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Noonan Syndrome and Related Disorders A Matter of Deregulated Ras Signaling

Monographs in Human Genetics


Vol. 17

Series Editor

Michael Schmid

Wrzburg

Noonan Syndrome and Related Disorders A Matter of Deregulated Ras Signaling


Volume Editor

Martin Zenker

Erlangen

25 figures, 17 in color, and 16 tables, 2009

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

Martin Zenker
Institute of Human Genetics University Hospital Erlangen University of ErlangenNuremberg Schwabachanlage 10 D91054 Erlangen

Library of Congress Cataloging-in-Publication Data Noonan syndrome and related disorders : a matter of deregulated ras signalling / volume editor, Martin Zenker. p. ; cm. -- (Monographs in human genetics, ISSN 0077-0876 ; v. 17) Includes bibliographical references and indexes. ISBN 978-3-8055-8653-5 (alk. paper) 1. Genetic disorders. 2. Ras oncogenes. 3. Ras proteins. I. Zenker, Martin. II. Series. [DNLM: 1. Noonan Syndrome--genetics. 2. Noonan Syndrome--physiopathology. 3. Signal Transduction. 4. ras Proteins--genetics. W1 MO567P v.17 2009 / WD 375 N817 2009] RB155.5.N66 2009 616 .042--dc22 2008035626

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, CH4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel ISSN 00770876 ISBN 9783805586535 e-ISBN 9783805586542

Contents

VII IX

Editorial Schmid, M. (Wrzburg) Preface Zenker, M. (Erlangen) History of Noonan Syndrome and Related Disorders Noonan, J.A. (Lexington, Ky.) The Clinical Phenotype of Noonan Syndrome Allanson, J.E. (Ottawa) Molecular Genetics of Noonan Syndrome Tartaglia, M. (Rome); Gelb, B.D. (New York, N.Y.) Genotype-Phenotype Correlations in Noonan Syndrome Sarkozy, A.; Digilio, M.C.; Marino, B.; Dallapiccola, B. (Rome) LEOPARD Syndrome: Clinical Aspects and Molecular Pathogenesis Sarkozy, A.; Digilio, M.C.; Zampino, G.; Dallapiccola, B.; Tartaglia, M. (Rome); Gelb, B.D. (New York, N.Y.) The Clinical Phenotype of Cardiofaciocutaneous Syndrome (CFC) Roberts, A.E. (Boston, Mass.) Molecular Causes of the Cardio-Facio-Cutaneous Syndrome Tidyman, W.E.; Rauen, K.A. (San Francisco, Calif.) The Clinical Phenotype of Costello Syndrome Kerr, B. (Manchester) The Molecular Basis of Costello Syndrome Sol-Church, K.; Gripp, K.W. (Wilmington, Del.) Endocrine Regulation of Growth and Short Stature in Noonan Syndrome Binder, G. (Tbingen) The Heart in Ras-MAPK Pathway Disorders Digilio, M.C.; Marino, B.; Sarkozy, A.; Versacci, P.; Dallapiccola, B. (Rome)

1 9 20 40 55 66 73 83 94 104 109

119

128 138 151

Myeloproliferative Disease and Cancer in Persons with Noonan Syndrome and Related Disorders Kratz, C. (Freiburg) Neurofibromatosis Type 1-Noonan Syndrome: Whats the Link? Denayer, E.; Legius, E. (Leuven) Animal Models for Noonan Syndrome and Related Disorders Araki, T.; Neel, B.G. (Toronto, Ont.) Towards a Treatment for RAS-MAPK Pathway Disorders Joshi, V.A.; Roberts, A.E.; Kucherlapati, R. (Boston, Mass.) Author Index Subject Index

165 166

VI

Contents

Editorial

This volume 17 of Monographs in Human Genetics is an in-depth discourse on the disorders of the Ras-MAPK pathway (Noonan-, cardio-facio-cutaneous-, Costello-, and LEOPARD syndromes). Like the two preceding volumes of this book series, it deals with important hereditary diseases with high clinical impact, and whose molecular causes have been unravelled in recent years. Noonan syndrome belongs to one of the most frequent monogenic disorders occurring in approximately one in 1,000 to 2,500 children and therefore has significant importance in public health genomics. Molecular analyses have led to the surprising result that all four syndromes can be traced back to specific mutations in genes coding for molecules that interact in the Ras-MAPK pathway. This exciting discovery does not only permit the precise diagnosis of the diseases, but also clears promising ways for potential therapies in the future. Martin Zenker, the Editor of the present volume, succeeded in bringing together the leading experts working on these diseases and received their contributions in a very short space of time. The articles treat both the clinical and molecular data exhaustively and give the reader a very timely update and outline of these related disorders. I thank Martin Zenker and all the authors for their time and effort to render possible the publication of this book. Furthermore, I gratefully acknowledge the constant promotion of this book series by Thomas Karger. Michael Schmid Wrzburg, August 2008

Preface

Noonan syndrome (NS), which is recognized as one of the most common monogenic disorders, was defined as a separate entity by Jacqueline Noonan in 1968. Thirty-three years later, the first gene for NS was identified by Marco Tartaglia and colleagues. Their discovery represented the spark for a series of new gene discoveries eventually showing that mutations that alter the function of molecules interacting in a common signalling cascade, the Ras-MAPK pathway, are responsible for NS and the clinically related disorders cardio-facio-cutaneous syndrome (CFCS), LEOPARD syndrome (LS), and Costello syndrome (CS). Together, these findings unexpectedly related this group of disorders to a signalling pathway which was previously known for its involvement in tumorigenesis. Thereby, the association of certain types of malignancies and tumor-like lesions with NS, LS, CFCS, and particularly CS has been elucidated. Vice versa, studies on the significance of somatic mutations in the same genes in sporadic tumors have been stimulated and yielded exciting new findings. Notably, the genes mutated in Neurofibromatosis 1 and a newly defined Neurofibromatosis 1-like phenotype encode negative regulators of the same pathway. Thus, the known clinical relations between all these conditions have become intelligible through the achievements of molecular research. The Editor of this volume of Monographs in Human Genetics greatly acknowledges the contributions of excellent experts in the field. Their comprehensive reviews provide most updated data on the various clinical and molecular aspects of known disorders of the Ras-MAPK pathway. Jacqueline Noonan herself is giving an historical overview in the first chapter. The book ends with a chapter on current and possible future treatment options for this group of disorders. Together the contributions to this volume nicely show the close relationship between clinical issues and molecular research and the mutual benefit for people working in either of these fields. It is of note that the previously established clinical entities are strongly correlated with certain mutated genes or in the case of LS specific functional consequences of certain mutations. The proposed term neuro-cardio-facial-cutaneous syndromes for all disorders caused by germline mutations in components of the Ras-MAPK pathway may be useful as a superordinate, but currently there is no need to replace the established nosology, which is also used in this book. The content of this volume certainly does not represent a story that has been completed, but it is much more than a progress report. The chase for genes for NS and related disorders seems to have reached a

IX

plateau, although it is obvious that there are still patients who do not have a mutation in the known genes. Following strict diagnostic criteria, the underlying mutation may now be found in more than 80% of patients with NS, 90% of patients with CFCS, and virtually all cases with CS. Future research will reach out for new goals by focusing on the refinement of genotype-phenotype correlations by studying larger cohorts, as well as on the development of model systems to explore the precise molecular pathogenesis of dysregulated Ras-MAPK signaling. One of the most fascinating prospects may be the possibility to invent treatment options for NS and related disorders by pharmacological modulation of Ras-MAPK signaling. Concerted international efforts will be required to reach these goals. Martin Zenker Erlangen, August 2008

Preface

Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 18

History of Noonan Syndrome and Related Disorders


J.A. Noonan
Department of Pediatrics, Division of Cardiology, College of Medicine, University of Kentucky, Lexington, Ky., USA

Abstract
Deregulation of the RAS pathway by some recently discovered germline mutations reveals that this pathway, known to play an impohrtant role in human oncogenesis, also plays an important role in fetal development, cognition and growth. In this volume, the clinical phenotype of Noonan syndrome and related disorders will be reviewed, the genes for these syndromes discussed and possible treatment options will be considered. This chapter will include a brief history of Noonan syndrome and related disorders, including LEOPARD, cardio-facio-cutaneous, Costello and Neurofibromatosis-Noonan syndrome. In addition, speculation as to the possible cause of the distinctive and similar facial phenotypes seen in infancy in these syndromes will be discussed. When Noonan syndrome (NS) was described, it was suspected that a genetic cause would be found. The exciting discovery that mutation of the PTPN11 gene was the cause of NS in about half the cases demonstrated that deregulation of the RAS pathway could cause a variety of congenital malformations. This important discovery showed that the RAS pathway plays a role not only in human oncogenesis but also in fetal development, cognition and growth. This chapter will briefly discuss the history of NS and related disorders including LEOPARD, cardio-facio-cutaneous, Costello and Neurofibromatosis-NS. In addition, speculation as to the possible cause of the distinctive and similar facial phenotypes seen in early infancy in these syndromes will be discussed.
Copyright 2009 S. Karger AG, Basel

Noonan Syndrome (NS)

In 1962, Noonan [1] presented at the Midwest Society for Pediatric Research a clinical study of associated noncardiac malformations in children with congenital heart disease and described nine patients who shared distinctive facial features which included hypertelorism, downslanting palpebral fissures, low set posteriorly rotated ears, ptosis and malar hypoplasia. In addition, most were short in stature, all had pulmonary stenosis and additional deformities included undescended testes and a chest deformity. In 1968 [2], she published these nine and an additional ten patients. Dr. John Opitz [3] proposed the eponym Noonan syndrome be given to this syndrome. He felt that she was the first to describe this condition to occur in both sexes, to be associated with normal chromosomes, to have a congenital heart defect and to be familial in some cases. Several authors have suggested that the first reported patient with what is now called NS was reported by Kobylinski [4] (1883). This was a 20-year-old male who had marked webbing of the neck. It was this feature that seemed to prompt most of the early reports. Funke [5] (1902) reported a patient with a webbed neck as well as short

stature, micrognathia, cubitus valgus and other minor abnormalities. This report was followed by Ullrich [6] (1930) who reported an 8-year-old girl with similar features. Turner [7] (1938) reported older females who had facies similar to Ullrichs but, in addition to short stature, had sexual infantilism. Before Turner syndrome was shown to be a sex chromosome abnormality, Flavell [8] (1943) introduced the term male Turner syndrome. This term led to considerable confusion in the literature for a number of years. Ullrich [9] (1949) reported a series of patients whom he had noted for over two decades. In that study, there was a 4:1 predominance of females over males. He noted the similarity between his patients and mice that had been bred by Bonnevie. Bonnevie was a mouse geneticist who had bred a mutant strain of mice with a webbed neck who also had lymphedema. The term Bonnevie-Ullrich syndrome became popular particularly in Europe. This term was used to describe children, some of whom would now be recognized as having NS while others would be recognized as having Turner syndrome. In 1959, Turner syndrome was found to have a 45, X chromosome pattern. Reports of male Turner Syndrome or Turner phenotype in males were reported throughout the 1960s and 70s. Heller [10] (1965) reviewed 43 cases from the literature and reported five additional cases of his own. These early reports were mainly by endocrinologists who used this term for patients with a variety of testicular problems with or without short stature. A vigorous attempt to find a chromosomal abnormality in the male Turner syndrome was unsuccessful. When chromosome studies became more widely available, it became clear that not all girls previously diagnosed with Turner syndrome had Turner syndrome but, in reality, had NS. Some, but certainly not all of the males previously called male Turner syndrome, fit the clinical description of NS. NS is one of the most common nonchromosomal syndromes seen in children with congenital heart disease.

It occurs worldwide. The estimated incidence of NS is reported to be 1:1,000 to 1:2,500. NS is an autosomal dominant disorder with complete penetrance but variable expressivity. Many cases however are sporadic. Some patients have such a mild phenotype that they are never recognized while individuals with severe manifestations can be recognized as abnormal in early infancy. Allanson et al. [11] made the important observation that the phenotype in NS changes significantly over time. Some cases previously felt to be sporadic were later recognized as familial when photographs of parents taken at a similar age to the affected child were compared. It is not uncommon for NS to be first recognized in a parent after an affected child is born. Noonan reported a high incidence of valvular pulmonary stenosis and noted that the valves were often dysplastic. Ehlers et al. [12], in 1972, reported the first case of hypertrophic cardiomyopathy and this report was followed by Hirsch et al. [13] in 1975. In 1992, Burch et al. [14] demonstrated that the microscopic findings are similar to those seen in nonsyndromic familial hypertrophic cardiomyopathy. In the 1970s, lymphatic problems were reported. Intestinal lymphangiectasia was reported by Vallet et al. [15] in 1972 and pulmonary lymphangiectasis by Baltaxe et al. [16] in 1975. In the 1970s and 1980s, there were several reports describing lymphangiograms showing lymphatic dysplasia. Lymphatic abnormalities are reported in less than 20% of patients but may present serious problems. Fetuses are commonly recognized to have cystic hygroma. Prolonged pleural effusions following heart surgery are common. Some infants are born with hydrops and chylous thorax. This may be difficult to manage and is a cause of death in some severely affected infants. Easy bruising is common in NS. Kitchens and Alexander [17] in 1983, described partial deficiency of Factor XI and others have described deficiencies of Factor VIII and XII as well as thrombocytopenia and platelet dysfunction. In the 1990s, the occurrence of myeloproliferative

Noonan

disorders, including juvenile myelomonocytic leukemia was reported in NS. In 1992 Sharland et al. [18] reported on a large clinical study of patients with NS and discussed the feeding problems which are so often a problem in infancy. He also called attention to the frequent eye findings. Growth hormone studies and the use of growth hormone began to be reported in the 1990s. In 1994 [19], the gene for NS was mapped to the long arm of chromosome 12. One family, however, did not link suggesting more than one gene was involved. The search for the mutant gene began but it was not found until 2001. Tartaglia et al. [20], found a mutation in the PTPN11 gene. This mutation is found in about half of the patients with NS. In the past three years, three additional genes hav e been identified, KRAS, SOS1 and RAF1. It is likely that additional genes will be found to represent the 2530% still without a known mutation. Mutations in the PTPN11 gene have a very high incidence of congenital heart disease of at least 80%. Pulmonary stenosis is most commonly found and there is a low incidence of hypertrophic cardiomyopathy. The most recently identified gene, RAF1, has a high incidence of hypertrophic cardiomyopathy. Patients with SOS1 have some cutaneous findings similar to Cardio-facio-cutaneous syndrome. Genotypephenotype correlations are discussed in a separate chapter of this book.

LEOPARD Syndrome

LEOPARD syndrome is a rare autosomal dominant disorder that shares many phenotypic features with NS. This syndrome was described by Gorlin et al. [21] in 1969. The facial features are similar to NS but in addition there are multiple lentigines and caf-au-lait spots as well as deafness. In 2002, two groups of investigators found PTPN11 mutations in LEOPARD syndrome and demonstrated that LEOPARD syndrome and NS are allelic disorders caused by different missense

mutations in the PTPN11 gene. About 90% of LEOPARD syndrome patients have a PTPN11 mutation but more recently a mutation in RAF1 has been shown to account for the remaining 10%. While NS most frequently has pulmonary stenosis and less commonly hypertrophic cardiomyopathy, LEOPARD syndrome has a very high incidence of hypertrophic cardiomyopathy and a lower incidence of pulmonary stenosis. These syndromes are very difficult to distinguish in infancy since lentigines do not appear until later in childhood and hearing loss may not be apparent in early infancy. Digilio et al. [22] recently reported 10 infants with suspected LEOPARD syndrome. Eight of these were found to have a mutation of the PTPN11 gene confirming the diagnosis of LEOPARD syndrome. They all had facies similar to NS although in some the findings were quite mild. Hypertrophic cardiomyopathy was present in seven of the eight infants and pulmonary stenosis in two of the eight. A single patient without hypertrophic cardiomyopathy had a dysplastic mitral valve. Although only one patient had lentigines, six of the eight did have caf-au-lait spots. This suggested to Digilio that caf-au-lait spots in early infancy in a patient with hypertrophic cardiomyopathy should strongly suggest the possibility of LEOPARD syndrome. It is of interest that, of the remaining two patients, one patient did have neurofibromatosis but had a Noonan phenotype. The second patient did not have a mutation of the PTPN11 gene. It will be interesting to see if that patient has a RAF1 mutation. LEOPARD syndrome is another example where the overlap between neurofibromatosis and NS exists.

Cardio-Facio-Cutaneous Syndrome (CFC)

CFC is a multiple anomaly syndrome with significant mental retardation. It occurs sporadically and is characterized by failure-to-thrive, macrocephaly, a distinctive face similar to NS with

History of Noonan Syndrome and Related Disorders

bitemporal constriction, hypertelorism, downward slanting palpebral fissures, depressed nasal root and low set ears. There is usually significant cutaneous involvement consisting of dry hyperkeratotic scaly skin, sparse or absent eyebrows and sparse or absent eyebrows and sparse slow growing curly hair. This syndrome was first described 20 years ago by Reynolds et al. [23] who described eight patients whom they felt represented a distinct syndrome. These reports were followed by considerable controversy in the literature. Many questioned whether CFC was a unique and separate condition or a variant of NS. Unlike NS, CFC is quite rare. About 100 cases have been confirmed so far. Although the facies are similar to NS in infancy, at older age, the face remains broad and coarse and does not develop the inverted triangular shape seen in NS. Cutaneous manifestations are prominent but may overlap with NS, especially NS with the SOS1 mutation. In 2002, Kavamura et al. [24] published a CFC index to aid in the diagnosis of this syndrome. Fortunately, in 2006, two groups of investigators found BRAF as well as MEK1, MEK2 and occasional KRAS mutations to be responsible for CFC. Earlier it has been determined that patients with CFC did not have a mutation in the PTPN11 gene so that by now it is clear that CFC is distinct from NS. The most common mutated gene appears to be BRAF followed by MEK1 and MEK2 and occasional patients with a KRAS mutation. Like NS, cardiovascular malformations are frequent. About 75% [25] have some kind of a cardiac malformation. Forty-five percent of those are pulmonary stenosis and 40% hypertrophic cardiomyopathy.

