciences Center 1122 NE. 13th Street, Suite 1400, Oklahoma City, OK 73117 (USA) Tel. +1 405 271 2767, Fax +1 405 271 3439, E-Mail [email protected]

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Phenotypic Aspects of Growth Hormoneand IGF-I-Resistant Syndromes Martin O. Savagea ⭈ Alessia Davida ⭈ Cecilia Camacho-Hübnera ⭈ Louise A. Metherella ⭈ Adrian J.L. Clarka Department of Endocrinology, William Harvey Research Institute, Barts and the London, School of Medicine and Dentistry, Queen Mary’s, University of London, London, UK, and Karolinska Institute, Stockholm, Sweden

Abstract Major advances in the diagnosis and characterisation of growth hormone (GH) and IGF-I resistant disorders have occurred during the past 15 years. With these advances has come the realisation that there is broad phenotypic variation within these diagnostic categories. We discuss the current status of endocrine and molecular evaluation, focussing on the phenotypic characteristics of genetic Copyright © 2009 S. Karger AG, Basel defects in the GH-IGF-I axis.

Introduction

Growth hormone (GH) resistance states may exist in a range of clinical situations, both congenital and acquired. This chapter will be limited to disorders caused by genetic defects in the GH-IGF-I axis. GH-resistant syndromes have been recognised for over 40 years [1, 2]; however, the emergence of impaired IGF-I production and action is more recent [3, 4]. This chapter describes the different genetic defects in the GH-IGF-I axis and assesses their effects on phenotypic features and endocrine mechanisms. These disorders will be discussed principally in the paediatric context because the main presentation of affected children concerns reduced linear growth which may be pre-natal or post-natal or both.

Classification of Growth Hormone and IGF-I Resistance

The genetic disorders with resistance to the actions of GH or IGF-I are shown in table 1.

Table 1. Aetiological classification of GH insensitivity states Primary (genetic) defects of the GH-IGF-I axis 1 GH receptor defects a Extracellular mutations b Transmembrane mutations c Intracellular mutations 2 GH signal transduction defects (STAT 5b mutations) 3 IGF-binding protein defect 4 Acid-labile subunit defect 5 Bioinactive GH 6 Bioinactive IGF-I 7 Primary defects of IGF-I production or action a IGF-I gene mutations b IGF-I receptor mutations Secondary (acquired) dysfunction of the GH-IGF-I axis 1 Malnutrition, parenchymal liver disease, type 1 diabetes 2 Catabolic states (e.g. intensive care, post-operative) 3 Chronic inflammatory and nutritional disorders (e.g. juvenile chronic arthritis, Crohn’s disease) 4 GH neutralizing antibodies in patients with GH gene deletion

Classical Growth Hormone Resistance GH resistance resulting from a homozygous or compound heterozygous mutations in the GH receptor (GHR) is usually associated with the most severe clinical phenotype, known as Laron syndrome, after Zvi Laron, who first described the condition in 1966 [1, 2]. This disorder was also discovered in two rural communities in Ecuador, carrying the same GHR mutation [2]. Laron syndrome is seen principally in populations with a high rate of consanguinity and is prevalent in the Middle East [2] and the Indian sub-continent. A more heterogeneous series of patients, mainly resident in European countries was reported [5] and brought to light the heterogeneous nature of the paediatric GH-resistant phenotype. The phenotype of Laron syndrome resembles severe congenital GH deficiency, but with high levels of circulating GH [1]. The natural history of this disorder is characterised by very abnormal post-natal growth, resulting in extreme short stature in adult life with height ranging from –5 to –12 SD [3]. Fetal growth is relatively unaffected, although abnormal craniofacial development may be present at birth. Classical patients have a characteristic facies with mid-facial hypoplasia, which is not specific for Laron syndrome, also being seen in genetic GH deficiency and results from severe IGF-I deficiency. Other findings include poor musculature, delayed motor development, prominent forehead, laryngeal hypoplasia, hip dyplasia, osteopenia, thin skin, sparse and thin hair, and microphallus [1, 2]. In patients

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with Laron syndrome, relative obesity is present, with a decreased ratio of lean body mass to fat mass. Puberty is delayed, but normal reproductive function has been reported. The endocrine system, specifically the GH-IGF-I axis, is very disturbed. Serum IGF-I, IGFBP-3 and ALS are severely decreased and GH secretion is elevated, both basally and after stimulation. There are severe deficiencies in the GH-dependent proteins IGF-I, IGFBP-3, and acid-labile subunit (ALS) [2] serum GHBP is usually undetectable, associated with mutations in the extracellular domain of the GHR [5, 6]. However in 25% of cases, GHBP is normal [5] or even elevated [6].

Growth Hormone Receptor Mutations without Laron Syndrome The study of a large population of patients with primary GH resistance has demonstrated that some patients have a less severe phenotype [5]. In fact, there is a variation of phenotype, with heights ranging from –5 to –12 SD, even in the population of severely affected subjects from Ecuador who share the common E180 sp mutation [2]. It is clear however that some homozygous GHR mutations are associated with residual receptor function leading to generation of IGF-I and a milder phenotype. The phenotypic spectrum was demonstrated in a study which assessed facial appearance in subjects with unequivocal GH resistance. A range from typical Laron syndrome to normal facies was seen [7]. Within the same family, a wide variation of phenotype was seen between two siblings, who both had homozygous intracellular mutations in exon 10 of the GHR [8]. but whose adult heights were –8.3 and –5.6 SD, respectively.

Pseudo-Exon Growth Hormone Receptor Mutation A further GHR mutation which can be associated with a highly variable phenotype is an intronic pseudo-exon mutation, first reported by Metherell et al. [9] in 4 members of a consanguineous Pakistani family. This homozygous mutation causes the insertion of a pseudoexon between exons 6 and 7. The 108-bp insertion caused the addition, in-frame, of 36 amino acids between codons 206 and 207. We predicted that this would affect dimerization of the receptor; however, crystal structure modelling of this mutant GHR showed no alteration of the dimerization domain and cell studies resulting in a defect in trafficking [Ross, pers. commun.]. The 4 patients had unequivocal GH resistance with deficiencies of IGF-I, IGFBP-3 and ALS, but of a lesser degree than is usually seen in Laron syndrome. GH secretion was also elevated [10]. Height SD values ranged from –4.0 to –5.6. A larger number of patients have recently been published emphasizing the phenotypic variation [11].

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SH2 domain

DNA-binding domain 141

323

486

pY699

589

787

STAT 5b protein

A630P: Kofoed E 2003 1680delG: Hwa V 2007 N398fsX413: Hwa V 2005 Q368fsX376: Vidarsottir S 2006 R152X: Bernasconi A 2006

Fig. 1. Molecular defects of the STAT5b gene.

Dominant Negative Heterozygous Growth Hormone Receptor Mutation In 1997, Ayling et al. [12] provided a new insight into the genetics of GH resistance, describing the first heterozygous mutation with a dominant negative effect. The mutation (IVS8as-1 G→C) was situated in the acceptor splice site of intron 8 resulting in the skipping of exon 9 and the production of a truncated GHR. The mutant GHR formed heterodimers with the wild-type GHR and exerted a dominant negative effect on the normal protein. A second mutation (IVS9ds+1 G→A) leading to the same consequence was described in 2 Japanese siblings. We have also studied such a patient with a similar mutation which was present in the child’s mother and grandfather. The degree of short stature is milder than in classical GH resistance, height SDS ranging from –3.0 to –4.3, and facial features are usually normal. There is also a milder degree of IGF-I deficiency, with Ayling’s subject having a serum IGF-I of –2.4 SD [12]. All 4 subjects had normal GHBP.

STAT5b Mutations (fig. 1) Kofoed et al. [13] reported the first case of a homozygous mutation in exon 15 of the STAT5b gene and demonstrated that the mutant protein could not be activated by GH, therefore failing to activate gene transcription. This child had features of severe GH resistance together with immunodeficiency consistent with a non-functional STAT5b. Several more patients have now been reported. Most have immune deficiency. STAT5b appears to have an important role in gene expression induced

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by cytokines, such as interferon gamma Human defects in the STAT5b gene have recently been reviewed [14].

Idiopathic Short Stature Idiopathic short stature (ISS) refers to a heterogeneous group of short children with undefined aetiology. However, there is evidence of some degree of GH resistance in a sizeable proportion of children with significant short stature [15]. This has been elegantly demonstrated in studies of the IGF-I generation test in ISS patients and normal controls [16] A recent therapeutic trial demonstrated that it was necessary to treat ISS children with large doses of GH to achieve serum IGF-I levels of 2.0 SD [17]. However, true mutations of the GHR with evidence of decreased receptor function are rare [6].

Acid Labile Subunit Defect IGF-I, the key GH-dependent effector protein regulating human growth, circulates as a ternary complex consisting of IGF-I, IGFBP-3 and acid labile subunit (ALS). An ALS knock-out (KO) animal model provided new insights in the role of ALS in the IGF-I system, with growth deficits were seen 3 weeks after birth. In 2004, Domene et al. [18] reported the first human case of an inactivating ALS mutation. The defect was a guanine deletion at position 1338, resulting in a frame-shift and the appearance of a premature stop codon (1338delG, E35fsX120). The patient had minimal post-natal growth impairment but basal GH levels were increased associated with reduction in IGF-I and IGFBP-3 and undetectable ALS, unresponsive to stimulation by GH. A normal puberty growth spurt and final height has been documented in these patients, despite extreme deficiency of all GH-dependent peptides [19]. The characteristics of homozygous human ALS mutations have been further defined by recent reports, which now establish insulin resistance as an integral part of the phenotype.

IGF-I Gene Defects (fig. 2) The first human case of an IGF-I gene mutation was reported by Woods et al. [20] in 1996. The patient was a 15-year-old male who had severe intrauterine growth retardation (IUGR) and dysmorphic features consisting of microcephaly, micrognathia, ptosis, severe sensorineural deafness and intellectual retardation. Post-natal growth was also abnormal with a lack of response to GH therapy. The endocrine profile was of interest because serum IGF-I was undetectable, but IGFBP-3 was normal, thereby excluding a GHR defect. GH secretion was increased and ALS also slightly increased.

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Deletion of exons 4, 5

IGF-I gene

1

2

3

4

5

5’

6 3’

IGF-I V44M Bioinactive IGF-I

T/A exon 6 3’ UTR

IGF-I R36Q

Fig. 2. Molecular defects of the IGF-I gene.

The patient had insulin resistance with elevated fasting serum insulin. He was treated with recombinant human IGF-I for 2 years and showed a good growth response as well as normalising his insulin sensitivity. Molecular analysis showed a homozygous partial deletion of the IGF-I gene. Almost 10 years later, the second human case was reported by Wit’s group in Leiden, the Netherlands [21]. This patient also had intellectual retardation, deafness and a history of IUGR. Interestingly, his serum IGF-I was elevated indicating a biologically inactive IGF-I molecule. The molecular defect was a valine to methionine substitution at residue 44 of the mature IGF-I molecule. A third case has also been reported [3].

IGF-I Receptor Defects The key role of IGF-I in prenatal and post-natal growth has been confirmed by reports of mutations in the human IGF-I receptor (IGF-IR). This defect was first reported in 2 patients from Cincinnati and Leipzig [22]. Both had IUGR and post-natal growth failure. Four further cases, two sets of mother and daughter, have been described, all with some degree of IUGR and post-natal growth failure. It is likely that this defect is a rare cause of IUGR, although the degree of severity may depend on the degree of IGF-I signalling, which in turn is likely to be related to the nature of the molecular defect of the IGF-IR.

Conclusions

A number of general conclusions can be made concerning the phenotypes of patients with either GH or IGF-I resistance. Firstly, homozygous GHR mutations usually

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cause the phenotype of Laron syndrome, characterised by essentially normal prenatal growth but severely abnormal post-natal growth and final adult height. There is, however, a spectrum of phenotypical severity with some GHR defects causing a milder phenotype and less disturbance of the GH-IGF axis. Post-GHR defects such as mutations of the STAT5b gene also cause severe post-natal growth failure but with a subtly milder phenotype, not associated with the classical Laron syndrome facies. ALS mutations cause a failure of formation of the circulating ternary complex with extreme deficiency of circulating IGF-I, IGFBP-3 and ALS; however, the phenotype is mild and normal pubertal growth and final height have been reported. This may be explained by the relative normality, and even upregulation, of locally produced IGF-I, specifically in the growth plate. When the GHR is functional GH secretion may also be up-regulated resulting in insulin resistance. The key message concerning defects of the IGF-I gene and IGF-IR is that fetal growth is impaired, although to a variable degree. Post-natal growth is also abnormal. All these molecular defects are rare and the journey is just beginning of the characterisation of the range of phenotypes seen with each defect. The next decade will add more details and clarification of molecular mechanisms as more cases are diagnosed and genetic investigation becomes more refined.