Costello Syndrome

Costello syndrome is a rare disorder. It was first described by Dr. Costello in 1971 [26] at a meeting. He described two patients with mental

retardation, high birth weight, feeding problems, coarse facies, nasal papillomata and loose integument of the back of the hands. In 1977 [27], he published these two cases in more detail. Following that, a number of authors reported patients who displayed the phenotype described by Costello but they were unaware of Costellos report. DerKaloustian et al. [28], first used the term Costello syndrome in 1991. In 1992, Johnson et al. [29] reported eight patients with Costello syndrome and reviewed 29 cases that had been previously reported under a variety of titles who undoubtedly also had Costello syndrome. These patients have a distinctive facial appearance which may be difficult to distinguish from NS and CFC in infancy. In 1994, Lurie [30] wrote that Costello was likely a sporadic autosomal dominant mutation. By 1991, malignancies were being reported in Costello patients, particularly bladder carcinoma and rhabdomyosarcoma. Costello syndrome is characterized by polyhydramnios, overgrowth and edema with postnatal feeding difficulties and failure-to-thrive. Characteristic facial features include macrocephaly, a high forehead, usually curly hair, hypertelorism, fleshy nasal tip, full lips, wide mouth, full cheeks and fleshy earlobes. In infancy, there is excessive skin wrinkling. The skin appears very loose. There is postnatal growth retardation and developmental delay. The characteristic skin disorder in Costello syndrome suggested that there might be an elastin fiber abnormality. Hinek et al. [31] in 2000 found that fibroblasts from Costello syndrome were able to produce normal levels of tropoelastin and to properly position the microfibrillar scaffold but they were unable to assemble elastin fibers because of a deficiency in the elastin binding protein. In addition, they found fibroblast cultures from Costello syndrome patients showed an increased rate of proliferation. They postulated that disturbed elastogenesis could explain the interesting skin findings and that increased proliferation of fibroblasts in tissue culture might explain

Noonan

the increased tumor rate in Costello syndrome. It now makes sense that deregulation of the RAS pathway could explain the functional deficiency of the 67-kDa elastin binding protein (EBP) proposed by Hinek. In 2005 [32], Aoki et al. reported that germline mutations in HRAS caused Costello syndrome. This finding was soon confirmed by a number of other investigators. Although this is a rare disease, as soon as the gene for Costello was discovered, 40 samples of DNA from Costello patients were available for confirmation. This DNA was available from patients who had been clinically diagnosed with Costello syndrome at the 2003 and 2005 International Costello Syndrome Meeting and through the Costello Syndrome Family network. Of the 40 patients with the clinical diagnosis of Costello syndrome, 33 were confirmed to have the HRAS mutation. Since it is often difficult to distinguish between CFC and Costello, it is not surprising that seven patients suspected of having Costello syndrome had mutations in either BRAF, KRAS, MEK1 or MEK2 which confirmed the phenotypic overlap between these disorders. It is now felt that the term Costello syndrome should be limited to those individuals who do have a mutation of the HRAS gene. Similar to NS and CFC, cardiovascular malformations are frequent, occurring in about 50% and include pulmonary stenosis in about 40% and hypertrophic cardiomyopathy in another 40%. Unlike NS or CFC, chaotic atrial arrhythmias are relatively frequent in Costello syndrome, particularly in infancy.

Neurofibromatosis-Noonan Syndrome (NF-NS)

have been recognized as the cause of neurofibromatosis Type 1. The NF1 gene product acts as a negative regulator to the RAS mediated signal transduction pathway. This finding provided the first direct evidence that the RAS pathway played an important role in human development. NF1 has a prevalence of about 1:3,000. Colley et al. [33] examined 94 patients with NF1 and found that 9.5% had findings that seemed similar to NS. It appears clear that there is a clinical overlap between both syndromes. So far, the etiology of NF-NS is unclear. There has been one report [34] of a patient showing features of both syndromes who was found to have two mutations, a PTPN11 mutation which was inherited from the father and a de novo NF1 mutation. This is the first and only report so far of molecular occurrence of both disorders in the same patient. Huffmeier et al. [35] recently reported seven patients from five unrelated families with variable phenotypes of the NF1-NS syndrome spectrum. Heterozygous mutations or deletions of NF1 were identified in all patients. No PTPN11 mutation was found. The NF1 mutation segregated with the phenotype in both familial cases. They felt this supported the hypothesis that variable phenotypes of the NF1-NS spectrum represent variants of NF1 mutation in the majority of cases. The NF1-NS facial phenotype is similar to NS but usually quite mild. Short stature is not common. Cardiac defects are less common but pulmonary stenosis is reported as well as a variety of other cardiac defects. Hypertrophic cardiomyopathy is also seen. Digilio et al. [22] as discussed under LEOPARD syndrome have reported the difficulty in distinguishing between LEOPARD syndrome and neurofibromatosis in infancy.

Since the mid 1980s, a number of investigators have written about the presence of Noonan phenotype associated with some patients with neurofibromatosis. Neurofibromatosis Type 1 (NF1) is an autosomal dominant disorder characterized by hamartomas in multiple organs. Mutations or deletions in the neurofibromin-1 gene (NF1)

Discussion

Bentires-Alj et al. [36] suggest that the phenotype overlap between NS, NF1 and the other related syndromes reflects a similar underlying

History of Noonan Syndrome and Related Disorders

pathogenesis, namely deregulation of the RAS pathway and proposed that all these syndromes be called Neuro-cardio-facio-cutaneous (NCFC) syndrome. There are clearly many phenotypic similarities in infancy in these syndromes. The facies typically show low set ears, downward slanting eyes and a short neck. Polyhydramnios is common and cystic hygroma is sometimes noted by fetal ultrasound. It is tempting to blame the cystic hygroma as a likely cause of the facial phenotype. However, an interesting study by Achiron et al. [37], causes some doubt at this explanation. They propose that NS has an evolving phenotype during in utero and postnatal life. Among 46,224 live born infants only seven newborn and four fetuses were found to have NS while some 3040 NS would be expected. Unlike Bekker et al. [38] who found cervical cystic hygroma in midtrimester to be a reliable sign for in utero diagnosis of NS Achiron noted none of his cases had evidence of septated cystic hygroma and only one of the fetuses had transient nuchal translucency. This observation indicates lymphatic abnormalities are not a sine qua non for a prenatal diagnosis of NS. Since the great majority of patients with these syndromes do not have nuchal translucency in utero it is necessary to propose another explanation for the typical facial phenotype. Some infants with NS and related disorders are clearly recognized as dysmorphic at birth. The four fetuses in Achirons report all developed bilateral hydrothorax and generalized edema. All had typical facies of NS. All were very ill and two died in the neonatal period. The seven infants diagnosed at birth or in early infancy had typical clinical findings of NS. This suggests that the other 30 to 40 NS expected among the 46,224 newborn delivered were mild enough to be unrecognized at least through the first year of life. It is not uncommon for a diagnosis of NS to be delayed past five to six years of age and sometimes into adulthood. Is it possible that the facial phenotype becomes more typical with time demonstrating that the RAS pathway continues to play a role in an evolving phenotype?

The RAS pathway must play a role in this common facial phenotype but it is still unknown. On the other hand it is also clear that the RAS pathway must play a role in early lymphatic development. Lymphatic problems are well recognized in NS. Chylous thorax may be present at birth or appear spontaneously later on or be a complication following heart surgery. Pulmonary and intestinal lymphangiectasia have been reported. Some cases occur in infancy but may be delayed until adulthood. Lymphedema may present for the first time in adulthood. Bloomfield et al. [39] reported the first neonatal case with lymphangiography. The infant was born at 33 weeks with severe edema and bilateral pleural effusions which proved to be chylous. The fluid was drained and the baby improved. On day 14 a lymphangiogram was carried out from the right foot. The six lymphatics that filled were dilated and saccular. There was no filling of lymph nodes in the groin or extension into the pelvic or para-aortic lymphatics or thoracic duct. Clearly the lymphatic system was dysplastic suggesting very abnormal development. Unfortunately the infant had two episodes of viral pneumonia and died at 5 months of age. I have personal knowledge of another infant with persistent chylous thorax who underwent lymphangiography. This demonstrated absence of the thoracic duct with multiple lymphatics draining directly into the chest cavity. There is need for further study of the role of RAS in lymphatic development. While lymphatic abnormalities are clinically recognized in only 20% of NS patients, they may be unrecognized clinically in many more. Few studies of the lymphatics have been carried out but the lymphangiograms so far performed have shown significant lymphatic abnormalities.

Conclusion

It is very exciting for me to learn that NS and these related disorders disturb the RAS pathway, demonstrating that this pathway plays an important role in development. Once we understand exactly

Noonan

how these mutations alter the pathway, it may be possible to develop strategies to treat at least the postnatal effects such as short stature and hypertrophic cardiomyopathy, to name a few. Already basic scientists are making important progress in understanding the effect of specific mutations on human development. Krenz et al. [40] have shown that a mutation of Q79R-Shp2 in NS results in increased activity of the extra cellular signal-regulated (ERK)1/2 and this results in hyperproliferation in valve primordia. Nakamura et al. [41] then generated a transgenic Q79R-Shp2 mouse model which showed again the role of enhanced ERK1/2 in cardiac malformation. They were able to prevent cardiac abnormalities by ERK1/2 modulation. Gauthier et al. [42] have demonstrated the important role of Shp2 in brain development. The normal Shp2 instructs cell precursors to make neurons and not astrocytes during the neurogenic period of development. A mouse knockin mutant Shp2 model is a phenocopy of human NS. This model was shown to inhibit basal neurogenesis and caused enhanced astrocyte formation. It will be very important for these patients to have continued long-term follow-up as they age. Follow-up of adults with syndromes is very
References
1 Noonan JA: Hypertelorism with Turner phenotype. Am J Dis Child 1968;116:373380. 2 Noonan JA, Ehmke DA: Associated noncardiac malformations in children with congenital heart disease. J Pediatr 1963;31:150153. 3 Opitz JM: Editorial comment: the Noonan syndrome. Am J Med Genet 1985;21:515518. 4 Kobylinski O: ber eine flughauthnliche Ausbreitung am Halse. Arch Anthropol 1883;14:342348. 5 Funke O: Pterygium colli. Dtsch Zeitschr Chir 1902;63:163167.

difficult. Registries to follow these patients will be important. In countries with established registries, it will be essential that these patients be followed long-term. As we follow these adults, we may be able to identify what role the RAS pathway plays in aging. There is plenty of exciting knowledge awaiting investigators as we continue to learn more about the RAS pathway. In the United States, without national registries, three support groups founded by mothers of affected children could play a role. The NS Support Group, Costello Family Network and Cardio-facio-cutaneous International are the three support groups. They hold international meetings every one to two years and families attend with their affected children. At these meetings, information about the syndrome is shared with families and the physicians attending always learn much from the families. The children are able to interact with affected peers which provides a lot of support. These groups have or are in the process of establishing registries which could play a very important role in long-term followup of patients with all these syndromes. Little is known of the natural history of these syndromes. These mutations likely continue to exert an effect on the RAS pathway throughout life.

6 Ullrich O: ber typische Kombinationsbilder multipler Abartungen. Z Kinderheilkd 1930;49:271276. 7 Turner HH: A syndrome of infantilism, congenital webbed neck, and cubitus valgus. Endocrinology 1938;25:566574. 8 Flavell G: Webbing of the neck with Turners syndrome in the male. Br J Surg 1943;31:150153. 9 Ullrich O: Turners syndrome and status Bonnevie-Ullrich; synthesis of animal phenogenetics and clinical observations on a typical complex of developmental anomalies. Am J Hum Genet 1949;1:179202.

10 Heller RH: The Turner phenotype in the male. J Pediatr 1965;66:4863. 11 Allanson JE, Hall JG, Hughes M: Noonan syndrome: the changing phenotype. Am J Med Genet 1985;21: 507514. 12 Ehlers KH, Engle MA, Levin AR, Deely WJ: Eccentric ventricular hypertrophy in familial and sporadic instances of 46, XX, XY Turner phenotype. Circulation 1972;45:639652. 13 Hirsch HD, Gelband H, Garcia O, Gottlieb S, Tamer DM: Rapidly progressive obstructive cardiomyopathy in infants with Noonans syndrome. Circulation 1975;52:11611165.

History of Noonan Syndrome and Related Disorders

14 Burch M, Mann JM, Sharland M, Shinebourne EA, Patton MA, McKenna WJ: Myocardial disarray in Noonan syndrome. Br Heart J 1992;68: 580585. 15 Vallet HL, Holtzapple PG, Eberlein WR, Yakovac WC, Moshang T Jr, Bongiovanni AM: Noonan syndrome with intestinal lymphangiectasia. J Pediatr 1972;80:269274. 16 Baltaxe HA, Lee JG, Ehlers KH, Engle MA: Pulmonary lymphangiectasia in 2 patients with Noonan syndrome. Radiology 1975;155:149153. 17 Kitchens CS, Alexander JA: Partial deficiency of coagulation factor XI as a newly recognized feature of Noonan syndrome. J Pediatr 1983;102:224227. 18 Sharland M, Burch M, McKenna WM, Patton MA: A clinical study of Noonan syndrome. Arch Dis Child 1992;67: 178183. 19 Jamieson CR, van der Burgt I, Brady AF, van Reen M, Elsawi MM, et al: Mapping a gene for Noonan syndrome to the long arm of chromosome 12. Nat Genet 1994;8:357360. 20 Tartaglia M, Kalidas K, Shaw A, Song X, Musat DL, et al: PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation and phenotypic heterogeneity. Am J Hum Genet 2002;70:15551563. 21 Gorlin RJ, Anderson RC, Blaw M: Multiple lentigenes syndrome. Am J Dis Child 1969;17:652662. 22 Digilio MC, Sarkozy A, de Zorzi A, Pacileo G, Limongelli G, et al: LEOPARD Syndrome: clinical diagnosis in the first year of life. Am J Med Genet A 2006;140:740746. 23 Reynolds JF, Neri G, Herrmann JP, Blumberg B, Coldwell JG, Miles PV, Opitz JM: New multiple congenital anomalies/mental retardation syndrome with cardio-facio-cutaneous involvement the CFC syndrome. Am J Med Genet 1986;25:413427. 24 Kavamura MI, Peres CA, Alchorne MM, Brunoni D: CFC index for the diagnosis of cardiofaciocutaneous syndrome. Am J Med Genet 2002;112:1216.

25 Roberts A, Allanson J, Jadico SK, Kavamura MI, Noonan J, et al: The cardiofaciocutaneous syndrome. J Med Genet 2006;43:833842. 26 Costello JM: A new syndrome. NZ Med J 1971;74:397. 27 Costello JM: A new syndrome: mental subnormality and nasal papillomata. Aust Paediatr J 1977;13:114118. 28 Der Kaloustian VM, Moroz B, McIntosh N, Watters AK, Blaichman S: Costello syndrome. Am J Med Genet 1991; 41:6973. 29 Johnson JP, Fried MH, Norton ME, Rosenblatt R, Feldman G, Yang S: Prenatal overgrowth with postnatal growth failure, dysmorphic facies, cutaneous features, and cardiomyopathy: overlap of AMICABLE, facio-cutaneous-skeletal (fcs) and Costello (cs) syndromes. Proc Greenwood Genet Cent 1992;12:98. 30 Lurie JW: Genetics of the Costello syndrome. Am J Med Genet 1994;52:358359. 31 Hinek A, Smith AC, Cutiongco EM, Callahan JW, Gripp KW, Weksberg R: Decreased elastin deposition and high proliferation of fibroblasts from Costello syndrome are related to functional deficiency in the 67-kD elastinbinding protein. Am J Hum Genet 2000;66:859872. 32 Aoki Y, Niihori T, Kawame H, Kurosawa K, Ohashi H, et al: Germline mutations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 2005;37:10381040. 33 Colley A, Donnai D, Evans DG: Neurofibromatosis/Noonan phenotype: a variable feature of type 1 neurofibromatosis. Clin Genet 1996;49:5964. 34 Bertola DR, Pereira AC, Passetti F, de Oliveira PS, Messiaen L, et al: Neurofibromatosis-Noonan syndrome: molecular evidence of the concurrence of both disorders in a patient. Am J Med Genet A 2005;136:242245.

35 Huffmeier U, Zenker M, Hoyer J, Fahsold R, Rauch A: A variable combination of features of Noonan syndrome and neurofibromatosis type I are caused by mutations in the NF1 gene. Am J Med Genet A 2006;140: 27492756. 36 Bentires-Alj M, Kontaridis MI, Neel BG: Stops along the RAS pathway in human genetic disease. Nat Med 2006;12:283285. 37 Achiron R, Heggesh J, Grisaru D, Goldman B, Lipitz S, Yagel S, Frydman M: Noonan syndrome: a cryptic condition in early gestation. Am J Med Genet 2000;92:159165. 38 Bekker MN, Go AT, van Vugt JM: Persistence of nuchal edema and distended jugular lymphatic sacs in Noonan syndrome. Fetal Diagn Ther 2007;22:245248. 39 Bloomfield FH, Hadden W, Gunn TR: Lymphatic dysplasia in a neonate with Noonans syndrome. Pediatr Radiol 1997;27:321323. 40 Krenz M, Yutzey KE, Robbins J: Noonan syndrome mutation Q79R in Shp2 increases proliferation of valve primordia mesenchymal cells via extracellular signal-regulated kinase 1/2 signaling. Circ Res 2005;97:813820. 41 Nakamura T, Colbert M, Krenz M, Molkentin JD, Hahn HS, Dorn GW 2nd, Robbins J: Mediating ERK 1/2 signaling rescues congenital heart defects in a mouse model of Noonan syndrome. J Clin Invest 2007;117:21232132. 42 Gauthier AS, Furstoss O, Araki T, Chan R, Neel BG, Kaplan DR, Miller FD: Control of CNS cell-fate decisions by Shp2 and its dysregulation in Noonan syndrome. Neuron 2007;54:245262.

Jacqueline A. Noonan Department of Pediatrics, Division of Cardiology, College of Medicine, University of Kentucky 800 Rose Street, MN470 Lexington, KY 40536 (USA) Tel. +1 859 323 5494, Fax +1 859 323 3499, E-Mail jnoonan@uky.edu

Noonan

Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 919

The Clinical Phenotype of Noonan Syndrome


J.E. Allanson
Childrens Hospital of Eastern Ontario, Ottawa, Canada

Abstract
Noonan syndrome is an autosomal dominant condition notable both for its common occurrence and phenotypic variability. It is characterized by short stature, congenital cardiac defects, unusual chest shape, broad or webbed neck, cryptorchidism, typical facial appearance and developmental delay of variable extent. It is frequently overlooked in the mildly affected individual and diagnosis in an adult often follows the birth of a child with more florid manifestations.
Copyright 2009 S. Karger AG, Basel

included hypertrophic cardiomyopathy, ischemic heart disease, breast cancer, and cerebral hemorrhage [5]. There are several excellent reviews [1, 39].