References 1

2

3

4

5

6

Laron Z: Laron syndrome (primary growth hormone resistance or insensitivity): the personal experience 1958–2003. J Clin Endocrinol Metab 2004;89: 1031–44. Rosenfeld RG, Rosenbloom AL, Guevara-Aguirre J: Growth hormone (GH) insensitivity due to primary GH receptor deficiency. Endocr Rev 1994;15:369– 390. Woods KA: Genetic defects of the growth hormoneIGF axis associated with GH insensitivity. Endoc Dev 2007;11:6–15. Walenkamp MJ, Wit JM. Genetic disorders in the GH IGF-I axis in mouse and man. Eur J Endocrinol 2007;157(suppl 1):S15–26. Woods KA, Dastot F, Preece MA, Clark AJ, PostelVinay MC, Chatelain PG. Ranke MB, Rosenfeld RG. Amselem S, Savage MO: Phenotype: genotype relationships in growth hormone insensitivity syndrome. J Clin Endocrinol Metab 1997;82:3529– 3535. Savage MO, Attie KM, Camacho-Hübner C, David A, Metherell LA, Clark AJL: Investigation and treatment of patients with characteristics of growth hormone insensitivity. Nat Clin Pract Endocrinol Metab 2006;2:395–407.

7 Burren CP, Woods KA, Rose SJ, Tauber M, Price DA, Heinrich U, Gilli G, Razzaghy-Azar M, Al-Ashwal A, Crock PA, Rochiccioli P, Yordam N, Ranke MB, Chatelain PG, Preece MA, Rosenfeld RG, Savage MO: Clinical and endocrine characteristics in atypical and classical growth hormone insensitivity syndrome. Horm Res 2001;55:125–130. 8 Milward A, Metherell L, Maamra M, Barahona MJ, Wilkinson IR, Camacho-Hubner C, Savage MO, Strasberger CJ, Clark AJL, Ross RJM, Webb SM: Growth hormone (GH) insensitivity syndrome due to a GH receptor truncated after Box1, resulting in isolated failure of STAT 5 signal transduction. J Clin Endocrinol Metab 2004;89:1259–1266. 9 Metherell LA, Akker SA, Munroe PB, Rose SJ, Caulfield M, Savage MO, Chew SL, Clark AJL: Pseudoexon activation as a novel mechanism for disease resulting in atypical growth hormone insensitivity. Am J Hum Genet 2001 69:641–646. 10 Bjarnason R, Banerjee K, Rose SJ, Rosberg S, Metherell L, Clark AJL, Albertsson-Wikland K, Savage MO: Spontaneous growth hormone secretory characteristics in children with partial growth hormone insensitivity. Clin Endocrinol 2002;57: 357–361.

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11 David A, Camacho-Hubner C, Bhangoo A, Rose SJ, Miraki-Moud F, Akker SA, Butler GE, Ten S, Clayton PE, Clark AJL, Savage MO, Metherell LA: An intronic growth hormone receptor mutation causing activation of a pseudoexon is associated with a broad spectrum of growth hormone insensitivity phenotypes. J Clin Endocrinol Metab 2007; 92;655–659. 12 Ayling RM, Ross R, Towner P, Von Laue S, Finidori J, Moutoussamy S, Buchanan CR, Clayton PE, Norman MR: A dominant-negative mutation of the growth hormone receptor causes familial short stature. Nat Genet 1997;16:13–14. 13 Kofoed EM, Hwa V, Little B, Woods KA, Buckway CK, Tsubaki J, Pratt KL, Besrodnik L, Jasper H, Tepper A, Heinrich JJ, Rosenfeld RG: Growth hormone insensitivity associated with a STAT5b mutation. N Engl J Med 2003;349:1139–1147. 14 Rosenfeld RG, Belgorosky A, Camacho-Hübner C, Savage MO, Wit JM: Defects in growth hormone receptor signalling. Trends Endocrinol Metab 2007; 18134–18141. 15 Wit JM, Reiter EO, Ross JL, Saenger PH, Savage MO, Rogol AD, Cohen P: Idiopathic short stature: management and growth hormone treatment. Growth Horm IGF Res 2008;18;111–136. 16 Selva KA, Buckway CK, Sexton G, Pratt KL, Tjoeng E, Guevara-Aguirre J, Rosenfeld RG: Reproducibility in patterns of IGF generation with special reference to idiopathic short stature. Horm Res 2003;60:237– 246. 17 Cohen P, Rogol AD, Howard CP, Bright GM, Kappelgaard AM, Rosenfeld RG, American Norditropin Study Group: Insulin growth factorbased dosing of growth hormone therapy in children: a randomized, controlled study. J Clin Endocrinol Metab 2007;92:2480–2486.

18 Domene H, Bengolea SV, Martinez AS, Ropelato MG, Pennisi P, Scaglia P, Heinrich JJ, Jasper HG: Deficiency of the circulating insulin-like growth factor system associated with inactivation of the acid-labile subunit gene. N Engl J Med 2004;350: 570–577. 19 Domené HM, Martínez AS, Frystyk J, Bengolea SV, Ropelato MG, Scaglia PA, Chen JW, Heuck C, Wolthers OD, Heinrich JJ, Jasper HG: Normal growth spurt and final height despite low levels of all forms of circulating insulin-like growth factor-I in a patient with acid-labile subunit deficiency. Horm Res 2007;67:243–249. 20 Woods KA, Camacho-Hübner C, Savage MO, Clark AJL: Intrauterine growth retardation and post-natal growth failure associated with deletion of the insulin-like growth factor-I gene. N Engl J Med 1996;355:1363–1367. 21 Walenkamp MJ, Karperien M, Pereira AM, HilhorstHofstee Y, van Doorn J, Chen JW, Mohan S, Denley A, Forbes B, van Duyvenvoorde HA, van Thiel SW, Sluimers CA, Bax JJ, de Laat JA, Breuning MB, Romijn JA, Wit JM: Homozygous and heterozygous expression of a novel insulin-like growth factor-I mutation. J Clin Endocrinol Metab 2005;90:2855– 2864. 22 Abuzzahab MJ, Schneider A, Goddard A, Grigorescu F, Lautier C, Keller E, Kiess W, Klammt J, Kratzsch J, Osgood D, Pfäffle R, Raile K, Seidel B, Smith RJ, Chernausek SD, Intrauterine Growth Retardation (IUGR) Study Group: IGF-I receptor mutations resulting in intrauterine and postnatal growth retardation. N Engl J Med 2003;349:2211–2222.

Prof. Martin Savage Department of Endocrinology John Vane Science Centre, Charterhouse Square London EC1M 6BQ (UK) Tel. +44 20 7882 6233, Fax +44 20 7882 6234, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 151–166

Double Diabetes: A Mixture of Type 1 and Type 2 Diabetes in Youth Paolo Pozzilli ⭈ Chiara Guglielmi Department of Endocrinology and Diabetes, University Campus Bio-Medico, Rome, Italy

Abstract The increase in the incidence of type 1 diabetes (T1D), especially in children 15 years) who are overweight and at least 400 million adults who are clinically obese. The outlook for

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the future is even bleaker, with more than 700 million people predicted to be obese by 2015 and a total of 380 million people predicted to be living with diabetes by the year 2025 [11, 12]. Each year a further seven million people develop diabetes; that is two people every 10 s [13].

Obesity in Childhood, Adolescence and Adulthood

Since the mid-1970s, the prevalence of overweight and obesity has increased sharply for both adults and children. Data from two NHANES surveys show that among adults aged 20–74 years the prevalence of obesity increased from 15.0% (in the 1976–1980 survey) to 32.9% (in the 2003–2004 survey). The two surveys also show increases in overweight among children and teens. For children aged 2–5 years, the prevalence of overweight increased from 5.0 to 13.9%; for those aged 6–11 years, the prevalence increased from 6.5 to 18.8%, and for those aged 12–19 years, the prevalence increased from 5.0 to 17.4%. These increasing rates raise concern because of their implications for Americans’ health [14]. Although one of the national health objectives for the year 2010 is to reduce the prevalence of obesity among adults to less than 15%, current data indicate that the situation is worsening rather than improving.

Definitions for Adults For adults, overweight and obesity ranges are determined by using weight and height to calculate a number called the ‘body mass index’ (BMI). BMI is used because, for most people, it correlates with their amount of body fat. An adult who has a BMI between 25 and 29.9 is considered overweight. An adult who has a BMI of 30 or higher is considered obese. It is important to remember that although BMI correlates with the amount of body fat, BMI does not directly measure body fat. Other methods of estimating body fat and body fat distribution include measurements of skinfold thickness and waist circumference, calculation of waist-to-hip circumference ratios, and techniques such as ultrasound, computed tomography, and magnetic resonance imaging (MRI).

Definition for Children A child’s weight status is determined based on an age- and sex-specific percentile for BMI rather than by the BMI categories used for adults. Classifications of overweight for children and adolescents are age- and sex-specific because children’s body

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Table 1. Prevalence of overweight among US children and adolescents (age 2–19 years) Age

2–5 years 6–11 years 12–19 years

Survey periods NHANES I 1971–1974

NHANES II 1976–1980

NHANES III 1988–1994

NHANES 2003–2004

5 4 6.1

5 6.5 5

7.2 11.3 10.5

13.9 18.8 17.4

Sex-and age-specific BMI ≥95th percentile based on the CDC growth charts. All values given in percent [24–26].

composition varies as they age and varies between boys and girls. The Center for Disease Control and Prevention defined overweight as at or above the 95th percentile of BMI for age and ‘at risk for overweight’ as between the 85th and 95th percentile of BMI for age [15, 16]. European researchers classified overweight as at or above the 85th percentile and obesity as at or above the 95th percentile of BMI [17]. Overweight children and adolescents are at risk for health problems during their youth and as adults. For example, during their youth, overweight children and adolescents are more likely to have risk factors associated with cardiovascular disease (such as high blood pressure, high cholesterol, and T2D) than are other children and adolescents [18]. Overweight children and adolescents are more likely to become obese as adults [19, 20]. For example, one study found that approximately 80% of children who were overweight at age 10–15 years were obese adults at age 25 years. Another study found that 25% of obese adults were overweight as children [21]. The latter study also found that if overweight begins before 8 years of age, obesity in adulthood is likely to be more severe.

Trends in Childhood Overweight

In the middle of the 1990s the average prevalence of overweight, including obesity, for youth in European countries was about 22%. The highest level was in Italy, with more than 30% affected children [22]. The highest prevalence of obesity among schoolage children (10–16 years of age) was in Malta (7.9%) and the lowest in Lithuania and Latvia (0.4%) [23]. Tables 1–3 and figures 1–3 show trends in childhood overweight based on NHANES data for various age groups, beginning with NHANES I (1971–1974) and ending with NHANES 2003–2004. Data from NHANES I (1971–1974) to NHANES 2003–2004 show increases in overweight among all age groups [24, 26]:

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Table 2. Adolescent boys prevalence of overweight by race/ethnicity (age 12–19 years) Survey periods

Non-Hispanic white Non-Hispanic black Mexican-American

NHANES III 1988–1994

NHANES 2003–2004

11.6 10.7 14.1

19.1 18.5 18.3

Sex-and age-specific BMI ≥95th percentile based on the CDC growth charts. All values given in percent [24–26].

Table 3. Adolescent girls prevalence of overweight by race/ethnicity (age 12–19 years) Survey periods

Non-Hispanic white Non-Hispanic black Mexican-American

NHANES III 1988–1994

NHANES 2003–2004

7.4 13.2 9.2

15.4 25.4 14.1

Sex-and age-specific BMI ≥95th percentile based on the CDC growth charts. All values given in percent [24–26].

• among preschool-age children (2–5 years), the prevalence of overweight increased from 5.0 to 13.9% • among school-age children (6–11 years), the prevalence of overweight increased from 4.0 to 18.8% • among school-age adolescents (12–19 years), the prevalence of overweight increased from 6.1 to 17.4%. Although overweight has increased for all children and adolescents over time, NHANES data indicate disparities among racial/ethnic groups. Figures 1–3 compare the prevalence for racial/ethnic groups of adolescent boys and girls aged 12 through 19 years. The prevalence rate of overweight was slightly higher among adolescent non-Hispanic white boys (19.1%) than among non-Hispanic black boys (18.5%) and MexicanAmerican boys (18.3%) [25]. Data from NHANES III (1988–1994) through NHANES 2003–2004 showed that adolescent non-Hispanic white and black boys experienced larger increases in the

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20 Percent

15 10 5

18.8

Age 2–5 years Age 6–11 years Age 12–19 years 5.0

4.0

6.1

17.4

13.9 11.3 10.5 5.0

6.5

7.2 5.0

0 NHANES I 1971–1974

NHANES II 1976–1980

NHANES III 1988–1994

NHANES 2003–2004

Survey period

Fig. 1. Prevalence of overweight among US children and adolescents (age 2–19 years). National Health and Nutrition Examination Surveys. Sex-and age-specific BMI >95th percentile based on the CDC growth charts [24–26].

prevalence of overweight (7.5% and 7.8%, respectively) compared to the increase among Mexican American boys (4.2%): • among non-Hispanic white boys, the prevalence of overweight increased from 11.6 to 19.1% • among non-Hispanic black boys, the prevalence of overweight increased from 10.7 to 18.5% • among Mexican-American boys, the prevalence of overweight increased from 14.1 to 18.3%. Non-Hispanic black girls had the highest prevalence of overweight (25.4%) compared to that of non-Hispanic white (15.4%) and Mexican-American (14.1%) girls [25]. Data from NHANES III (1988–1994) through NHANES 2003–2004 showed that non-Hispanic black adolescent girls experienced the largest increase in the prevalence of overweight (12.2%) compared to non-Hispanic white adolescent (8.0%) and Mexican-American adolescent (4.9%) girls [24, 26]: • among non-Hispanic white girls, the prevalence of overweight increased from 7.4 to 15.4% • among non-Hispanic black girls, the prevalence of overweight increased from 13.2 to 25.4% • among Mexican American girls, the prevalence of overweight increased from 9.2 to 14.1%.

Causes of Overweight and Obesity

Obesity is a complex condition with genetic, metabolic, behavioral and environmental factors all contributing to its development. However, the dramatic increase in the

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Non-Hispanic white

Fig. 2. Adolescent boys prevalence of overweight by race/ ethnicity (age 12–19 years). National Health and Nutrition Examination Surveys. Sex-and age-specific BMI >95th percentile based on the CDC growth charts [24–26].

Percent

30

Non-Hispanic black Mexican-American 19.1

20 11.6

10.7

14.1

18.5

18.3

10 0 NHANES III 1988–1994

NHANES 2003–2004 Survey period

Non-Hispanic white

Fig. 3. Adolescent girls prevalence of overweight by race/ ethnicity (age 12–19 years). National Health and Nutrition Examination Surveys. Sex-and age-specific BMI > 95th percentile based on the CDC growth charts [24–26]..

Percent

30

Non-Hispanic black Mexican-American

20 10

15.4

13.2 7.4

25.4 14.1

9.2

0 NHANES III 1988–1994

NHANES 2003–2004 Survey period

prevalence of obesity in the past few decades can only be attributed to significant changes in lifestyle influencing both children and adolescents [12]. These obesitypromoting environmental factors are usually referred to today under the general term of ‘obesogenic’ or ‘obesigenic’ [13]. The current changing nature of this ‘obesogenic’ environment has been well described in a WHO Technical Report as follows: ‘Changes in the world food economy have contributed to shifting dietary patterns, for example, increased consumption of energy-dense diets high in fat, particularly saturated fat, and low in unrefined carbohydrates. These patterns are combined with a decline in energy expenditure that is associated with a sedentary lifestyle, motorized transport, labor-saving devices at home, the phasing out of physically demanding manual tasks in the work-place, and leisure time that is preponderantly devoted to physically undemanding pastimes.’ Under the notion ‘lifestyle’ are included dietary changes and cultural, environmental, social and economic factors. Therefore, apart from genetic factors, the prerequisite for becoming obese is an imbalance between energy expenditure, modulated primarily by physical inactivity, and energy intake from excessive food and drinks.

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Consequences of Overweight and Obesity

Childhood overweight is associated with various health-related consequences. Overweight children and adolescents may experience immediate health consequences and may be at risk for weight-related health problems in adulthood. Being overweight or obese increases the risk of many diseases and health conditions, including the following: hypertension, osteoarthritis, dyslipidemia, T2D, coronary heart disease, stroke, gallbladder disease, sleep apnea and some cancers (endometrial, breast, and colon).

Double Diabetes

The attractive term ‘double diabetes’ (DD) applied to the pediatric diabetic population was first introduced by Libman and Becker and coworkers [27, 28] when referring to subjects with an atypical form of diabetes, also called hybrid diabetes, type 1.5 diabetes or LADY (latent autoimmune diabetes in youth). The issue of DD was recently revised by two investigators of the present application [29]. The presence of autoimmune markers towards β cells, namely glutamic acid decarboxylase (GAD), thyrosine phosphatase antibodies (IA-2) and insulin autoantibodies (IAA), typically define cases of DD in patients with T2D [30, 31]. It is difficult to define what type of diabetes these subjects suffer from under the current classification, being classified as affected by T2D because they are obese and insulin resistant but also as affected by T1D because of presence of autoantibodies to β cells. There is no doubt that these subjects present with an overlapping phenotype of both T2D and T1D. In the adult population, these subjects are usually defined as affected by latent autoimmune diabetes in the adult (LADA) [32–34]. Such definition is generally based on autoantibody positivity, age at onset (>35 years) and insulin-independence following diagnosis of hyperglycemia for a period of at least 6 months. Several studies have demonstrated a more aggressive course of the disease in LADA subjects characterized by failure of oral hypoglycemic therapy and progressive β cell loss leading to insulin dependency usually within 5 years of diagnosis in subjects with more than one autoantibody to β cells and when aged 35–45 years [35]. LADA is found in approximately 10% of all cases of T2D; however, insulin resistance and obesity are not main features of LADA subjects [36] whereas they are in DD. Therefore, it looks like LADA represents one end of a rainbow of autoimmune diabetes which is distinguishable from classical T1D only because it is diagnosed in adulthood and presents with some clinical, anthropometric and metabolic features usually associated with T2D [35]. Despite obesity and metabolic syndrome being on the increase, in particular among Hispanic and African-Americans but also in Caucasian youths [37], we know very little about the prevalence of DD. The prevalence and significance of autoimmune markers in children who clinically present with T2D needs to be established in different populations.

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In particular, it needs to be established whether autoantibody-positive youths with DD progress more rapidly to insulin dependence than those with T2D without autoantibodies to β cells. This is also relevant because these youths may be at increased risk for complications associated with loss of β cell function, including hypoglycemia, ketoacidosis, difficult management of the disease and microvascular complications [38], in addition to macrovascular complications typically associated with T2D.

Role of Genes in Double Diabetes

It is accepted that both genetics and the environment play a role in T1D as demonstrated by the observation that disease concordance among identical twins is 30–50% [39, 40]. It has been calculated that the HLA region accounts for approximately 40% of the genetic risk, although other genes have an additional risk [41]. Amongst the putative environmental factors for T1D viral infections, infant dietary exposure, deficiency in vitamin D supplementation, and reduced number of infections accounting for the hygiene hypothesis, have been proposed [42–44]. In T2D, the role of genetic causative factors, although they have been only recently elucidated in determining the disease, is probably limited without the ‘weight’ of the environmental factors which include overnutrition, sedentary lifestyle and obesity [45]. The role of genes in DD is certainly of interest. One may argue that the major genes independently associated with susceptibility to either T1D, e.g. HLA, insulin gene VNTR, protein tyrosine phosphatase non-receptor (PTPN22), cytotoxic T -lymphocyte-associated antigen-4, (CTLA-4) [reviewed in 46], or T2D, e.g. adiponectin gene (APM1), plasma cell membrane glycoprotein 1 (PC-1) gene, transcription factor 7-like 2 (TCF7L2) gene [reviewed in 47] can represent genetic determinants for DD. We can speculate that in DD the frequency of the major T1D genetic susceptibility gene (HLA) may be reduced whereas an association exists with genes associated with T2D risk. Most interesting are genes that in view of their function may influence the pathogenic processes operating both in T1D and T2D potentially leading to DD.

Role of Environmental Factors in Double Diabetes

The environmental factors which influence both T1D and T2D disease processes may indeed play an important role in DD. A worldwide obesity epidemic as a consequence of current lifestyle influences the T2D process, but how such a condition or other factors associated with obesity affect or modulate the process leading to β cell destruction is unknown. Surprisingly, an increase in body mass index (BMI) has been reported in one third of children at diagnosis of diabetes [48], a form of clinical presentation which was never reported in the past for diabetes diagnosed in childhood. Several hypotheses have been put forward, the most widely accepted being linked to

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the increased risk for developing T1D in subjects who are overweight during childhood or experienced rapid early growth. The ‘accelerator hypothesis’ argues that T1D and T2D are the same disorder of insulin resistance set against different genetic background and predicts a general inverse relationship between BMI (surrogate for insulin resistance) and age at diagnosis of T1D [49]. The accelerator hypothesis identifies three accelerators that determine the rate at which β cell mass fails during life represented by constitution (genes/gestation), insulin resistance (lipotoxicity/antigenicity) and immune response genotype (HLA) [49]. There is still controversy about the accelerator hypothesis, the major concern being at what point obesity accelerates the onset of T1D (i.e. earlier or at a later stage in the natural history of the autoimmune process after substantial destruction of β cells has taken place) [50]. This controversy is particularly relevant for DD as these subjects are persistently obese and insulin resistant whereas the immunological markers of T1D represent an additional co-existent factor. To summarize, the greater the BMI and associated insulin resistance, the greater the damage to β cells in T1D, similar to what occurs for T2D. Follow-up studies are definitely necessary to sustain the accelerator hypothesis in DD.

Role of Autoimmunity to Beta Cells in Double Diabetes

Evaluation of autoantibodies to islet cell antigens and loss of C-peptide secretion is currently used for the prediction of T1D. Several studies have confirmed that the combination of three autoantibodies (GAD65, IA-2, and IAA) predicts T1D within 5 years in nearly all cases [51, 52]. One autoantibody alone confers low risk for progression; its positive predictive value should be calculated together with the occurrence of high risk human leukocyte antigens (HLA) genotypes as well as with the presence of a first-degree relative affected by the disease. Autoantibody affinity together with epitope analysis and their association with inter- and intra-molecular epitope spreading may help to refine the predictive value [53–55]. Thus, most GAD antibody-positive sera at the time of T1D diagnosis recognize conformationdependent epitopes located in the middle and the C-terminus of the GAD molecule, whereas few recognize the N-terminus. The appearance of GAD antibodies specific for conformational epitopes has been demonstrated longitudinally in individuals at risk of progression to T1D. The increase of GAD binding to the T1D-associated middle epitopes was observed in 72% of the high-risk children (of whom >40% developed diabetes during the follow-up period), whereas only 10% of children who do not progress to T1D showed this dynamic epitope change, which means a positive predictive value of 80% [55]. GAD antibodies can possess therefore a prognostic value based on their affinity and epitope analysis for determining an individual’s risk for progression to T1D. This rule applies for prediction of T1D as DD data have shown the presence of one single autoantibody, implying that progression to β cell destruction may be slower [56] although this has not been demonstrated. More

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studies are required to determine whether epitope change and its prognostic significance follows the same pattern in subjects with DD as it does in lean prediabetic individuals.