Craniofacial Features

The cardinal features of Noonan syndrome are short stature, congenital heart defects, broad or webbed neck, characteristic pectus deformity, a particular facial appearance which changes with age and, in some cases, mild intellectual handicap. This pattern of features was recognized and reported more than 40 years ago by Noonan and Ehmke [1], however it is likely that Kobylinski, in 1883, was the first to publish on the condition [2]. Birth prevalence is estimated to be between 1/1,000 and 1/2,500 although mild expression is said to occur in 1 in 100 [3]. Average age at diagnosis is 9 years [4]. Life expectancy is likely to be normal in the absence of serious cardiac defects. In one natural history study, non-accidental mortality was 7%, with half of all deaths occurring in adulthood. Cause of adult death

Facial appearance changes with age (fig. 1) [4, 10]. In the newborn, key features include tall forehead, widespaced and down-slanting palpebral fissures, ptosis or thickened eyelids, epicanthal folds, depressed nasal root with upturned nasal tip, deeply grooved philtrum with high, wide peaks of the vermilion border (so-called cupids bow shape), low-set and posteriorly angulated ears with thick helices, small chin, and excessive nuchal skin with a low posterior hairline. During infancy, the head is relatively large in comparison to face size, with a tall and prominent forehead. Hypertelorism, ptosis or thick hooded eyelids remain characteristic. The nose is short and wide with a depressed root. During later childhood, the face may appear coarse or even myopathic. With increasing age, the face lengthens and becomes more triangular in shape with a broad forehead tapering to a small and

Fig. 1. Female with Noonan syndrome at different ages, showing how facial features change with time. (a) Baby, (b) child and (c) young adult.

pointed chin. In adolescence and young adulthood, the nose has a thin, prominent bridge and a wide base. The neck is longer with accentuated webbing (pterygium colli) or a prominent trapezius. In older adults, nasolabial folds are exaggerated and the skin appears thin and transparent [6, 10, 11]. The hair may be wispy or sparse during infancy and curly or woolly in older childhood and adolescence. Despite this subjective impression of age-related facial change, detailed measurements demonstrate the opposite. There is a Noonan-specific pattern of craniofacial widths, lengths, depths and arcs that is maintained over time. This is superimposed on normal changes that occur in face shape/size with age and is perceived as a change in gestalt [12]. Features likely to be seen irrespective of age include blue-green irides, frequently out of keeping with family eye colour, arched or diamond-shaped eyebrows, and low-set posteriorly angulated ears with thickened helices [4, 6]. Malocclusion is common and likely is related to the small chin and oral cavity [6, 10]. Relative or absolute macrocephaly is usual. Mean adult head circumference in males is 56.4 cm and in females is 54.9 cm [5].

Cardiovascular Anomalies

Congenital heart defects occur in between 50 and 90% of affected individuals [6, 13]. Since this feature may prompt diagnosis, and because many published reports come from tertiary and quaternary medical centres which place emphasis on serious structural manifestations, there may be bias of ascertainment. The most common anomaly, seen in up to half, is a dysplastic and/or stenotic pulmonary valve [5, 8, 1315]. It may be isolated or associated with other defects. Other common structural cardiac anomalies include atrial or ventricular septal defects and tetralogy of Fallot. Many other cardiac defects have been reported less commonly, including atrioventricular septal defect, aortic stenosis or dysplasia, coarctation of the aorta [1618], bicuspid aortic valve, double chambered right ventricle, mitral valve anomalies [19], Ebstein anomaly, total anomalous pulmonary venous return, supravalvular pulmonary stenosis, coronary artery dilatation, coronary artery fibromuscular dysplasia causing ischemia [20], and giant aneurysms of the sinuses of Valsalva caused by deficiency of medial elastin [21].

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Hypertrophic cardiomyopathy, both obstructive and non-obstructive, occurs in 20 33% [5, 13, 14, 18, 22, 23]. Hypertrophy may be mild or severe, and may present before or at birth, in infancy or childhood. It is histologically, echocardiographically and clinically indistinguishable from non-syndromic hypertrophic cardiomyopathy, except that arrhythmia and sudden death appear to be less common. Nonetheless, mortality appears to be higher in the children with Noonan syndrome and hypertrophic cardiomyopathy, with progression to cardiac failure in 25% [5]. Restrictive cardiomyopathy and dilated cardiomyopathy are reported but uncommon [24 26]. The electrocardiogram is abnormal in almost 90%. Extreme right axis deviation with superior counter-clockwise frontal QRS loop is likely related to asymmetric septal hypertrophy. Left axis deviation may occur secondary to a conduction abnormality; there may be left anterior hemiblock or an RSR pattern in lead V1. Abnormal findings may occur in a structurally normal heart. In older individuals, arrhythmia and congestive cardiac failure may be more common than previously suspected [27].

Growth and Feeding

Birth weight is usually normal but may be increased due to subcutaneous edema. In this situation there is rapid loss of weight in the neonatal period. Feeding difficulties occur in 77% of infants [4, 5, 28], and may be mild (15%), characterized by poor suck, or severe (38%), requiring tube feeding [5]. They are usually related to hypotonia and poor coordination of oral musculature, however immature gut motility and delayed gastrointestinal motor development are documented in some individuals [28]. Failure to thrive occurs in 40%. It is self-limited and usually resolves by 18 months of age.

Average birth length is 47 cm. Childhood growth tends to follow the general population third centile, with normal growth velocity. The pubertal growth spurt is frequently reduced or absent. Delayed bone maturation is common and allows prolonged growth potential into the 20s. Average adult height in males is 162.5 169.8 cm and in females is 152.7153.3 cm [5, 29]. Noonan syndrome growth curves are published [29, 30]. Growth hormone production is usually normal but a variety of physiological abnormalities are described which may or may not have consequences for growth or responsiveness to growth hormone [31]. One study suggested that children with more f lorid facial, thoracic and cardiac features of Noonan syndrome had higher peak growth hormone levels [32]. These children did not seem to differ in pre- or post-growth hormone treatment height when compared to children with a milder Noonan syndrome phenotype. Genotype was not reported but may be germane, because higher spontaneous and stimulated growth hormone secretion has been noted in children with PTPN11 mutations [33]. There is a growing body of lite rature on the use of growth hormone therapy in Noonan syndrome [3438]. Growth velocity is clearly enhanced in the first year of treatment, and, to a lesser extent, in year 2. Growth velocity gradually seems to fall after three years of treatment. The accelerating effect on bone maturation may compromise final height prognosis, although gain in height of 1 SD appears to be sustained. Several studies show improvement in intermediate and final adult height [36, 39, 40]. Use of growth hormone treatment also varies from country to country. Considerable enthusiasm for use remains in the United States. In Canada, growth hormone is only prescribed if growth hormone deficiency is proven. Response to growth hormone therapy may be better in those individuals without PTPN11 mutations [33, 41, 42]. The inferior response to growth hormone, greater likelihood of short stature, and

The Clinical Phenotype of Noonan Syndrome

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higher (compensatory) growth hormone levels in individuals with a PTPN11 mutation may be explained by the fact that SHP2 normally downregulates growth hormone receptor signaling. Gain-of-function mutations in PTPN11 will enhance this effect [33, 43].

Development and Behaviour

Early developmental milestones are often delayed, with average age of sitting at 10 months, first unsupported walking at 21 months and simple two-word phrases at 31 months. Joint laxity and hypotonia clearly contribute to the motor delay. Most children will do well in a normal school setting but 1040% will require additional help [4]. A large cohort of affected individuals, followed for many years, has demonstrated a strong association between significant feeding difficulties in infancy and intellectual handicap requiring special education [5]. Mild mental retardation occurs in up to 35%, however, IQ ranges from 64 to 127 [3, 4, 6, 44]. In one study of 48 affected British children, detailed psychometric testing demonstrated a mean fullscale IQ of 84 and 25% likelihood of learning disability [45]. Verbal IQ was slightly higher than performance IQ. Mild to moderate clumsiness and coordination problems were noted in about half the children. Other publications report learning disability with specific visual-constructional problems and verbalperformance discrepancy [44, 46, 47], language delay [4] and strengths in abstract reasoning and social awareness [48]. Studies of behaviour in Noonan syndrome have been somewhat contradictory. One study has suggested an increased likelihood of stubbornness and mood disorders [49]. Another found a majority of a group of 26 individuals to be impulsive, hyperactive and irritable [47]. A more recent study of 48 affected children has shown good self-esteem and has failed to identify a behavioral phenotype [45]. Notably few children are reported with autism,

sleep difficulties, severe aggression or anxiety [50]. Few details of psychological health are reported. In a cohort of 51 adults, 23% had depression and there was occasional substance abuse and bipolar disease [27]. In males, short stature, hypotonia and reduced athleticism appeared to be predisposing factors. Similar findings were not reported by Shaw et al. [5] although this natural history study had few questions on self-esteem and mental health. Detailed psychological assessment of 10 young adults demonstrated variable levels of intelligence and suggests moderate impairment of social cognition in terms of emotion recognition and alexithymia. In some individuals there were mild signs of anxiety and lowered mood. Key elements of this behavioral phenotype are deficiencies in social and emotional recognition and expression [51].

Ocular Anomalies

Ocular anomalies are among the most common findings in Noonan syndrome and have been well studied in two large cohorts [5, 52]. Strabismus and refractive errors are present in a majority, amblyopia in about one third, and nystagmus in about 10%. Anterior segment changes (prominent corneal nerves, anterior stromal dystrophy, cataracts, and panuveitis) are frequently found, while coloboma [52, 53], retinitis pigmentosa [54], congenital fibrosis of extraocular muscles [55] and spontaneous corneal rupture [53, 56] are rare associations. Optic nerve hypoplasia is occasionally reported, in contrast to cardiofaciocutaneous syndrome, which shares many characteristics with Noonan syndrome, and in which optic nerve hypoplasia appears fairly common.

Hearing

The hearing loss described in Noonan syndrome is usually a mild conductive loss secondary to recurrent otitis media, however sensorineural and

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mixed hearing loss, though quite rare, do occur [5, 57]. Qui et al. [58] found progressive high tone loss in 50% of 20 affected individuals. Temporal bone anomalies are reported [59].

Musculoskeletal Findings

The thorax usually displays pectus carinatum superiorly and pectus excavatum inferiorly due to precocious closure of sternal sutures (fig. 2). The chest is also broad with wide-spaced nipples. Shoulders are rounded and the upper chest appears long; with low-set nipples and axillary webbing. This chest phenotype provides a good clue to diagnosis. Cubitus valgus, brachydactyly and blunt fingertips are frequently found. There are less common reports of talipes equinovarus, joint contractures, scoliosis, vertebral and rib anomalies, and radio-ulnar synostosis. Joint hyper-extensibility occurs in 30% [4, 5]. The association between Noonan syndrome and malignant hyperthermia is poorly understood. Malignant hyperthermia has been linked to a Noonan phenotype and designated as King syndrome [6064]. The possibility of malignant hyperthermia is of greater concern in individuals with significant muscular pathology or elevated creatine kinase. Giant cell lesions of the jaws identical to those found in cherubism are described [6571]. This combination has been called Noonan-like/multiple giant-cell lesion syndrome. Cherubism may occur as an isolated autosomal dominant disorder caused by mutations in SH3BP2 [72], or as part of neurofibromatosis. In Ramon syndrome cherubism is associated with juvenile rheumatoid arthritis (polyarticular pigmented villonodular synovitis). Giant cell granulomas, and bone and joint anomalies that include polyarticular pigmented villonodular synovitis, are now recognized to be part of the Noonan syndrome spectrum, and have been reported in individuals with PTPN11 and KRAS mutations [66, 73, 74]. Polyarticular pigmented villonodular synovitis

Fig. 2. The chest phenotype showing wide-spaced and low-set nipples, pectus deformity with pectus carinatum superiorly and pectus excavatum inferiorly, and rounded shoulders.

is histologically identical to the consequences of peri-articular bleeding caused by hemophilia (K. Reinker, personal communication). This is intriguing given the bleeding diathesis that can accompany Noonan syndrome.

Central Nervous System

Seizures of varied types are found in 10% of individuals, with mean age of onset of 11 years [5]. Structural anomalies of the central nervous system are unusual. Hydrocephalus is reported in about 5% [7578]. Communicating hydrocephalus is generally described and Clericuzio and colleagues hypothesize that this may be related to extra-cranial lymphatic dysplasia [75]. They refer to studies documenting the drainage of cerebrospinal fluid from the subarachnoid space along the olfactory nerves to the nasal lymphatics, and from there to cervical lymph nodes [79]. There are several reports of Chiari I malformation in Noonan syndrome and additional individuals are known to the author [80, 81]. Other less common structural brain anomalies include schwannoma, Dandy-Walker malformation, and lateral meningocele [82]. Cerebrovascular anomalies have been described in a few individuals [8388].

The Clinical Phenotype of Noonan Syndrome

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Genitourinary System

Renal anomalies are commonly reported (10%), generally mild, and include dilatation of renal pelvis (most common), duplex systems, minor rotational anomalies, distal ureteric stenosis, and renal hypoplasia/aplasia [89]. In males, pubertal development varies from normal virilization with subsequent fertility, to delayed but normal pubertal development, to inadequate sexual development associated with early cryptorchidism and deficient spermatogenesis [90]. Mean age of onset of puberty is 14.5 years in males and 14 years in females [5]. Most females are fertile [6, 90].

but are more common in LEOPARD syndrome [95, 96]. Rare findings include xanthomas of the skin and oral mucous membranes, leukokeratosis of the lips and gingiva, molluscoid scalp skin, and vulvar angiokeratoma.

Lymphatics

Gastrointestinal System

Both splenomegaly (50%) and hepatomegaly (25%) are said to be common, although in this authors experience these figures seem high. The cause is unknown, but one might suspect an association with congestive heart failure or myelodysplasia on occasion. Rarely reported anomalies include choledochal cyst and midgut rotation [89].

Postnatally, a lymphatic abnormality is found in less than 20%; it may be localized or widespread; it is most commonly appreciated at birth but may not appear until adulthood [97]. Dorsal limb lymphedema is the most common finding. It may contribute to increased birth weight, and usually resolves in childhood. Less common abnormalities include generalized lymphedema, pulmonary lymphangiectasia, chylous effusions in pleural or peritoneal spaces, intestinal or testicular lymphangiectasia, and localized lymphedema of scrotum or vulva. Adolescent or adult onset does occur. Lymphangioma is a rare complication [98, 99]. The most common underlying pathology is lymph vessel hyperplasia with or without a thoracic duct abnormality. Lymphatic aplasia, hypoplasia and megalymphatics are also described.

Hematology Oncology Skin

Various skin manifestations are seen in Noonan syndrome including caf-au-lait spots, pigmented nevi, and lentigines [91]. Keratosis pilaris atrophicans has been noted in several instances, predominantly over extensor surfaces and the face [92]. On occasion facial keratosis is severe enough to cause absence of eyebrows and lashes, as seen in cardiofaciocutaneous syndrome. Ectodermal features seem to be more prevalent when Noonan syndrome is caused by mutations in SOS1 [93, 94]. Prominent fetal fingertip pads are often seen [4]. Multiple subcutaneous granular cell schwannomas are occasionally reported

Several different coagulopathies may occur, either alone or in combination [100, 101]. They affect about one third of individuals, however, many more will have a history of abnormal bleeding or easy bruising. The range of manifestations is broad, from severe surgical hemorrhage to asymptomatic laboratory abnormalities. There is poor correlation between bleeding history and actual defect. Laboratory findings include factor XI deficiency, factor XII deficiency, factor VIII deficiency [100, 102106], von Willebrand disease, and platelet dysfunction, which may be associated with trimethylaminuria or acyclooxygenase deficiency [101, 107]. Some factor deficiencies

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seem to improve with age. There is no evidence of hepatic dysfunction or vitamin K-dependent coagulation factor deficiency. Aspirin-containing medications should be avoided. Congenital bone marrow hypoplasia, congenital hypoplastic anemia, and pancytopenia have been reported rarely [108, 109]. A low frequency association with myeloproliferative disorders (MPD) exists. These include, in particular, juvenile myelomonocytic leukemia (JMML), but, more rarely, acute lymphoblastic leukemia [110113], chronic myelomonocytic leukemia [114, 115] and proliferation of erythroid precursors [116]. JMML associated with Noonan syndrome tends to have an earlier onset and milder presentation than sporadic JMML and spontaneous remission may occur [117]. One particular PTPN11 mutation, The73Ile, is found in almost half the children with Noonan syndrome and MPD, but is uncommon in individuals with Noonan syndrome without MPD [117, 118]. Somatic mutations in PTPN11 are a common cause of MPD unassociated with Noonan syndrome [117, 119]. Solid tumours such as pheochromocytoma, malignant schwannoma, vaginal and orbital rhabdomyosarcoma, and neuroblastoma have been reported rarely [120126].
References
1 Noonan JA, Ehmke DA: Associated noncardiac malformations in children with congenital heart disease. J Pediatr 1963;63:468470. 2 Kobylinski O: ber eine flughauthnliche Ausbreitung am Halse. Arch Anthropol 1883;14:342348. 3 Mendez HMM, Opitz JM: Noonan syndrome: A review. Am J Med Genet 1985;21:493506. 4 Sharland M, Burch M, McKenna WM, Patton MA: A clinical study of Noonan syndrome. Arch Dis Child 1992;67: 178183.

Immunological Findings

Autoimmune thyroiditis occurs in 5% of individuals with Noonan syndrome [127130]. Other autoimmune disorders, such as lupus, celiac disease, vitiligo, anterior uveitis and vasculitis are described infrequently [4, 130]. In addition, levels of anti-thyroglobulin and anti-microsomal thyroid antibodies seem to be higher than in the general population [130]. Antiphospholipid syndrome with Moyamoyalike vascular changes is reported [131, 132].

Prenatal Period

During pregnancy certain features may suggest the diagnosis of Noonan syndrome. The commonest of these are polyhydramnios, seen in 33%, and cystic hygroma [4, 133136]. Lack of septation of the cystic hygroma and regression prior to mid second trimester are associated with more favorable prognosis than those with later regression [134, 135]. Other ultrasonographic markers include scalp edema, pleural or pericardial effusion, ascites and/or hydrops [133, 137]. Chorioangiomas are described and may contribute to formation of edema through decreased fetal oncotic pressure secondary to loss of alpha-fetoprotein into amniotic fluid.

5 Shaw AC, Kalidas K, Crosby AH, Jeffrey S, Patton MA: The natural history of Noonan syndrome: a long-term follow-up study. Arch Dis Child 2007;92;128132. 6 Allanson JE: Noonan syndrome. J Med Genet 1987;24:913. 7 Char FC, Rodriguez-Fernandez HL, Scott CI, Borgankoar DS, Bell BB, Rowe RD: The Noonan-syndrome: A clinical study of forty-five cases. Birth Defects 1972;8:110118. 8 Noonan JA: Noonan syndrome: Update and review for the primary pediatrician. Clin Pediatr 1994;33:548555.

9 Yoshida R, Hasegawa T, Hasegawa Y, Nagai T, Kinoshita E, et al: Proteintyrosine phosphatase, non-receptor type 11 mutation analysis and clinical assessment in 45 patients with Noonan syndrome. J Clin Endocrinol Metab 2004;89:33593364. 10 Allanson JE: Time and natural history: the changing face. J Craniofac Genet Dev Biol 1989;9:2128. 11 Allanson JE, Hall JG, Hughes HE, Preus M, Witt RD: Noonan syndrome: the changing phenotype. Am J Med Genet 1985;21:507514. 12 Allanson JE: Noonan syndrome: the changing face. Proc Greenwood Genet Ctr 2001;20:7879.