Diagnosis of Double Diabetes

If a teenager or child with all the typical clinical features of T2D, excess body weight, acanthosis nigricans, high blood pressure, high cholesterol, polycystic ovary syndrome (PCOS) (for girls), positive family history of T2D, belonging to an ethnic/ racial minority group, and show autoantibodies against insulin-producing β cells, we would say he/she has elements of both kinds of diabetes. A number of reports have described that as many as 15–20% of teens with the typical symptoms of T2D have autoantibodies circulating in their blood. Because of the presence of these autoantibodies, they can no longer be considered a pure T2D case. The following clinical and biochemical parameters can pave the way to diagnose a youth with DD: 1 Presence of clinical features of T2D, hypertension, dyslipidemia, increased BMI with increased cardiovascular risk compared with children with classical T1D. Family history for T2D and T1D may be present. 2 Presence of reduced number of clinical features typical of T1D such weight loss, polyuria/polydipsia, insurgence of ketoacidosis; insulin therapy is not the first line of therapy compared with subjects with classical T1D. 3 Presence of autoantibodies to islet cells but reduced number and titre compared to T1D, and probably reduced risk at HLA locus compared with subjects with T1D. As compared with T1D where insulin resistance and obesity are not common features, DD is always characterized by an obese phenotype with the addition of coexistent β cell autoimmunity.

Prevention

While relatively little is known about the long-term outcome of DD since it is a newly identified condition, we do know that being overweight and having diabetes genes in one’s family are the main causes. Since we cannot do anything about our genes, managing weight is the most important way to prevent DD. To prevent the development of DD, it is important to be physically active and prevent excessive weight gain or obesity. A healthy lifestyle that includes good eating habits and lots of physical activity benefits all children and adults regardless of their risk for diabetes. For a person with T1D that shows signs of DD, achieving and maintaining a healthy weight may resolve their body’s problem with insulin resistance (the basic feature of T2D and obesity). As weight decreases, the amount of insulin required to control glucose levels decreases

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and blood pressure and lipid levels also return to more normal (or optimal) levels. In women and girls, the polycystic ovary syndrome may also improve. Since DD is a new problem, there is not enough experience to decide what treatment approaches are most effective. There are a number of research studies underway which will provide guidance within the next few years. In the meantime, maintaining glucose control, achieving weight loss, being physically active and eating a nutritious diet are essential to managing DD and decreasing complications [57].

Therapy of Double Diabetes

Given the rapidly increasing problems associated with obesity, attempts to prevent the development of a condition such as DD may be highly relevant. Considering that in DD consistent β cell function is still present at the time of diabetes diagnosis and its decline may be slower than in classical T1D, an intervention capable of interfering with some of the putative mechanisms involved in the disease process may be worthwhile. Therefore, an immunomodulatory trial similar to one of those considered for T1D might be considered in DD. Although prevention of T1D is still far from being achieved, prevention of T2D has been shown to be feasible. Changes in lifestyle are relevant in halting or delaying the progression to overt disease in subjects at risk for T2D, probably as a consequence of an improvement in insulin sensitivity, as shown in the Diabetes Prevention Program [58]. Despite the increased prevalence of T2D in the pediatric population, there is limited information about the relative effectiveness of any treatment approach and pediatric diabetologists have had to rely on treatment paradigms derived from research and experience in the care of adults with T2D. Treatment Options for T2D in Adolescents and Youth (TODAY) is a randomized parallel group trial designed to test the hypothesis that aggressive reduction in insulin resistance early in the course of T2D is beneficial for prolongation of glycemic control, as well as improvement in associated abnormalities and risk factors [59]. Participants meeting eligibility criteria at the end of run-in are randomized to (1) metformin alone, (2) metformin plus rosiglitazone, or (3) metformin plus an intensive lifestyle intervention called TODAY Lifestyle Program. TODAY is the first large-scale, systematic study of treatment effectiveness for T2D in youth [59]. Whether such an approach may be successful in DD where, in addition, autoimmune phenomena also play a major role is unknown. New trials aimed at reducing insulin resistance using metformin and glitazones have been proposed for adult diabetic subjects with autoantibodies to β cells (LADA) to prevent the decline of β cell function [60, 61]. A treatment capable of interfering with the putative mechanisms involved in the disease process should be considered. In DD, a consistent β cell function is still present at the time of diagnosis, the decline of β cell function may be slower than in classical T1D and insulin resistance is present.

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Diet and Physical Activity Diet and physical exercise regimens have shown significantly greater effect in preventing the onset of T2D compared to placebo (58% decrease in incidence), and changes in lifestyle were even superior to metformin therapy (31% decrease in incidence) [62]. Whether such an approach may be successful in DD where autoimmune phenomena also play a major role is unknown Metformin Adjunctive metformin in overweight young people with T1D improves glycemic control without the weight gain expected with insulin therapy. In the long term, insulin treatment was associated with a fall in BMI [63]. Insulin In the Tokyo Study, 54 GAD-positive patients were randomly divided into 2 groups: one group received insulin (Ins group), the other a sulfonylurea (SU group). The study showed that small doses of insulin effectively prevent β cell failure in slowly progressive T1D, specifically in those patients with preserved β cell function and a high GAD titer at the initiation of therapy [64]. Other pharmacological potential drugs for treatment of DD include orlistat, sibutramine, glucosidase inhibitors, thiazolidinediones and glucagon-like peptide-1 [65–68].

Conclusions

Over the past decade, it has become apparent that more cases of T1D are diagnosed in children and adolescents who were overweight or even obese before hyperglycemia has developed. Sometime even diagnosis of T1D is not easy to place because of the phenotypic features typically associated with T2D. In addition, the increase of obesity observed in children may contribute to speed up the process of β cell destruction in subjects genetically susceptible to T1D. It is therefore necessary to investigate this new emerging form of DD in childhood and adolescence because it contributes overall to the increasing incidence of T1D amongst different populations. It is very likely that with increasingly greater effects of the environment on the onset of autoimmune disease – particularly in the group of subjects with DD – lifestyle modifications, including diet and exercise, may be relevant for the prevention of T1D in some populations, just as they are for the prevention of T2D. In conclusion, all forms of diabetes have shown a rapid increase over the past decade associated with a concomitant increase of overweight and obesity as well an earlier age of onset of the disease. Additionally, new diabetes phenotypes appear (DD) which render difficult both diagnosis and consequently the therapeutic approach to these patients who are mostly in the pediatric age group. Urgent action is needed to reverse this trend.

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60 Pozzilli P, Guglielmi C: Immunomodulation for the prevention of SPIDDM and LADA. Ann NY Acad Sci 2006;1079:90–98. 61 Zhou Z, Li X, Huang G, Peng J, Yang L, Yan X, Wang J: Rosiglitazone combined with insulin preserves islet beta cell function in adult-onset latent autoimmune diabetes (LADA). Diabetes Metab Res Rev 2005;21:203–208. 62 Knowler WC, Barrett-Connor E, Fowler SE, Hamman RF, Lachin JM, Walker EA, Nathan DM, Diabetes Prevention Program Research Group: Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002;346:393–403. 63 Moon RJ, Bascombe LA, Holt RI: The addition of metformin in type 1 diabetes improves insulin sensitivity, diabetic control, body composition and patient well-being. Diabetes Obes Metab 2007; 9:143–145. 64 Maruyama T, Shimada A, Kanatsuka A, Kasuga A, Takei I, Yokoyama J, Kobayashi T: Multicenter prevention trial of slowly progressive type 1 diabetes with small dose of insulin (the Tokyo study): preliminary report. Ann NY Acad Sci 2003;1005:362– 369. 65 Drew BS, Dixon AF, Dixon JB: Obesity management: update on orlistat. Vasc Health Risk Manag 2007;3:817–821. 66 Neovius M, Johansson K, Rössner S: Head-to-head studies evaluating efficacy of pharmaco-therapy for obesity: a systematic review and meta-analysis. Obes Rev 2008;9:420–427. 67 Kjems LL, Holst JJ, Vølund A, Madsbad S: The influence of GLP-1 on glucose-stimulated insulin secretion: effects on beta-cell sensitivity in type 2 and nondiabetic subjects. Diabetes 2003;52:380–386. 68 Miller JL, Silverstein JH: The treatment of type 2 diabetes mellitus in youth: which therapies? Treat Endocrinol 2006;5:201–210.

Prof. Paolo Pozzilli Department of Endocrinolgy and Diabetes University Campus Bio-Medico, Via Alvaro del Portillo, 21 IT–00128 Rome (Italy) Tel. +39 06 22541 9184, Fax +39 06 22541 456, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 167–173

Cryptorchidism as Part of the Testicular Dysgenesis Syndrome: The Environmental Connection K.M. Maina ⭈ N.E. Skakkebæka ⭈ J. Topparib a

University Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark; Departments of Physiology and Paediatrics, University of Turku, Turku, Finland

b

Abstract Cryptorchidism is part of the testicular dysgenesis syndrome (TDS), which includes other male reproductive disorders such as hypospadias, testis cancer and reduced semen quality. These diseases appear to be linked by common pathogenic mechanisms, interfering with normal fetal testis development. Testis development and descent is dependent on androgens and thus on an intact hypothalamus-pituitary-gonadal axis. Although cryptorchidism occurs in rare syndromes and genetic disorders, in the majority of children the etiology remains open. Many maternal and fetal risk factors have been previously identified but recently, scientific focus has also been directed to environmental hormone disrupting chemicals and lifestyle, as the prevalence of testis cancer and cryptorchidism has increased and semen quality decreased over few decades in several countries. Some persistent environmental chemicals, e.g. polychlorinated pesticides and polybrominated flame retardants, were associated with testicular maldescent and testis cancer. In addition, prenatal exposure to phthalates was negatively correlated to testosterone levels and anogenital distance as a measure of androgen effect in infant boys. Alcohol consumption and maternal smoking during pregnancy also appeared to be a risk factor for cryptorchidism. Thus, current evidence suggests that the development of the male reproductive tract may be susceptible to adverse effects of environmental horCopyright © 2009 S. Karger AG, Basel mone disrupters.

Testicular development and descent is regulated by numerous genetic factors, and is dependent on hormones produced by the Leydig cells, e.g. insulin-like factor 3 (Insl3) and testosterone [1]. Although several complex syndromes and genetic disorders are linked with testicular maldescent and other genital malformations [1], in the majority of boys with cryptorchidism no distinct etiology can be ascertained. Cryptorchidism is one of the most frequent congenital malformations in newborn boys. The reported prevalence of approximately 2–9% for congenital cryptorchidism differs considerably between studies and varies depending on diagnostic criteria, age

at examination, selection of study population, country of origin, ethnicity, as well as the experience of the observer [1]. Some countries, e.g. Great Britain and Denmark, have observed an increase in the prevalence of cryptorchidism over three to four decades [2], suggesting that environmental factors may play a role. A synchronous increase in the prevalence of male reproductive disorders over recent decades has been reported for hypospadias [3–5] and testicular cancer in several areas [6]. Semen quality appears to decline in many Western countries [7, 8]. These disorders have common pathogenic risk factors, they can present in combination and they are also risk factors for each other. Thus, we brought forward the hypothesis suggesting that male reproductive disorders were interlinked in a testicular dysgenesis syndrome (TDS) that originates from fetal life [9]. Many risk factors for cryptorchidism have been previously described and encompass paternal, maternal and fetal characteristics, e.g. intra-uterine growth restriction, low birth weight, prematurity, placental insufficiency, pre-eclampsia, maternal diabetes and siblings with cryptorchidism [1]. However, recent studies have highlighted the potential adverse role of environmental chemicals with hormone disrupting properties and lifestyle factors on fetal testicular development and subsequent male reproductive disorders [10].

Trends in the Prevalence of Male Reproductive Disorders

Registry data for congenital malformations such as cryptorchidism and hypospadias are of limited value to study time trends, as there are considerable variations in the degree of reporting and ascertainment [1]. Thus, only few prospective population-based studies are available, which document an increase in the prevalence of cryptorchidism [2] and hypospadias [3–5]. At the same time, there appears to be a decrease in semen quality parameters in many Western countries [7]. Testicular germ cell cancer, in turn, has been thoroughly registered in many countries and shows a considerable increase over the past few decades in many countries [6]. Genetic factors alone cannot explain such an increase, but this observation points towards other environmental factors or an influence by lifestyle. There is now considerable scientific evidence that testis cancer originates from carcinoma in situ testis, which is linked to prenatal dysgenetic development of the primordial germ cells in the testis [11]. Thus, an increase in testis cancer may be a general indicator of male reproductive health in a population [12].

Environmental Factors and Lifestyle

Chemical compounds with estrogenic or anti-androgenic properties can adversely affect the development of the testis [13]. Chemicals with endocrine disrupting

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properties are ubiquitously found in the environment and exposure routes are many, e.g. diet, air, cosmetics, cleaning substances, electronic equipment [14]. It is difficult to establish causal links between exposure to environmental chemicals and adverse outcomes in humans, as the human exposure situation is complex and interlinked with lifestyle factors and genetic susceptibility. Some chemical substances such as halogenated compounds bio-accumulate and are therefore present for many generations, whereas others, e.g. phthalates, appear to be rapidly metabolized. Thus, human evidence is usually derived from epidemiological studies or nested case control investigations within larger populations. These studies are laborious to perform and chemical analyses of trace amounts of environmental chemicals are expensive. It has often been experiences from wildlife and animal experiments that have given rise to concern about human health issues. In addition, toxicological studies in animals provide crucial information on mechanisms of hormone disruption. Thus, many scientific disciplines supplement each other and need to collaborate, in order to strengthen research in this area. Exposure to phthalates and their metabolites is an example, where interdisciplinary cooperation contributed to the discovery that the development of the foetal testis can be adversely affected by perinatal exposure. Although phthalates are rapidly metabolized, the exposure to them is ubiquitous and phthalate metabolites have thus been found in many biological fluids, including amniotic fluid and breast milk [15]. Prenatal exposure to phthalates was related to a reduced anogenital distance in boys, which in turn correlated with an increased prevalence of congenital cryptorchidism, smaller scrotal size and smaller penis volume [16]. Another study showed that the concentration of phthalate metabolites in breast milk was negatively correlated to serum free testosterone levels in 3-month-old boys [17]. In rats, in utero exposure to dibutyl phthalate causes a picture similar to that of TDS in man, especially if exposure was during the most sensitive developmental window [18]. This effect included cryptorchidism, hypospadias, decreased sperm count and decreased testosterone and Insl-3 production. Several modes of actions for phthalates and their metabolites have been proposed [15], but the predominant effect of phthalates on the developing testis appears to be a reduction in testosterone biosynthesis. Exposure to phthalate esters appeared to reduce the expression of genes involved in cholesterol transport and steroidogenesis and the availability of cholesterol for steroidogenesis. In epidemiological studies, associations have been reported between parental occupations in agricultural work with higher than average exposure to pesticides and a higher prevalence of cryptorchidism, and in part also hypospadias, in their male offspring. There is a higher rate of orchidopexies in areas of high pesticide use versus unexposed regions [19]. Few studies are available which employ individual measurements of pesticide levels in a biological matrix. Fat tissue samples from boys during operation for cryptorchidism showed higher concentrations of heptachloroepoxide and hexachlorobenzene than controls [20]. Some nested case-controls studies have found significant associations between the level of exposure to pesticides [19, 21]

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and polybrominated flame retardants [22] and congenital cryptorchidism. Although many of the investigated substances have been banned for use in Western countries, their persistency in the food chain may still contribute to the overall cocktail exposure of current populations. A very recent study investigated children of mothers who had worked in greenhouses before pregnancy and during the first weeks of gestation. This study could not investigate which of the many pesticides used during the study period was responsible for the detected effects. It showed, however, a threefold increase of cryptorchidism in the exposed sons and several indicators of reduced reproductive function such as reduced penile length, serum testosterone and an increased gonadotropin drive in the 3-month-old children [23]. Maternal exposure to persistent environmental chemicals, including polybrominated flame retardants, could also be linked to the development of testicular cancer in their sons two decades later [24]. The mechanisms of these adverse effects are yet unknown. Flame retardants may exert their effect in combination with simultaneous exposure to other chemicals and lifestyle factors. Recently, a new approach to toxicological testing has been developed to address so-called ‘cocktail effects’ of combined chemical exposures in rodents. In one study, the authors used three different anti-androgenic chemicals in a combined oral exposure, which led to additive adverse effects, e.g. nipple retention and reduced anogenital distance in the outcome [25]. The most striking evidence from these studies, however, was the observation that the combined exposure at dose levels which for the individual chemical were below any observed adverse effect level, resulted in a high prevalence of hypospadias in these animals [26]. These results are of particular concern for humans as the study concept reflects more realistically the human exposure situation than traditional toxicological experiments. From fetal life until adulthood, a human is simultaneously exposed to many chemicals at low levels, many of which accumulate over the lifetime. It is yet unknown how genetic susceptibility, including regulating factors of metabolic processes, may in addition influence the individual outcome of exposures. The general increase in the prevalence of obesity among Western populations may contribute to reproductive health problems. Thus, it has been shown that both under and overweight in young men was associated with reduced semen quality [27]. Mild gestational diabetes and even impaired glucose tolerance test represented a risk for congenital cryptorchidism [28]. This may be of considerable importance for public health as the prevalence of maternal pre-pregnancy obesity and thus gestational diabetes is increasing in many Western countries. There are also indicators that suggest an association between maternal lifestyle during pregnancy such as moderate regular maternal alcohol consumption and mild disorders of testicular descent [29]. Another study found a correlation between persistent cryptorchidism and binge drinking [30], but not regular alcohol intake. Previous retrospective and registry-based studies did not find these associations, which may be due to underreporting of this malformation and variation in the ascertainment of

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cryptorchidism. The precise mechanisms by which alcohol intake may affect testicular development in humans are currently unknown. Also, maternal smoking during pregnancy was found to be an independent risk factor for bilateral versus unilateral congenital cryptorchidism [31] and reduced semen quality in adult offspring [27, 32]. As the Danish and Finnish women differed considerably in their alcohol consumption patterns, this lifestyle difference may contribute to the difference seen in the prevalence of cryptorchidism in the two Nordic countries [2]. Congenital and adult male reproductive disorders are interlinked in the testicular dysgenesis syndrome, which suggests common pathogenic factors acting in utero. Several chemicals with known endocrine disrupting effects in animal experiments or in vitro are also associated with an increased risk of congenital cryptorchidism in humans or with signs of subtle impairment of testicular function. There is emerging evidence that some chemicals may be associated with testis cancer development. In addition, modern lifestyle factors such as obesity and gestational diabetes, maternal smoking and alcohol consumption during pregnancy appear to constitute independent risk factors for gonadal descent and function in the male offspring. As human exposure is complex, research in this field should be considerably strengthened in order to provide more insight into the mechanisms of how the different factors are interlinked. In addition, genetic polymorphisms may alter the extent to which adverse factors cause a specific phenotype.

Acknowledgements This work was supported by the Novo Nordisk Foundation, The European Commission, The Danish Medical Research Council, The Novo Nordisk Foundation, The Turku University Central Hospital, The Academy of Finland and the Sigrid Jusélius Foundation.

References 1

2

3

Virtanen HE, Toppari J: Epidemiology and pathogenesis of cryptorchidism. Human Reprod Update 2008;14:49–58. Boisen K, Kaleva M, Main KM, Virtanen HE, Haavisto A-M, Schmidt IM, Chellakooty M, Damgaard IN, Mau C, Reunanen M, Skakkebæk NE, Toppari J: Difference in the prevalence of congenital cryptorchidism in infants between two Nordic countries, Lancet 2004;363:1264–1269. Boisen KA, Chellakooty M, Schmidt IM, Kai CM, Damgaard IN, Suomi AM, Toppari J, Skakkebæk NE, Main KM: Hypospadias in a cohort of 1072 Danish newborn boys: prevalence and relationship to placental weight, anthropometrical measurements at birth and reproductive hormone levels at 3 months of age. J Clin Endocrinol Metab 2005;90:4041–4046.

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5

6

7

Paulozzi LJ, Erickson JD, Jackson RJ: Hypospadias trends in two US surveillance systems. Pediatrics 1997;100:831–834. Pierik FH, Burdorf A, Nijman JM, de Muinck Keizer-Schrama SM, Juttmann RE, Weber RF: A high hypospadias rate in the Netherlands. Hum Reprod 2002;17:1112–1115. Purdue MP, Devesa SS, Sigurdson AJ, McGlynn KA: International patterns and trends in testis cancer incidence. Int J Cancer 2005;115:822–827. Swan SH, Elkin EP, Fenster L: The question of declining sperm density revisited: an analysis of 101 studies published 1934–1996. Environ Health Perspect 2000;108:961–966.

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8 Jørgensen N, Asklund C, Carlsen E, Skakkebæk NE: Coordinated European investigations of semen quality: results from studies of Scandinavian young man are a matter of concern. Int J Androl 2006;29: 54–61. 9 Skakkebæk NE, Raipert-de Meyts E, Main KM: Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod 2001;16:972–978. 10 Skakkebæk NE, Jørgensen N, Main KM, Rajpert-De Meyts E, Lefferes H, Andersson AM, Juul A, Carlsen E, Mortensen GK, Jensen TK, Toppari J: Is human fecundity declining? Int J Androl 2006;29:2–11. 11 Rajpert-De Meyts E: Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects. Hum Reprod Update 2006;12:303–323. 12 Skakkebæk E, Rajpert-de Meyts E, Jørgensen N, Main KM, Leffers H, Andersson AM, Juul A, Jensen TK, Toppari J: Testicular cancer trends as ‘whistle blowers’ of testicular developmental problems in populations. Int J Androl 2007;30:198–2005. 13 Toppari J, Larsen JC, Christiansen P, Giwercman A, Grandjean P, Guillette LJ Jr, Jégou B, Jensen TK, Jouannet P, Keiding N, Leffers H, McLachlan JA, Meyer O, Müller JR, Rajpert-De Meyts E, Scheike T, Sharpe RM, Sumpter J, Skakkebæk NE: Male reproductive health and environmental xenoestrogens. Environ Health Perspect 1996;104: 741–803. 14 Damgaard IN, Main KM, Toppari J, Skakkebæk NE: Impact of exposure to endocrine disrupters in utero and in childhood on adult reproduction: best practice and research, clinical endocrinology and metabolism. Baillières Endocrinol 2002;16:289– 309. 15 Lottrup G, Andersson AM, Leffers H, Mortensen GK, Toppari J, Skakkebæk NE, Main KM: Possible impact of phthalates on infant reproductive health. Int J Andrology 2006;29:172–180. 16 Swan SH, Main KM, Liu F, Stewart SL, Kruse RL, Calafat AM, Mao CS, Redmon JB, Ternand CL, Sullivan S, Teague JL, Study for Future Families Research Team: Anogenital distance – a marker of fetal androgen action – is decreased in male infants following phthalate exposure during pregnancy. Environ Health Perspect 2005;113:1056–1061. 17 Main KM, Mortensen GK, Kaleva MM, Boisen KA, Damgaard IN, Chellakooty M, Schmidt IM, Suomi AM, Virtanen HE, Petersen JH, Andersson AM, Toppari J, Skakkebæk NE: Human breast milk contamination with phthalates and alterations in endogenous reproductive hormones in three months old children. Environ Health Perspect 2006;114:270–276.