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24 Shimizu A, Oku Y, Matsuo K, Hashiba K: Hypertrophic cardiomyopathy progressing to a dilated cardiomyopathylike feature in Noonans syndrome. Am Heart J 1992;123:814816. 25 Wilmshurst PT, Katritsis D: Restrictive and hypertrophic cardiomyopathies in Noonan syndrome: the overlap syndromes. Heart 1996;75:9497. 26 Yu CM, Chow LT, Sanderson JE: Dilated cardiomyopathy in Noonans syndrome. Int J Cardiol 1996;56:8385. 27 Noonan JA: Noonan syndrome; in Goldstein S, Reynolds CR (eds): Handbook of Neurodevelopmental and Genetic Disorders in Adults. New York, Guilford Press, 2005, pp 308319. 28 Shah N, Rodriguez M, St Louis D, Lindley K, Milla PJ: Feeding difficulties and foregut dysmotility in Noonan syndrome. Arch Dis Child 1999; 81:2831. 29 Ranke MB, Heidemann P, Knupfer C, Enders H, Schmaltz AA, Bierich JR: Noonan syndrome: growth and clinical manifestations in 144 cases. Eur J Pediatr 1988;148:220227. 30 Witt DR, Keena B, Hall JG, Allanson JE: Growth curves for height in Noonans syndrome. Clin Genet 1986;30:150153. 31 Noordam C, van der Burgt I, Sweep CG, Delemarre-van de Waal HA, Sengers RC, Otten BJ: Growth hormone (GH) secretion in children with Noonan syndrome: frequently abnormal without consequences for growth or GH treatment. Clin Endocrinol 2001; 54:5359. 32 Noordam K, van der Burgt I, Brunner HG, Otten BJ: The relationship between clinical severity of Noonans syndrome and growth, growth hormone (GH) secretion and response to GH. J Pediatr Endocrinol Metab 2002;15:175180. 33 Binder G, Neuer K, Ranke MB, Wittekindt NE: PTPN11 mutations are associated with mild GH resistance in individuals with Noonan syndrome. J Clin Endocrinol Metab 2005;90: 53775381. 34 Ahmed ML, Foot AB, Edge JA, Lamkin VA, Savage MO, Dunger DB: Noonans syndrome: Abnormalities of the growth hormone IGF-1 axis and the response to treatment with human biosynthetic growth hormone. Acta Paediatr Scand 1991;80:446450.

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75 Clericuzio CL, Roberts A, Kucherlapati RS, Tworog-Dube E, Allanson JE: Communicating hydrocephalus in Noonan syndrome: A consequence of lymphatic dysplasia? Proc Greenwood Gen Ctr 2008;27:81. 76 Fryns JP: Progressive hydrocephalus in Noonan syndrome. Clin Dysmorphol 1997;6:379. 77 Henn W, Reichert H, Nienhaus Z, Zankl M, Lindinger A, et al: Progressive hydrocephalus in two members of a family with autosomal dominant Noonan phenotype. Clin Dysmorphol 1997;6:153156. 78 Heye N, Dunne JW: Noonans syndrome with hydrocephalus, hindbrain herniation, and upper cervical intracord cyst. J Neurol Neurosurg Psychiatry 1995;59:338339. 79 Walter BA, Valera VA, Takahashi S, Ushiki T: The olfactory route for cerebrospinal fluid drainage into the peripheral nervous system. Neuropathol Appl Neurobiol 2006;32:388396. 80 Ball MJ, Peiris A: Chiari (type I) malformation and syringomyelia in a patient with Noonans syndrome. J Neurol Neurosurg Psychiatry 1982; 45:753754. 81 Holder-Espinasse M, Winter RM: Type 1 Arnold-Chiari malformation and Noonan syndrome. A new diagnostic feature. Clin Dysmorphol 2003;12:275. 82 Hughes HE, Hughes RM, Summers A, Hochhauser L: Noonan syndrome and lateral meningoceles: another link with neurofibromatosis. Proc Greenwood Genet Ctr 1987;6:159. 83 Hara T, Sasaki T, Miyauchi H, Takakura K: Noonan phenotype associated with intracerebral hemorrhage and cerebral vascular anomalies: Case report. Surg Neurol 1993;39:3136. 84 Hinnant CA: Thromboembolic infarcts occurring after mild traumatic brain injury in a paediatric patient with Noonans syndrome. Brain Injury 1994;8:719727. 85 Hinnant CA: Noonan syndrome associated with thromboembolic brain infarcts and posterior circulation abnormalities. Am J Med Genet 1995;56:241244. 86 Robertson S, Tsang B, Aftimos S: Cerebral infarction in Noonan syndrome. Am J Med Genet 1997;71:111114.

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87 Schon F, Bowler J, Baraitser M: Cerebral arteriovenous malformation in Noonans syndrome. Postgrad Med J 1992;68:3740. 88 Tanaka Y, Masuno M, Iwamoto H, Aida N, Ijiri R, et al: Noonan syndrome and cavernous hemangioma of the brain. Am J Med Genet 1999;82:212214. 89 George CD, Patton MA, El Sawi M, Sharland M, Adam EJ: Abdominal ultrasound in Noonan syndrome: A study of 44 patients. Pediatr Radiol 1993;23:316318. 90 Elsawi MM, Pryor JP, Klufio G, Barnes C, Patton MA: Genital tract function in men with Noonan syndrome. J Med Genet 1994;31:468470. 91 Daoud MS, Dahl PR, Su WP: Noonan syndrome. Semin Dermatol 1995;14:140144. 92 Pierini DO, Pierini AM: Keratosis pilaris atrophicans faciei (ulerythema ophryogenes): A cutaneous marker in the Noonans syndrome. Br J Dermatol 1979;100:409416. 93 Tartaglia M, Pennacchio LA, Zhao C, Yadav KK, Fodale V, et al: Gain-offunction SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet 2007;39:7579. 94 Zenker M, Horn M, Wieczorek D, Allanson J, Pauli S, et al: SOS1 is the second most common Noonan gene but plays no major role in cardio-facio-cutaneous (CFC) syndrome. J Med Genet 2007;44:651656. 95 Lohmann DR, Gillessen-Kaesbach G: Multiple cutaneous granular cell tumours in a patient with Noonan syndrome. Clin Dysmorphol 2001;19:301302. 96 Sahn EE, Dunlavey ES, Parsons JL: Multiple cutaneous granular cell tumors in a child with possible neurofibromatosis. J Am Acad Dermatol 1997;36:327330. 97 Witt DR, Hoyme HE, Zonana J, Manchester DK, Fryns JP, et al: Lymphedema in Noonan syndrome: Clues to pathogenesis and premature diagnosis and review of the literature. Am J Med Genet 1987;27:841856. 98 Bloomfield FH, Hadden W, Gunn TR: Lymphatic dysplasia in a neonate with Noonans syndrome. Pediatr Radiol 1997;27:321323.

99 Evans DG, Lonsdale RN, Patton MA: Cutaneous lymphangioma and amegakaryocytic thrombocytopenia in Noonan syndrome. Clin Genet 1991;39:228232. 100 Sharland M, Patton MA, Talbot S, Chitolie A, Bevan DH: Coagulation-factor deficiencies and abnormal bleeding in Noonans syndrome. Lancet 1992;339:1921. 101 Witt DR, McGillivray BC, Allanson JE, Hughes HE, Hathaway WE, et al: Bleeding diathesis in Noonan syndrome: a common association. Am J Med Genet 1988;31:305317. 102 de Haan M, van der Kamp JJP, Briet E, Dubbeldam J: Noonan syndrome: partial factor XI deficiency. Am J Med Genet 1988;29:277282. 103 Emmerich J, Aiach M, Capron L, Fiessinger JN: Noonans syndrome and coagulation-factor deficiencies. Lancet 1992;339:431. 104 Kitchens CS, Alexander JA: Partial deficiency of coagulation factor XI as a newly recognized feature of Noonan syndrome. J Pediatr 1983;102:224227. 105 Massarano A, Wood A, Tait RC, Stevens R, Super M: Noonan syndrome: Coagulation and clinical aspects. Acta Paediatr 1996;85:11811185. 106 Singer ST, Hurst D, Addiego JE Jr: Bleeding disorders in Noonan syndrome: three case reports and review of the literature. J Pediatr Hematol Oncol 1997;19:130134. 107 Humbert JR, Hammond KB, Hathaway WE: Trimethylaminuria: the fishodour syndrome. Lancet 1970;2:770771. 108 Feldman KW, Ochs HD, Price TH, Wedgwood RJ: Congenital stem cell dysfunction associated with Turnerlike phenotype. J Pediatr 1976;88:979998. 109 Sackey K, Sakati N, Aur RJA, Shebib S, Sabbah RS, Rifai S: Multiple dysmorphic features and pancytopenia: a new syndrome? Clin Genet 1985;27:606610. 110 Attard-Montalto SP, Kingston JE, Eden T: Noonans syndrome and acute lymphoblastic leukaemia. Med Pediatr Oncol 1994;23:391392. 111 Johannes JM, Garcia ER, De Vaan GA, Weening RS: Noonans syndrome in association with acute leukemia. Pediatr Hematol Oncol 1995;12:571575.

112 Piombo M, Rosana C, Pasino M, Marasini M, Cerruti P, Comelli A: Acute lymphoblastic leukemia in Noonan syndrome: report of two cases. Med Pediatr Oncol 1993;21:454455. 113 Roti G, La Starza R, Ballanti S, Crescenzi B, Romoli S, et al: Acute lymphoblastic leukaemia in Noonan syndrome. Br J Haematol 2006;133:448450. 114 Bader-Meunier B, Tchernia G, Mielot F, Fontaine JL, Thomas C, et al: Occurrence of myeloproliferative disorder in patients with Noonan syndrome. J Pediatr 1997;130:885889. 115 Fukuda M, Horibe K, Miyajima Y, Matsumoto K, Nagashima M: Spontaneous remission of juvenile chronic myelomonocytic leukemia in an infant with Noonan syndrome. J Pediatr Hematol Oncol 1997;19:177178. 116 Kratz CP, Nathrath M, Freisinger P, Dressel P, Assmuss H-P, et al: Lethal proliferation of erythroid precursors in a neonate with a germline PTPN11 mutation. Eur J Pediatr 2006;165:182185. 117 Kratz CP, Niemeyer CM, Castleberry RP, Cetin M, Bergstrasser E, et al: The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and Noonan syndrome. Blood 2005;15:21832185. 118 Jongmans M, Sistemans EA, Rikken A, Nillesen WM, Tamminga R, et al: Genotypic and phenotypic characterization of Noonan syndrome: New data and review of the literature. Am J Med Genet A 2005;134:165170. 119 Tartaglia M, Niemeyer CM, Fragale A, Song X, Buechner J, et al: Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet 2003;34:148150. 120 Becker CE, Rosen SW, Engelman K: Pheochromocytoma and hyporesponsiveness to thyrotropin in a 46,XY male with features of Turner phenotype. Ann Intern Med 1969;70: 325333. 121 Cotton JL, Williams RG: Noonan syndrome and neuroblastoma. Arch Pediatr Adolesc Med 1995;149:1280 1281. 122 Jung A, Bechthold S, Pfluger T, Renner C, Ehrt O: Orbital rhabdomyosarcoma in Noonan syndrome. J Pediatr Hematol Oncol 2003;25:330332.

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123 Kaplan MS, Opitz JM, Gosset FR: Noonans syndrome: A case with elevated serum alkaline phosphatase levels and malignant schwannoma of the left forearm. Am J Dis Child 1968;116:359366. 124 Khan S, McDowell H, Upadhyaya M, Fryer A: Vaginal rhabdomyosarcoma in a patient with Noonan syndrome. J Med Genet 1995;32:743745. 125 Lopez-Miranda B, Westra SJ, Yazdani S, Boechar MI: Noonan syndrome associated with neuroblastoma: a case report. Pediatr Radiol 1997;27:324326. 126 Ijiri R, Tanaka Y, Keisuke K, Masuno M, Imaizumi K: A case of Noonans syndrome with possible associated neuroblastoma. Pediatr Radiol 2000;30:432433. 127 Chaves-Carballo E, Hayles AB: UllrichTurner syndrome in the male: review of the literature and report of a case with lymphocytic (Hashimotos) thyroiditis. Mayo Clin Proc 1966;41:843854.

128 Vesterhus P, Aarskog D: Noonans syndrome and autoimmune thyroiditis. J Pediatr 1973;83:237240. 129 Amoroso A, Garzia P, Vadacca M, Galluzzo S, Del Porto F, et al: The unusual association of three autoimmune diseases in a patient with Noonan syndrome. J Adol Health 2003;32:9497. 130 Lopez-Rangel E, Malleson PN, Lirenman DS, Roa B, Wiszniewska J, Lewis ME: Systemic lupus erythematosus and other autoimmune disorders in children with Noonan syndrome. Am J Med Genet A 2006;139:239242. 131 Ganesan V, Kirkham FJ: Noonan syndrome and Moyamoya. Pediatr Neurol 1997;16:256258. 132 Yamashita Y, Kusaga A, Koga Y, Nagamitsu S-I, Matsuishi T: Noonan syndrome, Moyamoya-like vascular changes, and antiphospholipid antibodies. Pediatr Neurol 2004;31: 364366.

133 Achiron R, Heggesh J, Grisaru D, Goldman B, Lipitz S, et al: Noonan syndrome: A cryptic condition in early gestation. Am J Med Genet 2000;92:159165. 134 Benacerraf BR, Greene MF, Holmes LB: The prenatal sonographic features of Noonans syndrome. J Ultrasound Med 1989;8:5964. 135 Donnenfeld A, Nazir MA, Sindoni F, Librizzi RJ: Prenatal sonographic documentation of cystic hygroma regression in Noonan syndrome. Am J Med Genet 1991;39:461465. 136 Zarabi M, Mieckowski GC, Mazer J: Cystic hygroma associated with Noonans syndrome. J Clin Ultrasound 1983;11:398400. 137 Bawle EV, Black V: Nonimmune hydrops fetalis in Noonans syndrome. Am J Dis Child 1986;140:758760.

Judith E. Allanson Department of Genetics, Childrens Hospital of Eastern Ontario 401 Smyth Road Ottawa, ON K1H 8L1 (Canada) Tel. +1 613 737 2233, Fax +1 613 738 4822, E-Mail allanson@cheo.on.ca

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Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 2039

Molecular Genetics of Noonan Syndrome


M. Tartagliaa B.D. Gelbb
di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanit, Rome, Italy; for Molecular Cardiology, Departments of Pediatrics and Genetics & Genomic Sciences, Mount Sinai School of Medicine, New York, N.Y., USA
bCenter aDipartimento

Abstract
Noonan syndrome (NS) is a genetically heterogeneous disorder that can result from mutations in the PTPN11, SOS1, KRAS, RAF1 and MEK1 genes, which encode transducers participating in the RAS-MAP kinase (MAPK) signaling pathway. The disorder is generally transmitted as an autosomal dominant trait, although many cases result from de novo mutations. Defects in the PTPN11 gene, which encodes the Src homology 2 (SH2) containing protein tyrosine phosphatase SHP-2, account for approximately 50% of cases. The more than 60 mutations that have been reported are almost all missense changes, and promote upregulation of protein function. Two additional distinct classes of missense PTPN11 mutations have been identified as somatic lesions in hematological malignancies and germline defects in LEOPARD syndrome (LS), which is clinically related to NS. While the former are generally more activating compared to the NS-causing mutations, the latter cause loss of catalytic activity of the phosphatase. Defects in the KRAS proto-oncogene account for roughly 2% of NS cases and engender gain of function in RAS signaling through reduced KRAS GTPase activity or increased GDP/GTP dissociation rate. As documented for PTPN11, the distributions of affected residues and amino acid substitutions in NS and cancer appear to be largely mutually exclusive. Missense mutations in SOS1 occur in approximately 10% of affected individuals. SOS1 is a RAS-specific guanine nucleotide exchange factor that catalyzes the release of GDP from RAS, facilitating the conversion of its inactive GDP-bound form to active GTP-bound RAS. NS-causing SOS1 mutations are activating and affect residues placed

in domains that stabilize the catalytically autoinhibited conformation of the protein. Finally, a small percentage of NS results from missense mutations in the RAF1 and MEK1 genes. RAF1 is a member of a small family of serine-threonine kinases, which are effectors of RAS that activate the dual specificity kinases MEK1 and MEK2. Activated MEK proteins, in turn, activate the MAPKs, ERK1 and ERK2. RAF1 gene mutations are observed in about 5% of NS cases and affect residues clustered in three regions of the protein with amino acid substitutions within the consensus 143 3 recognition sequence around Ser259 accounting for 75% of the mutations. Since 1433 binding at residue Ser259 stabilizes RAF1s catalytically inactive conformation and impairs its translocation to the plasma membrane, mutations affecting this motif promote increased RAF1 activity. Additional studies are required to fully understand the functional consequences of mutations affecting residues placed within the other two mutational hot spots within the activation segment region of the kinase domain and at the C-terminus. RAF1 gene mutations also account for approximately 3% of subjects with LS, and possibly a relevant fraction of pediatric cases with isolated hypertrophic cardiomyopathy. A single missense MEK1 mutation has been reported in two unrelated subjects with sporadic NS. MEK1 gene mutations are estimated to account for less than 2% of affected individuals. No data on the effect of the predicted amino acid change on MEK1 function and MAPK signaling is currently available.
Copyright 2009 S. Karger AG, Basel

Identification of the Noonan Syndrome Disease Genes: A Brief History

From a genetic point of view, Noonan syndrome (NS; OMIM 163950) was a poorly understood condition until recently. Autosomal dominant inheritance was apparent for the majority of families with the disorder, although evidence suggestive of an autosomal recessive form had been reported [1]. Genetic mapping studies for this disorder were performed with small kindreds with the first report appearing in 1992. Since NS shares some features with neurofibromatosis, markers flanking the NF1 and NF2 genes were tested and excluded allelism of NS to those traits [2, 3]. Next, Jamieson and co-workers studied a large Dutch kindred transmitting the trait to perform a genome-wide scan and observed linkage with several markers at chromosome 12q22-qter, which they named NS1 [4]. They also documented that NS was genetically heterogeneous, based on linkage exclusion to NS1 in some kindreds. The NS1 locus was refined to a region of approximately 7.5 cm using novel STRs [5]. Legius and co-workers studied a fourgeneration Belgian family transmitting the trait, achieving independent linkage to NS1, and refining the critical interval further to approximately 5 cm [6]. A positional candidacy approach was taken to identify the NS disease gene residing at NS1 [7], and Tartaglia and co-workers established PTPN11 as the NS1 disease gene a few years later [8]. PTPN11 was considered an excellent candidate because it mapped to the NS1 critical region and because its protein product, SHP-2, occupied a critical role in several intracellular signal transduction pathways controlling diverse developmental processes, including cardiac semilunar valvulogenesis [9]. Subsequent studies performed with large, clinically well-characterized cohorts provided an estimate of the relative importance of PTPN11 mutations in the epidemiology of NS, defined the spectrum of molecular defects in the disorder, and established genotype-phenotype correlations [1013]. Based on those efforts, it has

now been established that PTPN11 mutations account for approximately 50% of individuals with NS, are almost always missense changes that affect specific regions of the protein, and are more prevalent among subjects with pulmonary valve stenosis and short stature, and less common in individuals with hypertrophic cardiomyopathy (HCM) and/or severe cognitive deficits. Following the identification of PTPN11 as a NS disease gene, PTPN11 mutations have been identified in individuals with Noonan-like syndrome and multiple giant cell lesions in bone (NL/MGCLS, which is also known as NS with cherubism; OMIM 163955) [10, 14] and LEOPARD syndrome (LS; OMIM 151100) [15, 16], two developmental disorders known to be closely related to NS. Based on the higher prevalence of pediatric myeloproliferative disorders and leukemias in NS, Tartaglia and co-workers discovered that a different class of missense mutations in PTPN11 occurs as somatic events in myeloid and lymphoid malignancies [1719], and the identity of PTPN11 mutations conferring susceptibility to these hematological disorders was characterized [17, 20]. While the spectrum and distribution of NS-causing and leukemia-associated mutations provided the first hint about their possible consequences on SHP-2 function, biochemical characterization of a relatively large panel of germline or somatic mutations identified multiple mechanisms promoting SHP-2 gain of function [17, 2127]. Since SHP-2 has a critical positive role in RAS signaling (fig. 1), and the NS-causing PTPN11 mutations increased RAS-mediated signal flow, researchers hypothesized that mutations in other genes encoding proteins participating in this transduction pathway might underlie the half of NS cases without a mutation in PTPN11. This candidate gene approach represented the best available gene hunting strategy since no sufficiently informative PTPN11 mutation-negative family transmitting the trait had been identified to support a linkage study. Mutation analysis of candidate genes has allowed the identification of

Molecular Genetics of Noonan Syndrome

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SOS1: NS SOS1 SHC GRB2

HRAS: CS KRAS: NS, CFCS GDP-RAS

SHP2 PTPN11: NS, LS GDP-RAS

Neurofibromin

NF1: NF1, NFNS

RAF

RAF1: NS, LS BRAF: CFC MEK1: CFCS, NS MEK2: CFCS

Fig. 1. Schematic diagram showing the RAS-MAPK signal transduction pathway. The syndromes and their mutated proteins are as indicated. The double ovals in dark grey and the light grey ovals represent generic dimerized cell-surface receptors binding to their ligand.

MEK

ERK

Gene expression

three additional NS disease genes, KRAS, SOS1 and RAF1, in the last two years [2833]. KRAS codes for one of the three members of the RAS family, while SOS1 and RAF1 are, respectively, a RAS-specific guanine nucleotide exchange factor (GEF) and an effector of RAS with serine/ threonine kinase activity functioning as the upstream component of the RAS-associated MAPK cascade. The initial structural and biochemical characterization of mutations in these genes has provided evidence for their activating effects on protein function as well as on the hyperactivation of the RAS-MAPK transduction pathway [2934]. Genotype-phenotype correlation analyses have also documented that mutations in these genes are associated with distinct phenotypes. Specifically, KRAS defects were frequently found in children with a severe phenotype approaching cardiofaciocutaneous syndrome (CFCS; OMIM 115150) or Costello syndrome (CS; OMIM 218040) [28, 29, 35], two disorders clinically related to NS,

while the phenotypes associated with SOS1 and RAF1 mutations included ectodermal abnormalities, normal growth and absence of cognitive deficits [33, 36], and HCM and hyperpigmented cutaneous lesions [30, 31], respectively. Mutations in KRAS, SOS1 and RAF1 have been estimated to account for approximately 15% of affected individuals, indicating that other disease genes responsible for a relatively large portion of Noonan syndrome remain to be identified. These genes are likely to encode proteins with role in the RASMAPK signaling pathway. While other genes are expected to be identified in the next following years, mutational screening efforts focused on genes that encode transducers participating in the RAS-MAPK signaling pathway and are mutated in disorders clinically related to NS have allowed the identification of additional molecular lesions involved in NS pathogenesis. Indeed, a single missense mutation in MEK1, which encodes a dual specificity kinase

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ATG 1 2 3 4 5 6 7 8 9

TGA 10 11 12 13 14 15 16

N-SH2

C-SH2

PTP

104 112

216 221

524 Somatically acquired

Germline transmitted

b
Fig. 2. PTPN11 gene organization, SHP-2 domain structure and location of affected residues in human disease. (a) The PTPN11 gene and its encoded protein. The numbered, filled boxes at the top indicate the coding exons; the positions of the ATG and TGA codons are shown. The functional domains of the SHP-2 protein, consisting of two tandemly arranged SH2 domains at the N-terminus (N-SH2 and C-SH2) followed by a protein tyrosine phosphatase (PTP) domain, are shown below. The numbers below that cartoon indicate the amino acid boundaries of those domains. (b) Location of mutated residues in the three dimensional structure of SHP-2 in its catalytically inactive conformation (green, N-SH2 domain; cyan, C-SH2 domain; pink, PTP domain). Residues affected by germline (left) or somatically acquired (right) mutations are shown with their lateral chains colored according to the classification proposed by Tartaglia et al. (2006) (red, group I; yellow, group II; green, group III; cyan, group IV; orange, group V; violet, group VI; blue, unclassified).

that activates ERK proteins, has been identified [37]. According to this study, MEK1 gene mutations are estimated to account for approximately 3% of PTPN11- and SOS1-mutation negative NS cases. No data on the effect of this mutation on MEK1 function and MAPK signaling is currently available. Next, we will briefly review current knowledge on the molecular genetics of NS. Specifically, we discuss the function of the identified disease

genes, the diversity of disease-causing mutations and their consequences on protein function and intracellular signaling.

PTPN11

The PTPN11 gene (OMIM 176876) spans more than 90 kb, comprising 16 exons with an open reading frame of 1,779 bases. It encodes SHP-2,

Molecular Genetics of Noonan Syndrome

23

Table 1. Classification and relative distribution of germline and somatic PTPN11 mutations Mutation group Predicted effect on SHP-2 functiona Germline origin (n = 573) n (%) 243 (42.4) 66 (11.5) 27 (4.7) 195 (34.0) 28 (4.9) 12 (2.1) 2 (0.4) Somatic origin (n = 256) n (%) 217 (84.8) 3 (1.2) 27 (10.5) 4 (1.6) 5 (1.9)

I II III IV V VI others
a

A/I switching A/I switching and catalysis A/I switching and specificity A/I switching and/or catalysis SH2 pY-binding SH2 orientation or mobility

A/I = Active/Inactive conformation; SH2 = Scr homology 2 domain; pY = phosphotyrosyl-containing peptide.

a widely expressed cytoplasmic Src homology 2 (SH2) domain-containing, non-membranous protein tyrosine phosphatase functioning as an intracellular signal transducer that is required during development [3840]. SHP-2s structure is composed of two tandemly arranged aminoterminal SH2 domains (N-SH2 and C-SH2), a single catalytic domain (PTP) and a carboxyterminal tail containing two tyrosyl phosphorylation sites and a proline-rich stretch (fig. 2). Both the N-SH2 and C-SH2 domains selectively bind to short amino acid motifs containing a phosphotyrosyl residue and promote SHP2s association with cell surface receptors, cell adhesion molecules and scaffolding adapters. Crystallographic data indicate that the N-SH2 domain also interacts with the PTP domain using a separate site [41]. As these subdomains show negative cooperativity, the N-SH2 domain functions as an intramolecular switch controlling SHP-2 catalytic activation. Specifically, the N-SH2 domain interacts with the PTP domain basally, blocking the catalytic site. Binding of the N-SH2 phosphopeptide-binding site to a phosphotyrosyl ligand promotes a conformational change of the domain that weakens the auto-

inhibiting intramolecular interaction, making the catalytic site available to substrate, thereby activating the phosphatase. Although it has been demonstrated that SHP2 can either positively or negatively modulate signal flow depending upon its binding partner and interactions with downstream signaling networks, it is now established that SHP-2 positively controls the activation of the RAS-MAPK cascade induced by a number of growth factors and cytokines [3840]. In most cases, SHP-2s function in intracellular signaling appears to be distal to activated receptors and upstream to RAS. While the mechanisms of SHP-2s action and its physiological substrates are still poorly defined, accumulated evidence supports the view that both membrane translocation and PTPase activity are required for SHP-2 function. Available records based on more than 500 germline defects indicate that NS-causing PTPN11 mutations are almost always missense changes and are not randomly distributed throughout the gene [27]. Mutations have been classified into six major groups on the basis of their predicted effect on protein function (table 1 and fig. 2). Most of the mutations affect

24

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residues involved in the N-SH2/PTP interdomain binding network that stabilizes SHP-2 in its catalytically inactive conformation or are in close spatial proximity to them. These mutations are predicted to up-regulate SHP-2 physiological activation by impairing the switch between the active and inactive conformation, favoring a shift in the equilibrium toward the latter, without altering SHP-2s catalytic capability. Recent biochemical and molecular modeling data consistently support this view [24, 27, 42]. A number of mutations, however, affect residues contributing to the stability of the catalytically inactive conformation but also participating in catalysis or controlling substrate specificity. For a number of these defects it can be speculated that the individual substitution does not markedly perturb substrate affinity and/or catalysis, and that protein activation by N-SH2 dissociation might prevail. Finally, a few missense mutations affect residues located in the phosphopeptide binding cleft of each SH2 domain. Experimental evidence supports the idea that these amino acid substitutions promote SHP-2 gain of function by increasing the affinity of the protein for the phosphorylated signaling partners [24, 27] (our unpublished observations). Like many autosomal dominant disorders, a significant (but not precisely determined) percentage of cases results from de novo mutations. To investigate the parental origin of de novo mutations in NS, Tartaglia and co-workers studied 46 families, each consisting of an affected individual heterozygous for a PTPN11 mutation and unaffected parents [43]. Among the fourteen informative families identified in the study, the mutation was of paternal origin in all cases. Moreover, advanced paternal age was noted among fathers of sporadic NS cases with or without PTPN11 mutations, consistent with many, but not all, other autosomal dominant disorders with paternal origin of spontaneous mutations. Notably, a sex-ratio bias in transmission of the PTPN11 mutations was also observed within families transmitting

NS as well as for individuals with sporadic NS. This bias favored males by a factor of 2:1. The available data point to this bias being attributable to sex-specific developmental effects of PTPN11 mutations that favor survival of affected male embryos compared to female ones. Among families transmitting the trait, there were more transmitting mothers than fathers, a significant difference that can be ascribed to reduced fertility of male individuals with NS [44]. PTPN11 mutations have been identified in two phenotypes closely related to classic NS. An A-to-G transition at position 923 (Asn308Ser) was documented in a family with NL/MGCLS [10]. In this family, two siblings had lesions in the mandible while their mother only had typical features of NS [45]. The same mutation has been observed in individuals with sporadic NS and families segregating the condition without any bony involvement. More recently, mutational analysis of three unrelated families inheriting this disorder revealed PTPN11 mutations in two [14]. Both of the mutations, Asp106Ala and Phe285Leu, have also been observed in patients with NS. Thus, NL/MGCLS, which was introduced as a distinct nosologic entity characterized by the association of some cardinal features of NS with giant cell lesions of bone and soft tissue [46], should be considered as part of the NS phenotypic spectrum. Consistent with this view, this trait is genetically heterogeneous. Missense PTPN11 mutations have also been identified in LS [15, 16], a developmental disorder closely related to NS, with major features including multiple lentigines, short stature, distinctive face, cardiac defects and electrocardiographic conduction abnormalities, abnormal genitalia and sensorineural deafness [47, 48]. Analysis of several unrelated individuals with a phenotype fitting or suggestive of LS has confirmed the presence of a heterozygous PTPN11 mutation in the vast majority of cases. Tyr279Cys and Thr468Met represent the most common defects, even though additional mutations have been documented (see

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LEOPARD syndrome: Clinical aspects and molecular pathogenesis in this volume). The elucidation of the pathogenesis of NS, particularly with respect to the developmental perturbations, depends upon studies of animal models. Araki and co-workers generated and characterized a knock-in mouse bearing the Asp61Gly mutation in the Ptpn11 gene [49]. Consistent with biochemical data on human SHP-2 mutants expressed transiently in cell culture, embryo fibroblasts derived from Ptpn11D61G/+ mice exhibited enhanced Shp-2 activity and increased association of Shp-2 with Gab1 after stimulation with EGF. Cell culture and whole embryo studies revealed that increased Ras/Mapk signaling was variably present, appearing to be cell-context specific. Both homozygous and heterozygous mice had a conspicuous phenotype. The former genotype was an embryonic lethal. At day E13.5, these embryos were grossly edematous and hemorrhagic, had diffuse liver necrosis and severe cardiac defects. Among the Ptpn11D61G/+ embryos, approximately one half had ventricular septal defects, double-outlet right ventricle and increased valve primordia size. Myocardial development was grossly normal. The other half of these embryos had mitral valve enlargement. Other aspects of the NS phenotype were also observed in the heterozygotes, including proportional growth failure, cardiofacial dysmorphism, and a mild leukocytosis with increased neutrophils and lymphocytes in adult mice. Splenomegaly was present due to extramedullary hematopoiesis. There was a myeloid expansion in the bone marrow and spleen. Factor-independent myeloid colonies grew from the marrow and had increased sensitivity to IL-3 and GM-CSF. Hence, this genetic defect engendered a mild myeloproliferative disease similar to that observed in some NS patients. New information concerning gain-of-function Shp-2 and development has emerged through work with transgenic flies [50]. The Drosophila homolog of PTPN11, corkscrew (csw), acts downstream of several receptor tyrosine kinases

controlling developmental processes [51]. While ubiquitous expression of leukemia-associated csw transgenic alleles engendered embryonic or larval lethality, expression of an NS-causing allele, N308D, resulted in ectopic wing vein formation. Activation of Ras was necessary but not sufficient for the expression of these phenotypes. Since the ectopic wing vein phenotype closely resembled that observed with Egfr gain of function, epistatic studies with genes relevant for Egfr-Ras-Mapk signaling showed that the N308D allele interacted genetically with nearly all genes in the pathway, documenting dependence on the activation of the receptor by its ligand for ectopic wing vein formation [50]. Children with NS are predisposed to a spectrum of hematologic abnormalities, including juvenile myelomonocytic leukemia (JMML), a clonal myeloproliferative disorder of childhood characterized by excessive proliferation of immature myelomonocytic cells that infiltrate hematopoietic and non-hematopoietic tissues [52, 53]. The hallmark of JMML cells is the hypersensitive pattern of myeloid progenitor colony growth in response to GM-CSF, which is due to a selective inability to down-regulate RAS. Indeed, approximately 50% of children with JMML exhibit either oncogenic RAS mutations or neurofibromin loss of function, the latter is a GTPase activating protein (GAP) for RAS encoded by the NF1 tumor suppressor gene. PTPN11 mutation analysis on a relatively large number of children with NS and JMML has demonstrated the presence of germline mutations in the majority of cases, as well as the occurrence of genotype-phenotype correlations [17, 20, 22]. In particular, one mutation, a C-to-T transition at position 218 (Thr73Ile), was observed to occur in a large percentage of children, a striking finding since that lesion has a very low prevalence among NS-causing mutations. The association between this specific amino acid change and JMML in NS and the key-role of SHP-2 in RAS signaling and hematopoiesis raised the possibility that a distinct class of lesions in PTPN11, possibly

26

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acquired as a somatic event, might play a role in leukemogenesis. Indeed, somatic missense mutations in PTPN11 have been demonstrated to occur in approximately one-third of isolated JMML as well as variable proportions of other myeloid and lymphoid malignancies of childhood [1719, 22, 27, 54, 55]. The prevalence of PTPN11 mutations among adult patients with myeloid or lymphoid disorders appears to be considerably lower than observed among pediatric cases [27, 5659] (our unpublished data), even though SHP-2 overexpression has been documented in adult human leukemia [60]. Similarly, PTPN11 is only rarely mutated in non-hematologic cancers [59, 61]. As observed in NS, the vast majority of PTPN11 lesions identified in this heterogeneous group of hematologic malignancies are missense changes that alter residues located at the interface between the N-SH2 and PTP domains. Remarkably, the available molecular data indicate that specificity in the amino acid substitution is relevant to the functional deregulation of SHP-2 and disease pathogenesis (table 1 and fig. 2). Indeed, comparison of the molecular spectra observed with the NS and leukemias indicate a clear-cut genotypephenotype correlation, strongly supporting the idea that the germline transmitted PTPN11 mutations have different effects on development and hematopoiesis than those acquired somatically. Consistent with this, the biochemical behavior of SHP-2 mutants associated with malignancies tend to be more activating than observed with the NSassociated mutant proteins [24, 27, 42]. Moreover, the leukemia-associated PTPN11 mutations upregulate RAS signaling and induce cell hypersensitivity to growth factors and cytokines more than the NS defects do [17, 22, 23, 25]. Overall, the available genetic, modeling, biochemical and functional data support a model in which distinct gain-of-function thresholds for SHP-2 activity are required to induce cell-, tissue- or developmental-specific phenotypes, each depending on the transduction network context involved in the phenotype. According to this model, SHP-2

mutants associated with NS have relatively milder gain-of-function effects, which are sufficient to perturb development processes but inadequate to deregulate hematopoietic precursor cell proliferation. The PTPN11 mutations observed in isolated JMML and other hematologic malignancies produce mutant SHP-2 proteins with higher gains in function. Since these molecular lesions are observed almost exclusively as somatic defects, it is likely that they affect embryonic development and/or fetal survival. The PTPN11 mutations observed in NS with JMML produce SHP-2 with intermediate activity, which would explain the relatively benign clinical course of the leukemia compared to that observed in isolated JMML.

KRAS

Genetic linkage exclusion studies and PTPN11 genotyping established that one or more additional NS genes existed. Based on the link between the functions of SHP-2 and RAS, two groups independently used a candidate gene approach to discover that KRAS mutations can cause NS [28, 29]. Four missense heterozygous mutations in the KRAS gene were identified in seven individuals among 212 PTPN11 mutation-negative subjects with NS. Of note, a generally more severe NS phenotype was associated with KRAS mutations including one subject with JMML and craniosynostosis and two exhibiting a phenotype at the interface with CFCS and CS. Consistent with this, KRAS mutations were also identified in a small percentage of individuals diagnosed as having CFCS [29, 62]. The diversity of mutations associated with these developmental disorders as well as their phenotypic spectrum have been investigated further, refining the picture of a clustered distribution of germline disease-associated KRAS defects, and confirming the high clinical variability [35, 37]. On the whole, available data indicate that NS-causing KRAS mutations are missense and account for less than 3% of

Molecular Genetics of Noonan Syndrome

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PM1 PM2 PM3 G1 Switch I Switch II G domain

G2

G3

AUG

Isoform A

UAA

3 4

AUG Isoform B

UAA

Fig. 3. KRAS gene organization and protein domain structure. (a) Schematic diagram (above) and three dimensional representation (below) of the structural and functional domains defined within RAS proteins. The conserved domain (G domain) is indicated, together with the motifs required for signaling function (PM1 to PM3 indicate residues involved in binding to the phosphate groups, while G1 to G3 are those involved in binding to the guanine base). The hypervariable region is shown in grey, together with the C-terminal motifs that direct post-translational processing and plasma membrane anchoring (dark grey). The GTP/GDP binding pocket is shown in cyan (guanine ring binding surface) and yellow (triphosphate group binding surface) together with the Switch I (green) and Switch II (magenta) domains, according to the GTP-bound RAS conformation. (b) KRAS gene organization and transcript processing to produce the alternative KRAS isoforms A and B. The numbered black and grey boxes indicate the invariant coding exons and exons undergoing alternative splicing, respectively. KRASB mRNA results from exon 5 skipping. In KRASA mRNA, exon 6 encodes the 3-UTR.

affected individuals. As previously documented for PTPN11, the distributions of affected residues and amino acid substitutions in NS and cancer appear to be largely mutually exclusive (table 2 and fig. 3). The KRAS gene (OMIM 190070) spans more than 45 kb, is divided into 6 exons, and produces two transcripts through alternative splicing, resulting in two proteins called KRASA and KRASB (fig. 3) [63]. Exon 1 contains most of the 5 untranslated region, with the last few bases of it residing in exon 2 along with the translation initiation ATG shared by the two mRNAs. For the KRASA transcript, exon 5 contains the stop codon and a portion of the 3 untranslated region,

of which the remainder resides in exon 6. For the KRASB transcript, exon 5 is skipped so exon 6 comprises a portion of the coding region, the stop codon and the entire 3 untranslated region. As with the other members of the RAS family, KRAS isoforms use GDP/GTP-regulated molecular switching to control intracellular signal flow [64, 65]. They exhibit high affinity for both GDP and GTP, low GTPase activity, and cycle from a GDP-bound inactive state to a GTP-bound active state, the latter allowing signal flow by protein interaction with multiple downstream transducers (fig. 1). GDP/GTP cycling is controlled by GAPs, which accelerate the intrinsic GTPase activity, and GEFs, which promote release of GDP.