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18 Welsh M, Saunders PT, Fisken M, Scott HM, Hutchinson GR, Smith LB, Sharpe RM: Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism. J Clin Invest 2008; 118:1479–1490. 19 Damgaard IN, Skakkebæk NE, Toppri J, Virtanen HE, Shen H, Schramm KW, Petersen JH, Jensen TK, The Nordic Cryptorchidism Study Group, Main KM: Persistent pesticides in human breast milk and cryptorchidism. Environ Health Perspect 2006;114: 1133–1138. 20 Hosie S, Loff S, Witt K, Niessen K, Waag KL: Is there a correlation between organochlorine compounds and undescended testes? Eur J Pediatr Surg 2000;10: 304–309. 21 Fernandez MF, Olmos B, Granada A, Lopez-Espinosa MJ, Molina-Molina JM, Fernandez JM, Cruz M, OleaSerrano F, Olea N: Human exposure to endocrinedisrupting chemicals and prenatal risk factors for cryptorchidism and hypospadias: a nested case-control study. Environ Health Perspect 2007;115:8–14 22 Main KM, Kiviranta H, Virtanen HE, Sundqvist E, Tuomisto JT, Tuomisto J, Vartiainen T, Skakkebæk NE, Toppari J: Flame retardants in placenta and breast milk and cryptorchidism in newborn boys. Environ Health Perspect 2007;115:1519–1526 23 Andersen HR, Schmidt IM, Grandjean P, Jensen TK, Budtz-Jørgensen E, Kjærstad MB, Bælum J, Nielsen JB, Skakkebæk NE, Main KM: Impaired reproductive development in sons of women occupationally exposed to pesticides during pregnancy. Environ Health Perspect 2008;116:566–557. 24 Hardell L, Van Bavel B, Lindstrom G, Eriksson M, Carlberg M: In utero exposure to persistent organic pollutants in relation to testicular cancer risk. Int J Androl 2006;29:229–234. 25 Hass U, Scholze M, Christiansen S, Dalgaard M, Vingaard AM, Axelstad M, Metzdorff SB, Kortenkamp A: Combined exposure to anti-androgens exacerbates disruption of sexual differentiation in the rat. Environ Health Perspect 2007;115: 122–128. 26 Christiansen S, Scholze M, Axelstad M, Boberg J, Kortenkamp A, Hass U: Combined exposure to anti-androgens causes markedly increased frequencies of hypospadias in the rat. Int J Androl 2008; 1:241–248. 27 Jensen TK, Jørgensen N, Punab M, Haugen TB, Suominen J. Zilaitiene B, Horte A, Andersen AG, Carlsen E, Magnus Ø, Matulevicius V, Nermoen I, Vierula M, Keiding N, Toppari J, Skakkebæk NE: Association of in utero exposure to maternal smoking with reduced semen quality and testis size in adulthood: a cross-sectional study of 1770 young men from the general population in five European countries. Am J Epidemiol 2004;159:49–58.

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28 Virtanen HE, Tapanainen AE, Kaleva MM, Suomi AM, Main KM, Skakkebæk NE, Toppari J: Mild gestational diabetes as a risk factor for congenital cryptorchidism. J Clin Endocrinol Metab 2006;91: 4862–4865. 29 Damgaard IN, Jensen TK, the Nordic Cryptorchidism Study Group, Petersen JH, Skakkebæk NE, Toppari J, Main KM: Cryptorchidism and maternal alcohol consumption during pregnancy. Environ Health Perspect 2007;115:272–277 30 Jensen MS, Bonde JP, Olsen J. Prenatal alcohol exposure and cryptorchidism. Acta Paed 2007;96: 1681–1685.

31 Thorup J, Cortes D, Petersen BL: The incidence of bilateral cryptorchidism is increased and the fertility potential is reduced in sons of mothers who have smoked during pregnancy. J Urol 2006;176:734– 737. 32 Storgaard L, Bonde JP, Ernst E, Spanò M, Andersen CY, Frydenberg M, Olsen J: Does smoking during pregnancy affect sons’ sperm counts? Epidemiology 2003;14:278–286.

Katharina M. Main MD, PhD University Department of Growth and Reproduction Section 5064, Rigshospitalet Blegdamsvej 9, DK--2100 Copenhagen (Denmark) Tel. +45 3545 5085, Fax +45 3545 6054, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 174–180

Disorders of Sex Development in Developmental Syndromes Olaf Hiorta ⭈ Gabriele Gillessen-Kaesbachb a

Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics, and bInstitut für Humangenetik, Universität zu Lübeck, Germany

Abstract Disorders of sex development comprise an array of congenital conditions with atypical development of chromosomal, gonadal, and anatomical sex affecting the genitourinary tract and in most instances also the endocrine-reproductive system. While the molecular basis of some of these disorders has been well established, it remains elusive in others. This holds true especially for disorders of sex development that are associated with other congenital malformations and abnormalities in a ‘syndromic’ condition. Syndromic disorders of sex development may be due to monogenic defects, biochemical abnormalities of steroid synthesis, or cytogenetic abnormalities comprising microdeletions or duplications or unbalanced rearrangements. This review will focus on the clinical description of syndromic disorders of sex development guiding towards the genetic classification. The characterization of the underlying diagnosis will improve genetic counseling of the family including progCopyright © 2009 S. Karger AG, Basel nosis and recurrence risk.

‘Disorders of sex development (DSD)’ comprise a heterogeneous group of heritable disorders of sex determination and differentiation, formerly termed ‘intersexuality’. This includes chromosomal as well as monogenic disorders, which inhibit or change primarily genetic or endocrine pathways of normal sex development. The elucidation of the underlying cause may be extremely helpful in counseling patients and families with respect to gender assignment, overall prognosis and establishing an individualized management of the patient [1].

Interdisciplinary Approach to Syndromic DSD

In a subset of patients, DSD is part of a syndrome with other associated congenital malformations, Biochemical and genetic studies have elucidated common pathways and have identified the causative genes in a variety of DSD syndromes. In other

conditions, the etiology is yet unclear. It is very important to care for these patients in an interdisciplinary team consisting of pediatric surgeons/urologists, human geneticists, and pediatric endocrinologists. [2]. It is their task to develop diagnostic strategies helping to define the underlying genetic or biochemical disorder to allow for a stepwise and stringent management of the patient. In order to classify the underlying disorder, a detailed clinical examination and description, a biochemical age-related referenced endocrine profile, as well as genetic investigations are necessary. Clinical examination should reveal all abnormalities seen in the patient and include auxological measurements. It is very important to receive a detailed family history as well as precise data on the past medical history of the patient including birth data. In addition, imaging investigation by ultrasound of the morphological abnormalities of the urogenital tract are the best and fastest ways for an initial description and classification. A supplementary description of the anatomy and the type of gonadal abnormality may be needed, involving a pediatric surgeon’s description of the inner genitalia, namely the status of Wolffian duct or Mullerian duct development as well as the histological investigation of the gonads. During the newborn period and early infancy, endocrine investigations may be directed by the endogenous stimulation of the ‘mini-puberty’ to describe adrenal and gonadal disorders. Serum analysis can be complemented by a urinary steroid profile involving gas chromatography/ mass spectrometry, which will allow for identification of various adrenal abnormalities. Any biochemical analysis during infancy and childhood needs to be performed in a specialized pediatric-endocrine laboratory that has the special attire needed for these analyses and uses the respective age-related reference values. Cytogenetic analysis complemented by CGH array analysis may help to identify the underlying genetic defect. The results of this diagnostic approach may then allow classification of the syndromic disorder either as a distinct entity of monogenic origin, caused by a developmental gene defect or a biochemical abnormality as well caused by a single gene mutation.

Distinct Monogenic Syndromic Developmental Disorders with DSD

The initial, sexually indifferent phase of gonad formation begins at 5 weeks of gestation with the development of paired gonadal ridges. The process becomes sexually dimorphic after germ cell seeding at 6 weeks and bipotential gonad formation at seven weeks. Those gonads with a 46,XY karyotype begin expressing SRY (sex-determining region on the Y chromosome), the transcription factor which is thought to initiate the downstream molecular events of testis formation [3]. These transcription factors involved in bipotential gonad formation are necessary for developmental processes in other tissues, and their disruption often is associated with nongonadal malformations and diseases. An increasing variety of clinical syndromes with ambiguous

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genitalia are now elucidated on the molecular level. In the following a few examples are described in more detail (table 1). Mutations in the Wilms’ tumor 1 (WT1) gene lead to several clinical entities [4]. In addition to disruption of gonadal development in both sexes, the differentiation of the kidneys can be disturbed, leading to the formation of Wilms’ tumors during childhood. Moreover, chromosomal deletions of 11p13, the location of the WT1 gene, have been associated with aniridia, Wilms’ tumor and mental retardation as part of the WAGR-syndrome. Point mutations in the WT1 gene lead to DenysDrash and Frasier syndromes. In Denys-Drash syndrome, Wilms’ tumors are associated with severe kidney disease with mesangial sclerosis and gonadal dysgenesis. In Frasier syndrome, a complete gonadal dysgenesis is accompanied by a late-onset focal glomerulosclerosis and a high risk for gonadoblastoma instead of Wilms’ tumors. Of note, an abnormality of the external genitalia is only present in children with a 46,XY karyotype, because only there diminished androgen synthesis will cause a visible malformation. However, the developmental defects will be present in both sexes. In contrast to WT1 mutations, genetic alterations in the steroidogenic factor 1 (SF1) encoded by the NR5A1 gene will lead to gonadal dysfunction of variable extent and may be associated with severe adrenal failure due to a developmental defect of adrenal steroidogenesis [5]. While this is overt in some cases at birth, adrenal failure may develop later in others and has to be kept in mind [6]. Another example of a single gene involved in formation of the bipotent gonad in association with other malformations is the hand-foot-genital syndrome caused by mutation of the homeobox gene on chromosome 7 designated HOXA13. Females have duplication of the uterus and sometimes of the cervix, and might have a septated vagina. In males hypospadias are characteristic. The hands are small, the thumbs are malformed and the thenar eminence is flat. In the feet the first toe is small, and radiographs show a short first metacarpal and phalanx. Shortly after SRY expression, the SRY-related HMG-Box gene 9 (SOX9) gene, which is required for Sertoli cell differentiation and type II collagen production, begins to be expressed in the testes [3]. Patients with mutations in the SOX9 gene show bowing of the femur and tibia, a large head, a small jaw, a cleft palate and a flat nasal bridge. Congenital dislocation of the hip and heart defects are common. Characteristically, the scapulae are hypoplastic and the iliac wings are vertical and narrow. Ambiguous genitalia occur in the majority of patients with a 46,XY karyotype. A duplication of SOX9 can lead to a testis formation in individuals with a 46,XX karyotype, demonstrating the importance of gene dosis effects in gonadal differentiation. Other transcription factors like FGFR2 (10q26) have been associated with severe genital malformations in animals or humans. Mutations in the Fgfr2 gene lead to partial sex reversal in male mice; loss of the terminal 10q26 region also has been associated in humans with sex reversal, but a specific association with mutations in the Fgfr2 gene has not been described in humans. Similarly, loss of Fgf9, an inducer of Fgfr2, leads to disorders of sex development in male vertebrates but has not yet been

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Table 1. Genes known to be involved in syndromal forms of disorders of sex development Gene

Locus

Mullerian structures

External genitalia

Associated features

46,XY DSD single gene disorders of gonadal development WT1 11p13 AD dysgenetic

+/–

ambiguous to female

SF1

9q33

AD/AR

dysgenetic

+/–

SOX9

17q24-25

AD

+/–

ARX

Xp22.13

XR

dysgenetic or ovotestis dysgenetic

ambiguous to female ambiguous to female ambiguous

Wilms’ tumor, renal abnormalities, gonadal tumors Primary adrenal insufficiency Campomelic dysplasia

ATRX

Xq13.3

XR

dysgenetic

–

HOXA13

7p15

AD

unknown

–

Ambiguous to female Hypospadias

46,XX DSD single gene disorders of gonadal development RSPO1 1p34.3 AR ovotestis

+

ambiguous

WNT-4

–

male

1p35

Inheritance

AR

Gonad

testis

–

46,XY DSD with gene rearrangements, disorders of gonadal development DAX1 Xp21.3 duplication dysgenetic +/– ambiguous to female DMRT1+2 9p24.3 deletion dysgenetic +/– ambiguous to female WNT4 1p35 duplication dysgenetic +/– ambiguous to female

X-linked lissencephaly, epilepsy, temperature instability α-Thalassaemia, mental retardation Hand and foot abnormalities Keratoderma, hearing impairment, corneal opacities Renal, adrenal and pulmonary defects

Mental retardation, renal abnormalities, Mental retardation

46,XX DSD with gene rearrangements, disorders of gonadal development SOX9 17q24-25 duplication unknown – ambiguous to male SRY Yp11.3 translocation testis – ambiguous to female 46,XY DSD disorders of steroid synthesis with associated malformations DHCR7 11q12-13 AR testis –

POR

7q11.2

AR

testis

–

46,XX DSD disorders of steroid synthesis with associated malformations POR 7q11.d AR ovary +

Syndromic DSD

variable ambiguous

male to ambiguous ambiguous

Smith-Lemli-Opitz syndrome with facial abnormalities, clinoand syndactyly, other defect, mental retardation Antley-Bixler syndrome with craniosynostosis Antley-Bixler syndrome with craniosynostosis