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Table 2. KRAS affected residues and amino acid changes germinally transmitted or somatically acquired [28, 34, 35, 37, and 62] (germline mutations); catalogue of somatic mutations in cancer (COSMIC), http://www.sanger.ac.uk/perl/ genetics/CGP/cosmic?action=gene&amp;ln=KRAS (November 30, 2007) (somatic mutations). Amino acid Amino acid change Germline origin (n = 30) n (%) 1 (3.3) 1 (3.3) 1 (3.3) 6 (20.0) 1 (3.3) 1 (3.3) 1 (3.3) 1 (3.3) 1 (3.3) 1 (3.3) 3 (10.0) 2 (6.7) 1 (<0.01) 566 (5.2) 1319 (12.3) 3861 (35.9) 16 (<0.2) 3 (<0.1) 6 (<0.1) 479 (4.5) 654 (6.1) 2507 (23.3) 20 (0.2) 98 (<1.0) 928 (8.6) 23 (0.2) 41 (0.4) 13 (0.1) 2 (<0.1) 4 (<0.1) 1 (<0.01) Somatic origin (n = 10,754) n (%) 2 (<0.1)

Lys5

Asn Glu

Gly12

Ala Cys Asp Phe Leu Asn Arg Ser Val

Gly13

Ala Cys Asp Arg Ser Val

Val14 Gln22

Ile Lys Arg Glu

Pro34

Arg Leu Gln

Ile36 Thr58 Gly60

Met Ile Ala Arg

Molecular Genetics of Noonan Syndrome

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Table 2. (continued) Amino acid Amino acid change Germline origin (n = 30) n (%) 1 (3.3) 6 (20.0) 3 (10.0) Somatic origin (n = 10,754) n (%) 9 (<0.1) 75 (0.7) 13 (0.1) 22 (0.2) 11 (0.1) 22 (0.2) 58 (0.5)

Gln61

Glu His Lys Leu Pro Arg

Val152 Asp153 Phe156 other

Gly Val Ile

RAS proteins share a structure that includes a conserved domain (residues 1 to 165), known as the G domain, which is required for its signaling function, and a less conserved C-terminal tail, called the hypervariable region, that guides posttranslational processing and plasma membrane anchoring (fig. 3). Within this region, conserved sequence elements direct the GTP/GDP binding and exchange and GTP hydrolysis. Furthermore, two tracts, denoted as Switch I and Switch II, undergo major conformational changes upon GTP/ GDP exchange and mediate binding to effectors, GAPs and GEFs [64, 66]. As observed for the somatically acquired oncogenic NRAS, KRAS and HRAS mutations, some of the NS-causing KRAS defects were found to up-regulate protein function by impairing the switch between the active and inactive conformation [29]. In particular, biochemical characterization of the Val14Ile and Thr58Ile KRAS mutants documented an impaired intrinsic and GAP-stimulated GTPase activity compared to the wild type protein, which was, however, higher than that of the cancer-associated Gly12Asp mutant (that has negligible GTPase

activity). Interestingly, both the Val14Ile and Thr58Ile KRAS proteins were partially responsive to neurofibromin and p120 GAP, although to different extents. Consistent with these data, cells expressing each of the two mutants were hyper-responsive to hematopoietic growth factors. In a subsequent study, Schubbert and co-workers demonstrated that individual NS-causing KRAS mutations promote upregulation of KRAS by multiple mechanisms [34]. They showed that two mutants, Pro34Arg and Asp153Val, exhibited normal intrinsic rates of GTP hydrolysis, while a third mutant, Phe156Leu, had impaired GTPase activity that was similar to that observed for the cancer associated Gly12Asp KRAS mutants. Interestingly, Pro34Arg KRAS was insensitive to neurofibromin or p120 GAP stimulation while the Phe156Leu mutant had an intermediate level of responsiveness, and the Asp153Val KRAS protein exhibited a response to GAPs that was comparable to that observed for the wild type protein. These mutants also differed in their capability to bind guanine nucleotides with the Asp153Val protein exhibiting a normal GTP/GDP dissociation

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rate while Phe156Leu KRAS showed a dramatically increased rate of guanine nucleotide dissociation. Although these studies documented a complex pattern of intrinsic biochemical properties, expression of these mutants in COS-7 cells promoted higher levels of phosphorylated MEK and ERK proteins, indicating hyperactivation of the MAPK cascade [34]. Consistent with these findings, expression of these mutants in murine fetal liver cells conferred variable hyper-responsive behavior to GM-CSF. It should be noted that, different from what observed for cells expressing the cancer-associated Gly12Asp mutants, cell growth in these cells remained dependent on growth factor stimulation [34]. Mutations affecting exon 6 have been documented in one-third of NS or CFCS subjects with a KRAS germline mutation. This exon codes for residues at the C-terminus of KRASB but not KRASA (fig. 3). In general, the C-termini of RAS proteins are subjected to post-translational modifications, which have important implications for their functions [67]. Similar to HRAS and NRAS, KRASA is palmitoylated at cysteine residues upstream of the conserved CAAX motif, which is replaced with a polylysine stretch in KRASB. This differential processing of the two KRAS isoforms leads to alternative trafficking pathways to the plasma membrane and distinct membrane localization [63]. Moreover, recent evidence demonstrates that the two KRAS isoforms play distinct roles in development. While KRASB is ubiquitously expressed in embryonic and adult tissues, KRASA expression is restricted temporally and spatially and is not expressed in the adult heart [68]. Consistent with these data, loss of both the KRAS isoforms is embryonic lethal [69, 70], while absence of only KRASA does not perturb development [68]. Although KRAS mutations affecting domains shared by the two isoforms can cause NS and CFCS, the identification of exon 6 mutations documented that isolated KRASB gain of function is sufficient for disease pathogenesis, further evidence that isoform B plays the major role in development.

SOS1

Cell surface tyrosine kinase receptors activate RAS proteins by recruiting GEF proteins to the cytoplasmic side of the plasma membrane. These factors catalyze the release of GDP from RAS, facilitating the conversion of the inactive GDPbound form to active GTP-bound RAS [71]. Among the GEFs, two members of the son of sevenless (SOS) family, SOS1 and SOS2, promote guanine nucleotide exchange on RAS proteins, but not on RAS-related family members, such as the RAP and RHO proteins [72, 73]. Both the SOS proteins are constitutively bound to the Src homology 3 (SH3) domain of GRB2, and following growth factor stimulation, the GRB2-SOS complex binds directly to specific tyrosyl-phosphorylated motifs of the activated receptor or to an adaptor protein, such as SHC, through the SH2 domain of GRB2 [73, 74]. Based on their modulatory role in RAS signaling, two groups considered SOS1 and SOS2 as excellent candidate genes, and independently discovered that SOS1 is mutated in a relatively large percentage of subjects with NS [32, 33]. The SOS1 gene (OMIM 182530) spans more than 130 kb [75]. Exons 2 to 24 encompass the open reading frame that encodes a large multidomain protein of 1,333 residues [74]. The N-terminal portion of the protein contains a histone domain (HD; 200 residues) that is characterized by two tandemly arranged histone folds, and is followed by a Dbl homology (DH) domain (200 residues) and a pleckstrin homology (PH) domain (150 residues), which are implicated in the activation of RAC, a small GTPase of the RHO/CDC42 family [76]. The C-terminal half of the protein contains the RAS exchanger motif (REM) domain (200 residues) and the Cdc25 domain (300 residues), which are required for the RAS-specific nucleotide exchange activity of SOS1. Finally, the region at the C-terminus contains recognition sites for SH3 domains and mediates interaction of SOS1 with SH3 domain-containing adaptor

Molecular Genetics of Noonan Syndrome

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Fig. 4. SOS1 domain structure and location of affected residues in NS. (a) The predicted amino acid substitutions from the 22 SOS1 missense mutations are positioned below the cartoon of the SOS1 protein with its functional domains indicated above. Abbreviations: DH, Dbl homology domain; PH, plekstrin homology domain; Rem, RAS exchanger motif. (b) Location of the mutated residues on the three-dimensional structure of SOS1. The functional domains are color coded as follows: Histone folds, cyan; DH, magenta; PH, orange; Rem, green; Cdc25, yellow. Residues affected by mutations are indicated with their lateral chains (histone folds, violet; HD, blue; PH, green; helical linker, red; Rem, orange; Cdc25, cyan). Based on Sondermann et al. [78], which utilized structural data and computational modeling.

Histone DH PH Rem Cdc25 PxxP folds 1 198 404 550 750 1,050 1,200 1,333 W432R F623I E433K S548R W729L E846K G434R L550P I733F C441Y R552G D309Y R552K Y337C R552S T266K Y702H P478L M269R P478R M269T

E108K

proteins that deliver SOS1 to the membrane upon receptor activation (fig. 4). Additional anchorage sites on the membrane are provided by the phosphatidylinositol phosphate-binding site within the PH domain [77], and an extended positively charged surface of the HD domain [78]. Sos1 is widely expressed, and different from Sos2, which is dispensable for mouse development, loss of Sos1 function results in a range of embryonic defects, including cardiovascular abnormalities, causing mid-gestational lethality [79]. The available data indicate that SOS1 is the second most frequently mutated NS disease gene, accounting for approximately 2030% of subjects without a defect in PTPN11 or KRAS [32, 33, 36]. Thus far, all of the mutations identified are missense and affect multiple domains, clustering in specific regions of the protein (fig. 4). Approximately 40% of SOS1 defects affect three residues (Ser548, Leu550 and Arg552) located in a short helical linker connecting the PH and REM domains, with substitutions of residue Arg552

accounting for 30% of total mutations. A second mutation cluster is located within the PH domain (residues 432 to 434; 16% of mutations), while a third functional cluster resides at the interacting regions of the DH (Thr266 and Met269) and REM (Trp729 and Ile733) domains (14% of mutations). A single amino acid change (Glu846Lys) within the Cdc25 domain accounts for approximately 15% of defects. The GEF activity of SOS1 is controlled by two regulatory determinants: the RAS catalytic site and an allosteric site that stimulates exchange activity through the binding of nucleotide-bound RAS [80]. Whereas the former is located entirely within the Cdc25 domain, the allosteric site is bracketed by the Cdc25 domain and REM domains. The mechanism of activation of the protein is complex: in basal conditions, the interaction between the DH and REM domains stabilizes SOS1 in its catalytically inactive conformation by masking the allosteric binding site for RAS. Following SOS1 translocation to the membrane,

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the inhibitory effect of the DH domain is relieved by a still undefined event(s) allowing RAS binding to the allosteric site, which in turn promotes a conformational change of the REM and Cdc25 domains and RAS binding to the catalytic site [81]. Remarkably, most of NS-associated SOS1 mutations reside in regions within the molecule that are predicted to contribute structurally to the maintenance of the catalytically autoinhibited conformation. Specifically, structural data indicate that Arg552 interacts directly with the side chains of Asp140 and Asp169 in the histone domain of SOS1 [78]. Disruption of this interaction is expected to affect the relative orientation of the DH-PH unit and the REM domain. A similar perturbing effect is predicted for the other amino acid substitutions involving residues located in the helical linker connecting the PH and REM domains (fig. 4). While the mutation cluster affecting residues 432 to 441 may disrupt the autoinhibited conformation by destabilizing the PH domains conformation, the third cluster of mutations affects residues (M269R, W729L and I733F) located in the interacting surfaces of the DH and REM domains. Among them, Trp729 interacts directly with Met269, thereby positioning the DH domain in its autoinhibitory conformation. Biochemical data confirmed these predictions and demonstrated that NS-causing SOS1 mutations promote gain-of-function. Roberts and co-workers documented that transient expression of four mutants (Met269Arg, Asp309Tyr, Arg552Gly and Glu846Lys) in 293T cells induced sustained ligand-dependent ERK activation as well as enhanced and prolonged RAS activation [32]. A fifth mutant (Tyr337Cys) was unstable and did not accumulate significantly. Consistent with those findings, Tartaglia and co-workers observed a prolonged EGF-stimulated RAS activation in Cos-1 cells transiently expressing the Arg552Gly mutant and an essentially constitutive RAS activation in cells expressing the Trp729Leu SOS1 protein [33]. In starved cells, Arg552Gly and Trp729Leu expression resulted in modest

increases in ERK phosphorylation compared to wild type, while EGF-induced ERK activation did not differ among the SOS1 proteins. Since many of the SOS1 mutations alter residues related to SOS autoinhibition, either through interaction of the histone folds with the PH-REM linker or interaction of the DH domain at the allosteric RAS binding site, the predominant pathogenetic mechanism appears to be increased availability of the allosteric RAS binding site enhancing GEF activity and, as a consequence, increased RASGTP levels. It should be noted that the DH-PH module of SOS has also been implicated in the activation of the Rho GTPase RAC [76]. The extent to which SOS1 gain-of-function mutations affect different RAS-dependent or RAC-dependent signals remains to be determined.

RAF1

The proto-oncogene RAF1 (also known as CRAF) kinase was identified through its homology to the v-Raf oncogene contained in certain oncogenic murine and avian retroviruses [82]. It is a member of a small family of serine-threonine kinases that includes two additional members, ARAF and BRAF [8385]. These proteins are effectors of RAS that phosphorylate and activate the dual specificity kinases MEK1 and MEK2, which in turn promote the activation of the MAPKs, ERK1 and ERK2. The three members of the RAF family are likely playing different roles in the activation of the RAS-MAPK signaling cascade. Indeed, BRAF has a considerably higher MEK kinase activity compared to ARAF and RAF1, and these proteins also differ in their expression profiles as well as in the regulatory mechanisms controlling their function [83]. Furthermore, based on the murine knock-out models, they appear to have unique roles during development [8689]. The RAF1 gene (OMIM 164760) encodes a 74kDa protein characterized by three functional domains, known as conserved regions 1 to 3 (CR13)

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(fig. 5). The N-terminal CR1 contains the domain involved in GTP-RAS binding and a cysteinerich region that mediates RAF1 interaction with the cytosolic surface of the cell membrane. CR2 contains a negative regulatory domain controlling protein translocation to the membrane and its catalytic activation, while the C-terminal CR3 comprises the kinase domain of the protein [83]. RAF1 is required during development since loss of its function is embryonic lethal [87]. Different from BRAF, which is frequently mutated in colon, ovary and thyroid cancers and melanoma (COSMIC database, http://www.sanger.ac.uk/ genetics/CGP/cosmic/), RAF1 missense changes are observed rarely in malignancies [90]. Two studies recently identified missense mutations in RAF1 in subjects with NS who were negative for a mutation in PTPN11, KRAS or SOS1. Pandit and co-workers documented a heterozygous condition for a RAF1 lesion in 18 out of 231 individuals (8%) [30], while a higher prevalence (10/30) was reported by Razzaque and coworkers [31]. Of interest, RAF1 gene mutations were also identified in two of six subjects with LS without a mutation in PTPN11 [30]. RAF1 mutations affected residues clustered in three regions of the protein (fig. 5). The first cluster affects the consensus 1433 recognition sequence (Arg256Ser257Thr258pSer259Thr260Pro261) or an adjacent residue within the CR2 region. Of note, Arg256, Ser257, Ser259 and Pro261, which are the invariant residues within this motif, were all found to be mutated. Amino acid substitutions within this region account for approximately 75% of total RAF1 defects. The second cluster includes mutations affecting residues within the activation segment region of the kinase domain (Asp486 and Thr491), and constitute 13% of NS- or LS-causing RAF1 amino acid changes. Interestingly, several BRAF missense mutations detected in solid tumors alter the activation segment, including some (Asp594Gly and Thr599Ile) homologous to those identified in subjects with NS [83]. Finally, the third cluster (13% of RAF1 mutations) comprises

two adjacent residues (Ser612 and Leu613) located at the C-terminus in proximity of Ser621, a residue that undergoes phosphorylation and is important for the regulation of RAF1 catalytic activation. Although nearly none of the RAF1 residues mutated in NS and LS is altered in cancer, one somatic missense mutation (Ser259Ala) in RAF1 has been observed in an ovarian carcinoma [91]. Since a high prevalence of hypertrophic cardiomyopathy was observed among individuals with NS/LS and a mutated RAF1 allele, Pandit and co-workers also performed RAF1 mutation analysis in a relatively large cohort of unrelated subjects with isolated HCM who were without mutations in eight myofilament genes known to cause this disease. They identified a single missense mutation (Thr260Ile) in a male patient who had been diagnosed with HCM at age 3 years [30]. Since this cohort included only 10 individuals with HCM presenting before age 13, additional studies of pediatric HCM may be warranted. No RAF1 mutation was identified by Razzaque and co-workers in 100 cases of isolated HCM [31]. RAF1 catalytic activation is complex. In its inactive conformation, the N-terminal portion of the protein is thought to interact with and inactivate the kinase domain at the C-terminus. This autoinhibited conformation is stabilized by 14 33 protein dimers that bind to phosphorylated Ser259 and Ser621 [8385]. Dephosphorylation of Ser259, which is possibly mediated by protein phosphatase 2A (PP2A) or protein phosphatase 1C (PP1C), is required for stable interaction with GTP-RAS, allowing protein translocation to the plasma membrane and further interaction with other still uncharacterized proteins. Among them are serine/threonine kinases that phosphorylate regulatory residues that, in their unphosphorylated state, contribute to stabilizing the catalytically inactive conformation of the protein. To examine the functional consequences of RAF1 mutations, Pandit and co-workers expressed mutants from each of the three

34

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Fig. 5. RAF1 domain structure and location of affected residues in human disease. The domains of the RAF1 protein (above) are indicated (CR, conserved region; RBD, RAS binding domain, CRD, cysteine-rich domain) along with two serine residues (blue) that can be phosphorylated as part of RAF1s regulation. The mutations observed in Noonan and LEOPARD syndromes and those associated with cancer are shown above and below the cartoon, respectively. BRAF domain structure (below) is reported for comparison, together with location of residues altered in developmental disorders (black) or those more commonly mutated in human cancers (prevalence higher than 1.5%, according to COSMIC database, http://www. sanger.ac.uk/genetics/CGP/cosmic/) (red). The BRAF T599I substitution, which rarely occurs in cancer and is homologous to the Noonan syndrome-causing RAF1 T491I change, is also reported.

CR1

S259F L613V T491I/R S257L T260R/I P261S/L/A D486N/G R256S S612T V263A CR2 CR3

RAF1

Activation segment RBD CRD S427G S259 S621 P207S Q335H I448V E478K R226I S259A

Q257R A246P CR1


CR2

N581D G469E G534R F468S L485F F595L D638E S467A E501K/G K499F G596V CR3 BRAF

G464/E/R/V

K601E/N G469A/R/S/V V600A/D/E/G/K/L/M/R G466A/E/R/V T599l L597L/Q/R/S/V D594/G/E/K/V

clusters transiently in Cos-1 cells. Pro261Ser and Leu613Val RAF1 proteins, representative of the HCM-associated clusters, displayed increased kinase activity compared to wild type protein basally and after EGF stimulation [30]. Consistent with that study, Razzaque and coworkers observed enhanced kinase activity for the Ser257Leu, Pro261Ser, Pro261Ala, Val263Ala and Leu613Val RAF1 mutants [31]. In contrast, Asp486Asn and Thr491Ile, representing the mutation cluster in the activation segment and not associated with HCM, were observed to be kinase impaired [30]. Of note, while the expression of the Ser257Leu, Pro261Ser, Pro261Ala, Val263Ala and Leu613Val RAF1 mutants resulted in a constitutively increased activation of the MAPK cascade, expression of the Asp486Asn mutant, which was kinase dead, caused a reduced activation of the pathway while expression

of Thr491Ile, which was MEK kinase impaired, also resulted in constitutive ERK activation [30, 31]. It is interesting to note that the Thr599Ile BRAF mutant, which is homologous to the Thr491Ile RAF1 protein, has modestly increased kinase activity but substantially increased ERK activation, most likely through complexing with wild type BRAF and RAF1 [92]. Dimerization effects may similarly explain the modest reduction of MEK kinase activity but increased ERK activation observed with Thr491Ile RAF1. In contrast, cancer-associated Asp594Val BRAF and the NS-associated Asp486Asn RAF1 impair kinase activities and reduce ERK activation. Their pathogenetic mechanism awaits explanation. Pandit and co-workers also investigated the status of 1433 binding for the Pro261Ser and Leu613Val RAF1 mutants, and demonstrated that the increased activation promoted by the

Molecular Genetics of Noonan Syndrome

35

amino acid substitution was associated with a loss of 1433-mediated inactivation. This finding is consistent with the data obtained by Light and co-workers who engineered Ser257Leu Raf1 and demonstrated that this mutant protein had normal phosphorylation at Ser259 but failed to bind 1433 and had increased kinase activity [93]. On the contrary, the Leu613Val mutant bound to 1433 normally at Ser621 and had normal phosphorylation of Ser259 so the mechanism through which mutations in this cluster activated RAF1s MEK kinase activity remains to be explained.

MEK1

mouse models indicates that MEK1 function is required during embryonic development [95], while MEK2 is dispensable [96]. Of note, even though MEK kinase activity is necessary for cell transformation via the MAPK cascade [97], and constitutively active MEK mutants promote transformation [98], mutations in these genes have not been reported in human cancers [99]. A few missense mutations in MEK1 (Phe53Ser and Tyr130Cys) and MEK2 (Phe57Cys) had previously been reported in a small fraction of subjects with CFCS [100]. Functional characterization of the three mutants documented that their transient expression in 293T cells were more active than the wild type protein in stimulating ERK phosphorylation basally.

A missense mutation in MEK1, predicting the Asp67Asn amino acid substitution, has recently been reported in two unrelated subjects with sporadic NS [37]. One additional previously unreported variant (Glu44Gly), which was identified in a third sporadic case and her apparently clinically unaffected mother but not in 200 population-matching controls, is likely to represent a private polymorphism. According to this study, MEK1 gene mutations would account for approximately 3% of PTPN11- and SOS1-mutation negative NS cases. No data on the effect of the Asp67Asn change on MEK1 function and MAPK signaling is currently available. MEK1 (OMIM 176872) and the functionally related MEK2, belong to a family of dual specificity kinases that phosphorylate substrates at tyrosine and serine/threonine residues [94]. Their open reading frames encompass eleven exons, which code for proteins of 393 (MEK1) and 400 (MEK2) residues. The MEK proteins share a conserved structure including a negative regulatory domain at the N-terminus and a single protein kinase domain. MEK1 and MEK2 are both effectors of RAF proteins and activate ERK1 and ERK2, but appear to play non-redundant roles. In particular, genetic evidence from

Concluding Remarks

In the last few years, we have witnessed the elucidation of genetic causes of NS, which can now be viewed as a disorder of dysregulated RASMAPK signaling. Further studies are needed to identify the still missing genes, probably functionally related, responsible for the remaining 35% of affected individuals as well as to understand in detail NS disease pathogenesis. These steps are preludes to developing improved diagnostics and therapeutics for this genetic disorder.

Acknowledgements
The authors apologize to colleagues whose work was not cited due to limited space. Research in the authors laboratories is supported in part by grants from TelethonItaly (GGP07115), Programma di Collaborazione Italia-USA/malattie rare and from Associazione ONLUS Morgan Di Gianvittorio per la cura e la ricerca nei tumori e leucemie in et pediatrica (to M.T.), and from the National Institutes of Health (HL71207, HD01294 and HL074728) and March of Dimes (FY03 52) (to B.D.G.).

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Marco Tartaglia Department of Cell Biology and Neurosciences Istituto Superiore di Sanit 00161 Rome (Italy) Tel. 3906 4990 2569, Fax 3906 4938 7143, E-Mail mtartaglia@iss.it

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Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 4054

Genotype-Phenotype Correlations in Noonan Syndrome


A. Sarkozya,b M.C. Digilioc B. Marinod B. Dallapiccolaa,b
Casa Sollievo della Sofferenza, San Giovanni Rotondo and CSS-Mendel Institute, Rome, of Experimental Medicine, La Sapienza University, Rome, cMedical Genetics, Bambino Ges Hospital, Rome, and dPediatric Cardiology, La Sapienza University, Rome, Italy
bDepartment aIRCCS

Abstract
Noonan syndrome (NS) is an autosomal dominant disorder mainly characterized by short stature, distinct facial anomalies and congenital heart defects. The cumulative record of genotype-phenotype correlations clearly indicates that PTPN11 gene mutations, responsible for almost half of the cases, either sporadic or familial, are responsible for a wide clinical spectrum, characterized by a high prevalence of pulmonary valve stenosis, typical facial features, cryptorchidism and bleeding diathesis. Mutations in the SOS1 gene are associated with clinical features partly overlapping those found in PTPN11 mutation-positive individuals, but distinguished by a low frequency of short stature and mental retardation, and a high prevalence of macrocephaly, ptosis, and skin features similar to those of cardio-facio-cutaneous syndrome (CFCS). RAF1 gene mutations are strongly associated with hypertrophic cardiomyopathy, mental retardation, short stature, and skin features of LEOPARD syndrome. Patients with KRAS mutations are sporadic, affected by variable mental retardation and may manifest features overlapping those of Costello and CFCS, while MEK1 mutations have been found so far only in two unrelated NS individuals.
Copyright 2009 S. Karger AG, Basel

Noonan syndrome (NS) is an autosomal dominant disorder characterized by short stature, distinct facial anomalies, congenital heart defects (CHD),

developmental delay and several additional clinical features [reviewed in 1]. NS shows a wide phenotypic variability, ranging from mildly affected adults to severely affected newborns, occasionally representing a life-threatening or lethal condition, also in the prenatal life. NS is a common condition, occurring in approximately 1/1,0002,500 individuals [23]. However, mild expressions are likely to be overlooked. In particular, facial features are often subtle in adults, and in the absence of other features the diagnosis may be missed [4]. In fact, adults are frequently diagnosed after the birth of a more severely affected infant. Moreover, clinical course and severity of NS are varying both between and within families, suggesting a relatively weak, if any, genotype-phenotype correlation. NS shares many features with the less common Noonan-like/Multiple Giant Cell Lesions (NLS), LEOPARD (LS), Costello (CS), cardio-facio-cutaneous (CFCS) and Neurofibromatosis type 1Noonan (NFNS) syndromes [5]. Patients with these disorders present overlapping features, such as facial abnormalities, CHD and short stature, together with other common abnormalities such

as skin and genital anomalies and variable degree of mental retardation. Recently, the term neurocardio-facial-cutaneous (NCFC) syndrome has been introduced for all these conditions which are caused by germline mutations in some of the key components of the highly conserved RAS-MAPK cascade [5]. Missense PTPN11, KRAS, SOS1, RAF1 and MEK1 gene mutations occur in about 65% of the NS patients, and it is likely that other loci will soon be identified in the remaining 35% of negative cases [613]. In addition to genetic heterogeneity, NS also displays a wide allelic heterogeneity in all known genes. The wide clinical spectrum of NS and its overlap with other related disorders have been debated for a long time, and the discovery of genetic causes underlying these conditions is providing some insight into this complex clinical scenario. In particular, some current research is attempting to prove any relationship between the allelic and genetic heterogeneities and the different clinical presentations. We will illustrate current knowledge on the clinical presentations of NS patients due to mutations in the different disease-genes and try to establish possible genotypephenotype correlations, based on the analysis of personal and published data. Worthy of mention, the progressive nature of this condition and the overlap between similar disorders warrant additional prospective studies. Genotype-phenotype correlations are further complicated by the obvious heterogeneity of the mutational spectrum within each NS gene and the private character of some mutations. Advances in the understanding of biochemical and functional implications of different mutations are supporting the researchers in this difficult task.

PTPN11 gene mutations is higher in families than in sporadic patients (60 vs. 40%). Several reports of PTPN11 screening have been published worldwide and detailed clinical description of mutated patients provided [1523]. However, the frequency of mutations and clinical characteristics may have been biased not only by the different selection criteria, but also by the different clinical settings in which the patients have been enrolled (i.e. endocrinological vs. cardiological units). In addition, the clinical data are at times puzzling, since a few LS individuals have been included in the patients cohorts or have been misdiagnosed as NS, increasing the incidence of some LS features in the NS population [15, 17]. Altogether, more than 50 germline PTPN11 mutations have been reported in about 400 patients [1524]. PTPN11 gene mutations have been found also in LS and NLS [15, 25]. LS patients display mutations in exon 7, 12 and 13, different from those occurring in NS [2426]. Conversely, patients with NLS display mutations overlapping those reported in NS and LS [14, 15, 27]. Clinical Presentation of NS Patients with PTPN11 Gene Mutations In general, PTPN11-positive patients show a wide clinical variability. About 76% of PTPN11positive NS individuals show a stature lower than the 3rd centile. Of note, published data are mainly referring to young patients, and the follow-up studies of mutated individuals indicate a mean adult height of 167.4 cm in males and 152.7 cm in females [21]. Detailed facial characteristics have been provided only by a few reports, pointing to a high frequency of low-set ears (85%), downslanting palpebral fissures (68%) and ptosis (53%) [1618] (fig. 1a). Other studies have indicated the presence of characteristic facial features in up to 90% of the cases [17, 2122]. Macrocephaly has been noted in 39% of the patients [19]. Short neck and/or pterygium colli are common features (30 52%), as well as thoracic anomalies (65%) [15, 18, 20, 22]. Cardiac defects have been described in

NS and the PTPN11 Gene

Germline mutations in the PTPN11 gene, encoding the SHP2 protein, are responsible for about 4550% of NS patients [14]. The incidence of

Genotype-Phenotype Correlations in Noonan Syndrome

41

Fig. 1. Facial features of NS patients with mutation in the PTPN11 (a), SOS1 (b) or RAF1 (c) gene, respectively.

more than 80% of the PTPN11-positive NS patients. Pulmonary valve stenosis (PVS) is the most common cardiac defect (68%), followed by atrial septal defect (ASD; 24%) and hypertrophic cardiomyopathy (HCM; 9%). Atrio-ventricular septal defect (AVSD) has been reported only in 3% of the cases [15, 23], bleeding diathesis in 44% and cryptorchidism in up to 80% of males. Mental retardation of variable degree and learning difficulties have been noticed in about 40% of the cases. Prevalence of ectodermal features, such as facial keratosis pilaris (6%) and curly hair (34%), has been described by Zenker et al. [17, 28]. Genotype-Phenotype Correlation Germline PTPN11 mutations are spread in several exons, encoding different protein domains. Even in the presence of a wide allelic heterogeneity, available data indicate that a few mutations, in exons 2, 3, 7, 8 and 13, encoding the N-SH2 and the PTP domains, contribute to almost half of the total germline changes in NS [22]. Comparison between the clinical features of PTPN11 mutation-positive and mutation-negative patients has shown a significant association between PTPN11 mutations and a few clinical features (table 1). A consistent correlation was established between PVS and PTPN11 mutations,

and comprehensive analysis of the cardiological features in more than 350 patients supports this association (68 vs. 49%, p = 0.0007) [15]. In contrast, mutated patients have less HCM (9 vs. 28%, p < 0.0001), and a higher cardiac involvement (83 vs. 64%, p = 0.001), even though this association can be biased by the selection criteria. An association between PTPN11 mutation and short stature, distinct facial features, easy bruising, and pectus deformity had been reported in some studies [16, 17, 19]. However, the comprehensive review of available data at present does not support any consistent difference between PTPN11 mutations and short stature, macrocephaly, pectus deformities, short neck, mental retardation/learning difficulties (table 1). Conversely, typical facial features, cryptorchidism and bleeding diathesis are more common in the mutated patients (see table 1). Several studies have addressed the growth parameters and GH therapy in patients with PTPN11 mutations. A more severe mechanism acting on growth retardation in NS patients with PTPN11 mutation has been suggested [29]. Other studies have shown that mutated patients tend to be shorter in length at birth and more commonly small for gestational age [30]. This tendency was significant by the age of 6 years, when the mutated patients appear significantly shorter [30].

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Table 1. Comparison of clinical features of PTPN11 mutation positive (PTPN11+) NS patients with PTPN11 mutation negative (PTPN11) and unselected NS patients
Features PTPN11+ (%)a PTPN11 (%)b p-Value PTPN11+ (%)a All NS (%)c p-Value

Polyhydramnios
Fetal macrosomia Short stature (<3rd) Macrocephaly Downslanting palpebral fissures Ptosis Low-set ears with thickened helices Thick lips/macrostomia Pterygium colli Short neck Short/webbed neck Thorax abnormalities Cardiac defects Pulmonary valve stenosis Septal defect Hypertrophic cardiomyopathy Atrioventricular septal defect Facial keratosis pilaris Curly hair Cryptorchidism Mental retardation Bleeding diathesis Distinct facial features
a

no
no 169/223 (76) 9/23 (39) 39/57 (68) 31/58 (53) 61/72 (85) no 40/130 (31) 16/37 (43) 23/44 (52) 139/215 (65) 236/285 (83) 247/362 (68) 81/340 (24) 31/362 (9) 4/127 (3) (6) (34) 82/104 (79) 76/185 (41) 62/142 (44) 100/113 (88)

no
no 64/95 (67) no 4/8 (50) 3/8 (38) 14/31 (45) no 10/31(32) 5/8 (62) no 59/84 (70) 42/66 (64) 47/96 (49) 21/94 (22) 27/96 (28) no no no 33/58 (57) 38/82 (46) 1/23 (4) 15/23 (65) 0.0040 0.0001 0.0096 0.0012 0.0007 <0.0001 <0.0001

no
no 169/223 (76) 9/23 (39) 39/57 (68) 31/58 (53) 61/72 (85) no 40/130 (31) 16/37 (43) 23/44 (52) 139/215 (65) 236/285 (83) 247/362 (68) 81/340 (24) 31/362 (9) 4/127 (3) (6) (34) 82/104 (79) 76/185 (41) 62/142 (44) 100/113 (88)

43/130 (33)
no 84/115 (73) 19/151 (12) no no no no no no no 144/151 (95) 132/151 (87) 93/151 (62) 29/151 (19) 30/151 (20) no 21/151 (14) 44/151 (29) 64/83 (77) 32/105 (30) 37/151 (25) no 0.0605 0.0006 <0.0001 0.0005 0.0036

See references 1523. See references 15, 17 and 18. c See reference 31.
b

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These individuals display unremarkable GH secretion after pharmacological stimuli, low serum IGF-1 and ALS concentrations, but normal IgFBP-3 level, which argue for a post receptor signalling GH resistance, specific for IgF-1 and ALS and not for IgFBP-3 stimulation. Long term follow-up of mutated patients indicate that the distribution of mean height SDS is narrower in these patients [21]. Conversely, non mutated patients show a broader spread of height SDS, suggesting that they are genetically heterogeneous [21]. Patients with PTPN11 mutations show frequencies of short stature, cardiac involvement, PVS, cryptorchidism and mental retardation similar to those in the general NS population [31]. However, the prevalence of pectus anomalies and HCM is less common in the mutated patients compared to the general NS population (p < 0.0001 and p = 0.0005, respectively), while bleeding diathesis and macrocephaly are significantly more common (table 1). The partial overlap with the general NS population could be related to the fact that PTPN11 mutations are prevailing in NS patients, while differences in the frequency of some features, such as HCM, may suggest that mutations in genes other than PTPN11 could be responsible for these defects, which are quite rare in the PTPN11-positive patients. Clinical manifestations have been evaluated in a few cohorts of patients displaying PTPN11 mutations within the SH2 or PTP domain, and no distinct phenotype, distinguishing these subjects from the general NS population has emerged [15]. It has been suggested that patients carrying a C-SH2 domain mutation are more prone to some additional features unusual in NS. One newborn with the E139D mutation displayed short stature, PVS, hepatosplenomegaly and leukocytosis [16]. Jongmans et al. described this mutation in two patients, one of which with classical NS manifestations associated with hypothalamic glioma and multiple nevi [20]. Tartaglia et al. found the same change in an affected father and in his two children [15], showing distinct facial features of

NS, normal intelligence, slightly left ventricular thickening in one son and profound bilateral sensorineural deafness in both children. However, the number of reported cases is not adequate to reach some definite conclusion. We have shown an association between exon 8 mutations and PVS [19]. Of note, this association might be related to the high prevalence of both PVS and residue 308 mutations in the PTPN11 mutated NS patients. Other reports have found less mental retardation, speech delay and learning disabilities in the heterozygotes for the common exon 8 substitution (N308D) [15, 17]. Finally, some data are pointing to a relationship between ASD and exon 3 mutations [19]. The T411M mutation has been described in a young patient with short stature, triangular facies, dowslanting palpebral fissures, hypertelorism, webbed neck, PVS, bleeding diathesis, prominent corneal nerves, ectodermal involvement with sparse and coarse hair, sparse eyebrows and eyelashes, and developmental delay [32]. This mutation was also found in his mother and sister, who presented with a mild NS phenotype. Zenker et al. reported the G409A mutation in several members of a family segregating a variable and mild NS phenotype, comprising chest deformities, pterygium colli, growth retardation and mild craniofacial features [33]. The NS phenotype was not evident in the older sister and in her mother who were heterozygous for this mutation. It has been suggested that this partial phenotype resulted from incomplete penetrance of this mutation, which has been never documented so far in NS families. Altogether, these data indicate that PTPN11 amino acid exchanges in a specific region of the SHP-2 protein may give rise to variable phenotypes that at times could be so mild to be overlooked on clinical evaluation. The T73I mutation, the most common lesion among infants and young children with NS and myeloproliferative disease (about 50% of the cases), is rare in NS subjects without juvenile myelomonocytic leukaemia (JMML) (2%) [24]. These

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observations indicate that patients with the T73I substitution are at risk of developing myeloproliferative disorders. However, haematological tumours have been reported in NS patients carrying different mutations [14]. Worthy of mention, the spectra of somatic and germline PTPN11 mutations are quite different. Functional data have shown that germline mutations are gain-of-function alleles, but less active than the oncogenic mutants. This explains the relatively low frequency of malignancies in syndromic patients, with the only exception of those carrying tumour-associated mutations. A single case of compound heterozygosity for the Y63C and N308S missense PTPN11 mutations has been described. This rare event occurred in a hydropic foetus with cystic hygroma conceived by NS parents [34]. The pregnancy resulted in early spontaneous demise at 12 weeks. This observation points to a more severe perturbing effect on the development and/or foetal survival of compound heterozygotes and homozygotes for NScausing PTPN11 mutations and heterozygotes for PTPN11 activating mutations exhibiting stronger gain-of-function effects, as occur in the somatic leukemia-related mutations. PTPN11 gene mutations have also been described in LS and NLS [14, 15, 2527]. Allele specificity of LS-associated mutations is strongly supported by clinical differences between NS and LS related PTPN11 mutations. In particular, LS patients show a higher frequency of HCM, skin anomalies, hypotonia and deafness [25, 26, 35]. Conversely, occurrence of the same mutations in NLS patients as well as in NS or LS, indicate that NLS may represent an extreme variant of NS and LS, manifesting with additional features, including cysts and other skeletal anomalies [14, 15, 27]. Functional data on PTPN11 mutants document a close correlation between the identity of the lesion and the disease and demonstrate that NS-causative mutations are less effective in promoting SHP-2 gain of function, compared to those occurring in leukemias [24]. Furthermore,

recurrent LS mutations engender different SHP-2 catalytic activities compared to the NS-associated mutations, thus strengthening the genotypephenotype correlation results. However, it is still unclear why the gain-of-function and apparent loss-of-function of SHP-2 mutants result in similar disorders such as NS and LS. Detection of RAF1 gene mutation in some LS patients suggests that this condition could not be simply due to a reduced RAS signalling transduction [12]. Hence, individuals with PTPN11 mutations are characterized by high prevalence of PVS, typical facial dysmorphisms, cryptorchidism and bleeding diathesis, even though the phenotypic spectrum may be extremely wide, ranging from mild to full-blown or severely affected NS patients, as well as to NLS and LS. Allele specificity of PTPN11 mutations in NS and LS is useful in addressing the correct diagnosis in young and borderline individuals and provides some support for the clinical management and follow-up.