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reported in humans. Instead, mutations in these genes can induce various skeletal dysplasias. The association of mental retardation and anomalies of the brain with genital malformations affecting children with a 46, XY karyotype has been reported for two different X-linked disorders, namely the α-thalassemia-mental retardation (ATRX) syndrome (ATRX gene on Xp13) and disorders showing mutations in the aristalessrelated homeobox gene (ARX gene on Xp22). The latter is characterized by a wide phenotypic spectrum including the infantile epileptic-dyskinetic encephalopathy (West syndrome) or a conditon characterized by lissencephaly, hypoplastic corpus callosum and ambiguous genitalia (XLAG syndrome). The association of palmoplantar keratoderma, congenital bilateral corneal opacities, onychodystrophy, hearing impairment and ovotesticular DSD in a child with 46,XX karyotype was found to be caused by a homozygous mutation in R-spondin 1 (RSPO1 gene), which also points out the pivotal role of this gene in gonadal differentiation due to regulation of the Wnt-4 pathway [7]. Wnt-4 and Wnt-7a are signaling molecules found in Müllerian ducts and show XX-specific gonadal expression. In Sertoli and Leydig cells, Wnt-4 overexpression upregulates the dose-sensitive sex-reversal locus on the X chromosome (DAX-1) and this may explain the etiology of human XY sex reversal associated with 1p35-p31 duplication. However, recently a novel autosomal-recessive syndrome (Serkal syndrome) was described that consists of female to male sex reversal and renal, adrenal, and lung dysgenesis and is associated with additional developmental defects. Using a candidate-gene approach, a disease-causing homozygous missense mutation in the human WNT4 gene was identified. The mutation was found to result in markedly reduced WNT4 mRNA levels in vivo and in vitro and to down regulate WNT4dependent inhibition of β-catenin degradation [8]. DAX1 is a gonad-specific transcription factor upregulated in the ovary. Mutations in DAX-1 are responsible for adrenal hypoplasia congenita (AHC), a syndrome of adrenal hypoplasia and hypogonadotropic hypogonadism in 46,XY individuals with an unequivocal male phenotype. DAX1 is said to function as an anti-testis factor in the ovary but is not required for normal testicular function. In contrast, DAX1 duplication can repress SRY and causes 46,XY sex reversal [9]. While the defects of testicular/ovarian-specific transcription factors, such as SRY or DAX-1, are not usually associated with other anomalies, it largely depends on the size and localization of the duplication /deletion that other genes may be also be compromised and, hence, the resulting phenotype is a complex syndrome. Single gene mutations have not been documented as the cause for DSD in the genes DMRT1 and DMRT2; instead the deletion of the genes is often due to distal monosomy of chromosome 9p. Patients with a 46,XY karyotype often have completely female external genitalia, however, also the presence of ovotesticular DSD has been described. A mental retardation of varying degree has been described. Additional described features include severe growth retardation [10].

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Syndromic DSD as a Consequence of Defects in Biochemical Pathways

Two distinct biochemical disorders have been described that present with supplementary malformations in addition to DSD. Antley-Bixler syndrome was initially described a as trapezoidocephaly synostosis syndrome caused by mutations in the fibroblast growth factor receptor gene FGFR2. However, an Antley-Bixler-like phenotype exists, where patients have additional ambiguous genitalia with both 46,XX and 46,XY karyotypes and biochemical features of a combined 21-hydroxylase (CYP21) and 17α-hydroxylase (CYP17) deficiency [11]. Although androgen levels may be low in infants with a 46,XX karyotype after birth, they may be severely virilized and even their mothers can virilize, which pointed to an alternative steroid metabolism during pregnancy. In these cases, Antley-Bixler syndrome is caused by inactivating mutations in P450-oxidoreductase (POR gene), which is a flavoprotein and an electron donor to all microsomal P450 enzymes. Smith-Lemli-Opitz syndrome (SLOS) is an autosomal-recessive disorder with multiple congenital malformations and mental retardation of variable expression. The patients may exhibit microcephaly, hypotonia, facial dysmorphism and ambiguous genitalia. Also, congenital heart defects as well as skeletal abnormalities are found. The latter often consist of cutaneous syndactyly of toes 2 and 3 and postaxial polydactyly. SLOS is due to mutations in the gene encoding for the cholesterol biosynthesis enzyme 7-dehydrocholesterol reductase [12]. Hence, plasma cholesterol may be low, and the substrate 7-dehydrocholesterol is elevated. The biochemical findings, however, do not correlate with the phenotypic expression and severity.

Unclassified Syndromic DSD

A variety of disorders of sex development remain unclassified today and their cause elusive. This includes distinct entities like the Muller-Rokitansky-Kuster-Hauser syndrome with vaginal and uterine aplasia in association with renal malformations in females with a 46,XX karyotype, but also the bladder exstrophy and epispadias complex as well as many other complex malformations involving the genito-urinary tract. In these cases, a distinction has to be made between developmental syndromes involving the morphologic formation of the external and internal genitalia secondary to an overruling malformation of the pelvic region and syndromes that may inhibit genital malformation secondary to gonadal or endocrine involvement as described above. In any case, a stringent and stepwise clinical, endocrine and genetic differential diagnosis has to be established. Employing modern molecular genetic techniques like ultra high resolution CGH, an attractive powerful approach can be offered that can detect minor changes in the genome which could contribute to or be responsible for the phenotype. Using these modern techniques, further syndromes involving the genitor-urinary tract will be elucidated in the near future.

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Hughes IA, Houk C, Ahmed SF, Lee PA: LWPES Consensus Group, ESPE Consensus Group: Consensus statement on management of intersex disorders. Arch Dis Child 2006;91:554–563. Hiort O, Holterhus PM: Molecular and hormone dependent events in sexual differentiation; in Henry A, Norman A (eds): Encyclopedia of Hormones. Los Angeles, Academic Press, 2004, pp 349–356. Clarkson MJ, Harley VR: Sex with two SOX on: SRY and SOX9 in testis development. Trends Endocrinol Metab 2002;13:106–111 Pelletier J, Bruening W, Kashtan CE, Mauer SM, Manivel JC, Striegel JE, Houghton DC, Junien C, Habib R, Fouser L, Fine RN, Silverman BL, Haber DA, Housman D: Germline mutations in the Wilms’ tumor suppressor gene are associated with abnormal urogenital development in Denys-Drash syndrome. Cell 1991;67:437–447 Achermann JC, Ito M, Ito M, Hindmarsh PC, Jameson JL: A mutation in the gene encoding steroidogenic factor-1 causes XY sex reversal and adrenal failure in humans. Nat Genet 1999;22:125–126. Köhler B, Lin L, Ferraz-de-Souza B, Wieacker P, Heidemann P, Schröder V, Biebermann H, Schnabel D, Grüters A, Achermann JC: Five novel mutations in steroidogenic factor 1 (SF1, NR5A1) in 46,XY patients with severe underandrogenization but without adrenal insufficiency. Hum Mutat 2008;29: 59–64

7 Tomaselli S, Megiorni F, De Bernardo C, Felici A, Marrocco G, Maggiulli G, Grammatico B, Remotti D, Saccucci P, Valentini F, Mazzilli MC, Majore S, Grammatico P: Syndromic true hermaphroditism due to an R-spondin1 (RSPO1) homozygous mutation. Hum Mutat 2008;29:220–226. 8 Mandel H, Shemer R, Borochowith ZU, Okopnik M, Knopf C, Indelman M, Drugan A, Tiosano D, Gershoni-Baruch R, Choder M, Sprecher E: SERKAL syndrome: an autosomal-recessive disorder caused by a loss-of-function mutation in WNT4. Am J Hum Genet 2008;82:39–47. 9 Barbaro M, Oscarson M, Schoumans J, Staaf J, Ivarsson SA, Wedell A: Isolated 46,XY gonadal dysgenesis in two sisters caused by a Xp21.2 interstitial duplication containing the DAX1 gene. J Clin Endocrinol Metab 2007;92:3305–3313. 10 Raymond CS, Parker ED, Kettlewell JR, Brown LG, Page DC, Kusz K, Jaruzelska J, Reinberg Y, Fletjer WL, Bardwell VJ, Hirsch B, Zarkower D: A region of human chromosome 9p required for testis development contains two genes related to known sexual regulators. Hum Molec Genet 1999;8:989–996. 11 Arlt W, Walker EA, Draper N, Ivison HE, Ride JP, Hammer F, Chalder SM, Borucka-Mankiewicz M, Hauffa BP, Malunowicz EM, Stewart PM, Shackleton CH: Congenital adrenal hyperplasia caused by mutant P450 oxidoreductase and human androgen synthesis: analytical study. Lancet 2004;363:2128– 2135. 12 Porter FD: Smith-Lemli-Opitz syndrome: pathogenesis, diagnosis and management. Eur J Hum Genet 2008;16:535–541.

Prof. Dr. Olaf Hiort Division of Pediatric Endocrinology and Diabetes Department of Pediatrics, University of Lübeck Ratzeburger Allee 160, DE–23538 Lübeck (Germany) Tel. +49 451 500 2191, Fax +49 451 500 6867, E-Mail [email protected]

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Hiort · Gillessen-Kaesbach

Author Index

Accadia, M. 20 Ballarini, E. 114 Barberi, L. 29 Bergamaschi, R. 114 Bertini, E. 38 Bocciardi, R. 61 Calandra, E. 83 Camacho-Hübner, C. 143 Cerrato, F. 1 Chernausek, S.D. 135 Cicognani, A. 114 Citro, V. 1 Clark, A.J.L. 143 Cubellis, M.V. 1 D’Amico, A. 38 Dattani, M.T. 67 David, A. 143 De Crescenzo, A. 1 di Iorgi, N. 83 Dobrowolny, G. 29 Eggermann, T. 10 Ferrero, G.B. 1 Giacinti, C. 29 Gillessen-Kaesbach, G. 174 Guglielmi, C. 151 Gurrieri, F. 20 Hiort, O. 174 Hussain, K. 95

Kapoor, R.R. 95 Kelberman, D. 67 Larizza, L. 1 Maghnie, M. 83 Main, K.M. 167 Mazzanti, L. 114 Metherell, L.A. 143 Montanari, F. 114 Moscarda, M. 53 Musarò, A. 29 Napoli, F. 83 Neri, G. 53 Pelosi, L. 29 Pozzilli, P. 151 Ravazzolo, R. 61 Riccio, A. 1 Rossi, A. 83 Russo, S. 1 Savage, M.O. 143 Scarano, E. 114 Secco, A. 83 Silengo, M.C. 1 Skakkebæk, N.E. 167 Sparago, A. 1 Tamburrino, F. 114 Toppari, J. 167 Torella, M. 114 Verde, G. 1

James, C. 95

181

Subject Index

ABCC8, hyperinsulinemic hypoglycemia mutations 98, 99, 106 Achondroplasia clinical features 126 etiology 126 growth 126 growth hormone secretion and therapy 126, 127 Acid labile subunit (ALS), defects 147 Alpers-Huttenlocher syndrome, clinical features 47 Alpers syndrome clinical features 42 gene mutations 41 ALS, see Acid labile subunit Angelman syndrome (AS) clinical features 26 epidemiology 26 genetic counseling 27 imprinting regulation 22, 23, 26, 27 Antley-Bixler syndrome, gene mutations 179 AS, see Angelman syndrome ATRX, mutations in disorders of sexual development 178 Bannayan-Riley-Ruvalcaba syndrome (BRRS) clinical features 58 gene mutations 58 Barth syndrome, clinical features 48 BCS1L, mutation in mitochondrial diseases 46 Beckwith-Wiedemann syndrome (BWS) clinical features 2, 3, 53, 54, 97 epidemiology 53 gene mutations 3, 54, 55, 97, 98 hyperinsulinemic hypoglycemia 97–100 imprinting center 1 clinical effects of aberrations 4, 5

182

epigenetic mosaicism 5, 6 hypermethylation 4 IGF2-H19 imprinting loss 6, 7 microdeletions 3, 4 imprinting center 2 defects 98 BMI, see Body mass index Body mass index (BMI), obesity adults 153 children 153, 154 BRRS, see Bannayan-Riley-Ruvalcaba syndrome BWS, see Beckwith-Wiedemann syndrome CACNA1C, Timothy syndrome mutations 105 CDG, see Congenital defects of glycosylation CNP, see C-type natriuretic peptide Congenital defects of glycosylation (CDG), hyperinsulinemic hypoglycemia 96, 97, 106, 107 Costello syndrome gene mutations 102 hyperinsulinemic hypoglycemia 101, 102 COX, see Cyclooxygenase Cryptorchidism environmental and lifestyle risk factors 168–171 prevalence 167, 168 C-type natriuretic peptide (CNP) gene 63 mutations and stature 63 overexpression, growth aberrations, and mechanisms 63–66 signaling 62, 63 Cyclooxygenase (COX), deficiency 40, 41 DAX1, mutations in disorders of sexual development 178

de Toni-Debré-Fanconi syndrome, clinical features 43 DGUOK, mutation in mitochondrial diseases 47 Diabetes double diabetes beta cell autoimmunity 160, 161 diagnosis 161 environmental factors 159, 160 genes 159 latent autoimmune diabetes in the adult 158 prevalence in children 158, 159 prevention 161, 162 treatment 162, 163 obesity association 152, 153 type 1 versus type 2 151, 152 Disorders of sexual development (DSD) biochemical pathway defects and syndromic disorders 179 interdisciplinary approach to syndromic disorders 174, 175 monogenetic syndromic developmental disorders 175–178 unclassified syndromic disorders 179 Double diabetes, see Diabetes DSD, see Disorders of sexual development

growth 121 growth hormone secretion and therapy 121

Ectopic posterior pituitary (EPP) clinical features 85–87 endocrine consequences 90, 91 gene mutations 87–89 idiopathic forms 89, 90 stature implications 92 Ellis-van Crevald syndrome clinical features 129 etiology 129 growth 129 growth hormone secretion and therapy 129 Epigenetics, see also Imprinting reprogramming 21, 22 Silver-Russell syndrome chromosome 11 14, 15 imprinted gene networks 16, 17 EPP, see Ectopic posterior pituitary

Genomic imprinting, see Imprinting GH, see Growth hormone Glycosylation defects, see Congenital defects of glycosylation GPC3, Simpson-Golabi-Behmel syndrome mutations 55 Growth hormone (GH) insensitivity syndromes classification 143, 144 idiopathic short stature 147 insulin-like growth factor-I therapy adverse effects 140, 141 body composition effects 140 growth rates 137, 138 metabolic effects 139, 140 organ growth 138, 139 rationale 135, 136 Laron syndrome 136, 144, 145 receptor mutations dominant negative heterozygous receptor mutation 146 pseudo-exon receptor mutation 145 without Laron syndrome 145 STAT5b mutations 146, 147 treatment strategies 136, 137 secretion and therapy achondroplasia 126, 127 Ellis-van Crevald syndrome 129 Floating-Harbor syndrome 121 hypochondroplasia 128 Kabuki syndrome 122 Leri-Weill syndrome 130 Noonan syndrome 118 Pallister-Hall syndrome 123, 131 Prader-Willi syndrome 24, 25, 120, 131 pseudoachondroplasia 129 Silver-Russell syndrome 124, 125 Turner syndrome 116, 117 therapy benefits 132 complications 132 dosing 132 prospects for study 133

FGFR2, mutations in disorders of sexual development 176 Floating-Harbor syndrome clinical features 120, 121

H19, see Imprinting center 1 HESX1 ectopic posterior pituitary role 88 genotype-phenotype correlations 77

Subject Index

183

HESX1 (continued) hypopituitarism role 67 knockout mouse 85 septo-optic dysplasia mutations 68 HOXA13, mutations in disorders of sexual development 176 HRAS, Costello syndrome mutations 102 Hyperinsulinemic hypoglycemia Beckwith-Wiedemann syndrome 97–100 congenital defects of glycosylation 96, 97, 106, 107 Costello syndrome 101, 102 Kabuki syndrome 102, 103 overview of associated syndromes 96 Simpson-Golabi-Behmel syndrome 101 Sotos syndrome 100 Timothy syndrome 104, 105 transient versus persistent 95, 96 trisomy 13 103 Turner syndrome 103, 104 Usher syndrome 105, 106 Hypochondroplasia clinical features 127 etiology 127 growth 127 growth hormone secretion and therapy 128 IC1, see Imprinting center 1 Idiopathic short stature (ISS), management 147 IGF-1, see Insulin-like growth factor-1 IGF-2, see Insulin-like growth factor-2 Imprinting, see also Epigenetics Angelman syndrome 22, 23, 26, 27 epigenetic reprogramming 21, 22 overview 2, 20, 21 Prader-Willi syndrome 22–26 Imprinting center 1 (IC1) Beckwith-Wiedemann syndrome clinical effects of aberrations 4, 5 epigenetic mosaicism 5, 6 hypermethylation 4 IGF2-H19 imprinting loss 6, 7 microdeletions 3, 4 genes 2 Wilms’ tumor and absence of microdeletions 7, 8 Insulin double diabetes management 163 hyperinsulinemia, see Hyperinsulinemic hypoglycemia

184

Insulin-like growth factor-1 (IGF-1) acid labile subunit defects 147 gene defects 147, 148 gene structure 30, 31 isoforms clinical importance 32, 33 local effects on muscle homeostasis and regeneration 33, 34 transcription 31, 32 transgenic mouse studies of muscular dystrophy and muscle wasting 34, 35 resistance syndromes classification 143, 144 receptor defects 148 structure 30 supplementation rationale 34–36 therapy for growth hormone insensitivity syndromes adverse effects 140, 141 body composition effects 140 growth rates 137, 138 metabolic effects 139, 140 organ growth 138, 139 rationale 135, 136 transcription 30–32 Insulin-like growth factor-2 (IGF-2), see Imprinting center 1 ISS, see Idiopathic short stature Kabuki syndrome clinical features 102, 121 etiology 121 gene mutations 102 growth 121 growth hormone secretion and therapy 122 hyperinsulinemic hypoglycemia 102, 103 KCNJ11, hyperinsulinemic hypoglycemia mutations 98, 99 Kearn-Sayre syndrome (KSS) clinical features 42, 43, 45, 48 gene mutations 40 KSS, see Kearn-Sayre syndrome Laron syndrome, see Growth hormone Latent autoimmune diabetes in the adult, see Diabetes Leber’s hereditary optic neuropathy (LHON) clinical features 49 gene mutations 40 Leigh syndrome clinical features 42, 49, 50

Subject Index

gene mutations 40 Leri-Weill syndrome clinical features 129 etiology 130 growth 130 growth hormone secretion and therapy 130 LHON, see Leber’s hereditary optic neuropathy LHX3 genotype-phenotype correlations 77 hypopituitarism role 67, 74 structure 72, 73 LHX4 ectopic posterior pituitary role 89 genotype-phenotype correlations 77 hypopituitarism role 67, 73, 74 MDDS, clinical features 49 MELAS, clinical features 42, 43, 45 MERRF, see Myoclonus epilepsy and ragged red fibers Metformin, double diabetes management 162, 163 Mitochondrial diseases, see also specific diseases clinical presentation 39 diagnosis 39, 40 DNA abnormalities 38 genetics 40, 41 syndromes in cytopathies blood 48, 49 central nervous system 42, 43 endocrine system 45 gut 44 heart 44, 45 kidney 43, 44 liver 46–48 myopathy 41, 42 skin 49, 50 symptoms 42 MLASA, clinical features 48 MNGIE, clinical features 41, 44 Myoclonus epilepsy and ragged red fibers (MERRF), clinical features 42, 43, 49 Navajo neurohepatopathy (NNH), gene mutations 48 NNH, see Navajo neurohepatopathy Noonan syndrome clinical features 117 etiology 117 growth 118 growth hormone secretion and therapy 118

Subject Index

NR5A1, mutations in disorders of sexual development 176 NSD1, Sotos syndrome mutations 56, 100 Obesity consequences 158 definitions adults 153 children 153, 154 diabetes association 152, 153 etiology 156, 157 prevalence 153 trends in children 154–156 Pallister-Hall syndrome clinical features 122 etiology 122 growth hormone secretion and therapy 123, 131 Pallister-Killian syndrome (PKS) clinical features 59 prenatal diagnosis 59 Patau syndrome, see Trisomy 13 Pearson syndrome clinical features 48, 50 gene mutations 48, 49 PEO, see Progressive external ophthalmoplegia Perlman syndrome clinical features 57 genetics 57, 58 PIT-1, see POU1F1 Pituitary organogenesis 83, 84 posterior lobe development abnormalities 84, 85 ectopic posterior pituitary 85–92 endocrine consequences of disorders 90, 91 phenotypes of developmental syndromes 86 transcription factors, see HESX1, LHX3, LHX4, POU1F1, PROP1, SOX2, SOX3 PKS, see Pallister-Killian syndrome POLG, mutation in mitochondrial diseases 47, 48 POLIP, clinical features 44 POU1F1 genotype-phenotype correlations 77 hypopituitarism role 67, 76–78 mouse mutants 76

185

Prader-Willi syndrome (PWS) clinical features 23–25, 119 epidemiology 23 etiology 119 genetic counseling 27 growth 119 growth hormone secretion and therapy 24, 25, 120 imprinting regulation 22–26 Progressive external ophthalmoplegia (PEO) clinical features 42, 43 gene mutations 41 PROP1 Ames dwarf mouse mutant 74, 75 genotype-phenotype correlations 77 hypopituitarism role 67, 75, 76, 78 recessive inheritance of mutations 75 Pseudoachondroplasia clinical features 128 etiology 128 growth 128 growth hormone secretion and therapy 129 PTEN, Bannayan-Riley-Ruvalcaba syndrome mutations 58 PWS, see Prader-Willi syndrome RSPO1, mutations in disorders of sexual development 178 SCO1, mutation in mitochondrial diseases 46 Septo-optic dysplasia (SOD), HESX1 mutations 68 Sexual development, see Cryptorchidism, Disorders of sexual development SGBS, see Simpson-Golabi-Behmel syndrome SHANK3, deletion 59 Silver-Russell syndrome (SRS) chromosomal aberrations chromosome 7 12, 13 chromosome 11 14, 15 chromosome 15 11, 12 chromosome 17 11, 12 clinical features 11, 123 genetics 11, 123 genotype-phenotype correlations 17, 18 growth 123, 124 growth hormone secretion and therapy 124, 125 imprinted gene networks 16, 17 Simpson-Golabi-Behmel syndrome (SGBS) clinical features 55, 101

186

gene mutations 55 hyperinsulinemic hypoglycemia 101 SLOS, see Smith-Lemli-Opitz syndrome Smith-Lemli-Opitz syndrome (SLOS), gene mutations 179 SOD, see Septo-optic dysplasia Sotos syndrome clinical features 56, 100 gene mutations 56, 100 hyperinsulinemic hypoglycemia 100 SOX2 developmental expression 71 genotype-phenotype correlations 77 hypopituitarism role 67, 71, 72 SOX3 alanine expansions 71 duplication 70 ectopic posterior pituitary role 89 genotype-phenotype correlations 77 hypopituitarism role 67, 70, 71 knockout moue 69, 70 X-linked mental retardation mutations 69 SOX9, mutations in disorders of sexual development 176 SRS, see Silver-Russell syndrome STAT5b, mutations 146, 147 SURF-I, deficiency 40 Timothy syndrome clinical features 104 gene mutations 105 hyperinsulinemic hypoglycemia 105 TK2, mutation in mitochondrial diseases 46, 47 Trisomy 13 clinical features 103 hyperinsulinemic hypoglycemia 103 Turner syndrome clinical features 103, 104, 115 etiology 115 growth 115 growth hormone secretion and therapy 116, 117 hyperinsulinemic hypoglycemia 103, 104 22q13 deletion syndrome clinical features 58, 59 gene mutations 59 UBE3A, Angelman syndrome and loss of function 22 Ucx4.1, null mutation 85

Subject Index

Uniparental disomy (UPD), Silver-Russell syndrome 13 Angelman syndrome 26 Prader-Willi syndrome 25 UPD, see Uniparental disomy Usher syndrome clinical features 105 hyperinsulinemic hypoglycemia 105, 106

Subject Index

Weaver syndrome clinical features 57 gene mutations 57 Wilms’ tumor, imprinting center 1 absence of microdeletions 7, 8 WNT4, mutations in disorders of sexual development 178 WT1, mutations in disorders of sexual development 176

187