NS and the KRAS Gene

Germline KRAS mutations account for less than 3% of NS patients [7, 8, 11, 36]. Up to now, 9 different mutations have been described in 18 sporadic NS individuals, including a few with a phenotype overlapping CS and two with CFCS [7, 8, 11, 36]. Mutations are located in exons 2, 3 and 6 (the first, second and fourth translated exons of isoform B). KRAS mutations have been identified also in CFCS and CS, in which the mutational spectrum overlaps in part that of NS, arguing for a wide clinical variability with a weak genotypephenotype correlation [7, 11, 36]. Clinical Presentation of NS Patients with KRAS Gene Mutation In general, the phenotype of NS patients carrying a KRAS mutation is variable although often severe, being characterized by short stature and developmental delay in almost all individuals. No

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familial case has been described, suggesting a reduced fitness of the patients with KRAS mutation. Some individuals also display some distinct features of CS and CFCS, possibly in relation with the molecular spectrum overlap [7, 8, 11, 36]. Schubbert et al. identified a missense KRAS mutation (T58I) in a 3-month-old female with a severe NS and a JMML-like myeloproliferative disorder (MPD) [7]. Subsequently, they identified three patients with the V14I mutation and one with the D153V change. On clinical ground, their phenotype was milder compared to that in the T58I individual, and none had a history of MPD or cancer [7]. Carta et al. identified KRAS mutations in two NS patients with a severe NS, overlapping in part CS and CFCS [8]. Zenker et al. found 2% KRAS gene mutations in a wide NS cohort (8/236 patients) [36]. Mutated patients exhibited typical craniofacial features, short stature, macrocephaly, short/webbed neck, thorax deformity and characteristic ocular anomalies. These individuals had a mild to moderate mental retardation and three of them also presented with additional cerebral abnormalities, including mild hydrocephalus, intracranial vascular anomalies and Dandy-Walker malformation [36]. Nava et al. described four additional NS patients with KRAS mutations [11]. All of them showed typical NS facies, no major skin involvement, marked developmental delay and short stature, while PVS was present in two, and mitral valve defect associated with HCM and isolated HCM in single patients. Three of them showed failure to thrive; two had sparse hair and one sparse eyebrows. Accordingly, they concluded that these patients were at the severe uppermost end of the NS spectrum [11]. Genotype-Phenotype Correlation The KRAS mutations reported in NS so far cluster in three hot spots located in exon 2, 3 and 6. Considering the wide clinical variability and the low number of KRAS mutation-positive patients, it is difficult to delineate genotype-phenotype

correlations. However, it seems clear that patients with KRAS mutations display mild to moderate mental retardation, which is found only in one third of the general NS population (p < 0.0001) [31]. Interestingly, 10 out of these 18 NS patients exhibited a change in exon 2, while the CFCS and CS phenotypes were homogeneously distributed among the three hot spots. Although somatic KRAS missense mutations are among the most common molecular lesions in human cancers, NS patients with KRAS mutations usually are not affected by tumours. Similar to the PTPN11 gene, there is great divergence between the spectrum of somatic and germline KRAS mutations. However, functional data are showing that NS-associated KRAS mutants, which are less active than their oncogenic counterpart, may have different intrinsic GTPase activities that might underlie the extreme phenotype in a few of these patients [7].

NS and the SOS1 Gene

Germline mutations in the SOS1 gene, encoding a GEF protein, cause about 1015% of all NS cases (table 2) [9, 10, 28]. This figure changes in the different studies, possibly because of non homogeneous inclusion criteria. In fact, the frequency of SOS1 gene mutations, in patients without mutation in the PTPN11 and KRAS genes, was 28% in the cohorts selected according to strict clinical criteria and 5% in a group of more clinically heterogeneous patients [28]. Up to now, 23 germline missense SOS1 mutations, clustering in three different protein regions, have been found in 56 NS individuals, either sporadic or familial [9, 10, 28]. Clinical Presentation of NS Patients with SOS1 Gene Mutation Tartaglia et al. provided a detailed clinical description of 16 SOS1 mutation-positive individuals [9]. They showed a high prevalence of

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Table 2. Comparison of clinical features between patients with SOS1 gene mutations, the unselected NS population and NS patients with PTPN11 gene mutation
Features SOS1+ (%)a All NS (%)b p-Value SOS1+ (%) PTPN11+ (%)c p-Value

Polyhydramnios
Fetal macrosomia Short stature (<3rd)

8/15 (53)
9/15 (60) 19/53 (36) 9/16 (56) 15/16 (94) 16/16 (100)

43/130 (33)
no 84/115 (73) 19/151 (12) no no no no no no no 144/151 (95) 132/151 (87) 93/151 (62) 29/151 (19) 30/151 (20) no 21/151 (14) 44/151 (29) 64/83 (77) 32/105 (30) 37/151 (25) no 0.0015 <0.0001 0.0194 0.08125 0.0110 0.0002

8/15 (53)
9/15 (60) 19/53 (36) 9/16 (56) 15/16 (94) 16/16 (100) 16/16 (100) 14/16 (88) no no 15/16 (94) 33/40 (83) 23/28 (82) 41/56 (73) 10/56 (18) 6/56 (11) no 8/16 (50) 14/16 (88) 16/30 (53) 10/51(19) 13/56 (23) no

no
no 169/223 (76) 9/23 (39) 39/57 (68) 31/58 (53) 61/72 (85) no 40/130 (31) 16/37 (43) 23/44 (52) 139/215 (65) 236/285 (83) 247/362 (68) 81/340 (24) 31/362 (9) 4/127 (3) (6) (34) 82/104 (79) 76/185 (41) 62/142 (44) 100/113 (88) <0.0001 <0.0001 0.0093 0.0050 0.0090 0.0280 <0.001 <0.0001 0.0537 0.0003

Macrocephaly Downslanting palpebral fissures Ptosis

Low-set ears with thickened helices 16/16 (100) Thick lips/macrostomia Pterygium colli Short neck Short/webbed neck Thorax anomalies Cardiac defects Pulmonary valve stenosis Septal defect Hypertrophic cardiomyopathy Atrioventricular septal defect Facial keratosis pilaris Curly hair Cryptorchidism Mental retardation Bleeding diathesis Distinct facial features
a

14/16 (88) no no 15/16 (94) 33/40 (83) 23/28 (82) 41/56 (73) 10/56 (18) 6/56 (11) no 8/16 (50) 14/16 (88) 16/30 (53) 10/51(19) 13/56 (23) no

See references 9, 10 and 28. See reference 31. c See references 1523.
b

CHDs (81%), primarily PVS (62%) and septal defects (25%), pectus deformities (100%), short and webbed neck (94%) and facial dysmorphisms, in particular ptosis and low set malformed ears

and downslanting palpebral fissures (fig. 1b). Ectodermal features, including facial keratosis pilaris (50%) and curly hair (88%) were quite common, as well as macrocephaly (56%). Conversely,

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mental retardation was present in only one individual (6%) while in only 2 of 15 patients height was below the 3rd centile (13%). Tartaglia et al. concluded that the SOS1-associated phenotype, although clearly within the NS spectrum, was overlapping in part that of CFCS as far as macrocephaly and ectodermal manifestations were concerned, but was clearly different, because both the mental development and linear growth were preserved [9]. However, Roberts et al. have not confirmed this conclusion [10]. In fact, their 15 SOS1 mutated individuals displayed both facial characteristics and other features overlapping those in the general NS population. CHDs were present in 83% of the patients, including PVS in 73%, followed by HCM (20%) and ASD (13%). Short stature was present in 31%, cryptorchidism in 50% and bleeding disorder/easy bruising in 1740% of them. Five individuals needed special education (45%), but mental retardation was not reported in any of them. Roberts et al. did not comment on the cutaneous features. However, by a closer scrutiny of published pictures of the SOS1 mutated patients, both sparse eyebrows and curly hair could be appreciated in a number of them [10]. The high frequency of ectodermal anomalies in SOS1 positive patients was confirmed by Zenker et al. [28]. They identified 22 additional unrelated patients and three affected mothers with SOS1 mutations, including 18 from a group of individuals with a NS diagnosis based on strict criteria, and 4 from a group of subjects with less strict criteria. Among the second group, three SOS1-positive patients had typical NS, while one female presented an atypical phenotype, characterized by mild facial features and short stature. Accordingly, most of the SOS1 mutation-positive patients presented a clear NS phenotype. Facial keratosis pilaris was detected in 58% of the cases, sparse eyebrows and curly hair in 78% and ichthyosiform skin changes in 4%. Up to 80% of them presented PVS, 52% short stature, 71% thoracic anomalies and 45% cryptorchidism. Zenker et al. found a high frequency of ocular ptosis (80%). Minor subsets of

patients showed mental retardation (21%), septal defects (16%), bleeding diathesis (12%) and HCM (4%) [28]. Comparison of these three patient cohorts points to a similar spectrum of CHD, with high prevalence of PVS, but different frequencies of mental retardation or need for special education, easy bruising, cryptorchidism and skin anomalies. Differences in patient selection and clinical evaluation may account for some of these discrepancies. In fact, Zenker et al. recruited several patients from a paediatric endocrinology department where they had been referred for growth retardation [28]. Moreover, 25% of the patients reported by Roberts et al. were not examined directly, but rather evaluated retrospectively on photographic materials and clinical records [10]. Accurate direct clinical inspection of the patients heterozygous for mutations in novel genes is mandatory in order to delineate the eventual phenotype specificities and the differences in respect to the general NS population. In table 2, the cumulative frequencies of the 56 SOS1-positive patients are reported. The overall prevalence of short stature was 36%, mental retardation 19%, bleeding diathesis 23% and cryptorchidism 53%. Keratosis pilaris and curly hair have been reported in about 50 and 90% of the patients. Based on these results it appears that NS patients with SOS1 mutations display a rather distinctive form of NS with ptosis, ectodermal symptoms, normal intelligence and low frequency of short stature. Genotype-Phenotype Correlation Germline SOS1 mutations are spread in several exons, encoding different protein domains. Even in presence of wide allelic heterogeneity, a few mutations are recurring, such as those involving the R552 residue, with three major mutation clusters. Detailed clinical description was not reported for all patients, and accordingly, genotype-phenotype correlation is not available at present between the different protein domains, exons or single mutations.

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The clinical spectrum associated with SOS1 gene mutations is broad, and although clearly within the NS phenotype, resembles in part CFCS for some dysmorphisms, including macrocephaly and ectodermal manifestations, but is clearly different in respect to development and linear growth, which are preserved. The comparison between SOS1 mutation-positive cohorts and either the PTPN11-positive or the general NS population, displays a number of consistent differences (table 2). The frequencies of short stature in SOS1-positive and PTPN11-positive patient cohorts are significantly different (36 vs. 76%, p < 0.001). Similarly, the prevalence of ptosis, thoracic anomalies, facial keratosis pilaris and curly hair is significantly higher in the SOS1-mutated individuals. Conversely, SOS1-positive patients show a lower frequency of cryptorchidism, mental retardation and bleeding diathesis. A statistically significant difference for short stature was observed also comparing SOS1-positive individuals with the general NS population (p = 0.01), while mental retardation and bleeding diathesis disclosed overlapping figures. SOS1 mutations abrogate autoinhibition of the protein, increasing RAS activation and downstream signalling. Accordingly, NS-associated mutants are hypermorphs. Comparison of the effect of five representative mutants on EGF stimulated RAS-ERK activation has indicated that the M269R mutation had the greatest effect [10]. Interestingly, two reported individuals with this mutation had PVS in association with ASD or HCM. The stature was lower than the 3rd centile and both had cryptorchidism. The second most highly active mutant tested was E846K which was associated with ASD. It has been suggested that these findings were similar to those of the knockin NS animal model with the D61G change in the PTPN11 gene [37]. In fact, this model discloses a higher frequency of ASD compared to the model with the weaker N308D allele. Roberts et al. concluded that the degree of activation of SOS1 or SHP2 proteins, as well as the level of ERK

hyperactivation could be the key determinants of ASD in NS [10].

NS and the RAF1 Gene

Germline RAF1 mutations have been recently reported by two independent groups in less than 5% of sporadic and familial NS patients [12, 13]. Prevalence of RAF1 mutations, although somehow discordant in the two cohorts (8 vs. 33%, total prevalence of 10% in patients without PTPN11 and SOS1 mutations), suggests that the RAF1 gene could be the third cause of NS. RAF1 mutations have also been identified in 2 of 6 LS individuals without PTPN11 gene mutations [12]. Altogether, 14 different germline missense RAF1 mutations, involving exons 7, 14 and 17, have been detected in 28 unrelated NS patients [12, 13]. Clinical Presentation of NS Patients with RAF1 Gene Mutation Pandit et al. offered detailed clinical description of 23 RAF1 mutation-positive NS patients [12]. HCM was present in 16/21 (76%) individuals, while PVS was less common (6/21, 29%), and in only three cases was not associated with HCM (3/23). Short stature (86%) and macrocephaly (76%) were frequent as well. The most common dysmorphic features were downslanting palpebral fissures, ptosis and low set ears with thickened helices (about 90% each) (fig. 1c). The neck was short (76%) and the thorax abnormal in up to 71% of the cases. Cryptorchidism was present in 6 of the 9 males, while mental retardation/ learning difficulties were diagnosed in 42% of the mutated patients. Skin anomalies, in particular nevi and caf-au-lait spots were found in 8/21 patients (38%), while curly hair and hyperkeratosis were found only in a subset of these cases (19 and 10%). Pandit et al. also identified a single missense RAF1 mutation (T260I) in a male subject in a cohort of 241 individuals with isolated HCM, who developed ventricular hypertrophy at age of

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3 years, in the absence of other features of NS or LS [12]. Razzaque et al. found RAF1 mutations in 11 NS patients [13]. HCM was present in 80% of them, septal defects in 55%. PVS was not reported in any individual. Two of these patients died of severe HCM, while one had an atrial tumor. Two patients were treated with GH, but one of them developed HCM, and the treatment was interrupted. Short stature was present in 91%, typical facial features, as well as short or webbed neck in all, chest deformities in 71%, cryptorchidism in 11% and mental retardation in 80% of them. Curly hair was present in 27% of the patients, but no data relative to hyperpigmented skin lesions have been provided and no picture of the mutated patients published. Taken together, RAF1 mutations result in a phenotype characterized by CHD (93%), in particular HCM (78%), short stature (90%) and mental retardation (55%) (table 3). NS patients also present typical facial features (100%), in particular ptosis and downslanting palpebral fissures (90%), short and webbed neck (83%), thoracic anomalies (71%), macrocephaly (76%) and cryptorchidism (39%). Skin anomalies include curly hair (22%) and hyperpigmented lesions in 1/3 of the patients in the Pandit et al. series [12]. Genotype-Phenotype Correlation Germline RAF1 mutations cluster in three exons, encoding the CR2 and CR3 protein domains. Even in presence of a wide allelic heterogeneity, mutations occur in a few positions, such as residue S257, T260-P261 and L613. Clinical description of single mutated patients was provided and accordingly, some genotype-phenotype correlation can be drawn. Individuals with RAF1 mutations show a wide clinical spectrum (NS and LS), distinct from that observed in PTPN11, KRAS and SOS1 mutated patients. In particular, HCM is significantly more common and PVS significantly less frequent in RAF1 mutation-positive patients compared to those with PTPN11 or SOS1 mutations, or in the

general NS population (table 3). Short stature is extremely common, but comparison with other NS cohorts has shown a significant difference only with the SOS1 mutation-positive cohort (p < 0.001). Mental retardation is more common in RAF1 mutation-positive individuals than in the general NS cohorts or in the patients with SOS1 mutations. Compared to PTPN11 mutation-positive individuals, RAF1 mutated patients display more frequently macrocephaly, ptosis and short or webbed neck, but, similarly to SOS1 mutated patients, less cryptorchidism and bleeding diathesis (table 3). Multiple nevi, lentigines and cafau-lait spots occur in 1/3 of the RAF1 positive NS individuals, a figure much higher than in the general NS population, which suggests a predisposition to hyperpigmented lesions in patients with RAF1 mutation. Allele specificity has been demonstrated, as shown by the presence of HCM only in 1/6 of the subjects with mutated D486 and T491 residues, 3 of whom had PVS. Conversely, 25/28 NS individuals with mutations clustering to residues S259 and S612 had HCM, and only two of them had PVS [12, 13]. This allele specificity was in part supported by functional studies indicating a difference in kinase activity in the mutants from the HCM-associated clusters (P261S and L613V) compared to the non HCM-associated cluster. The former disclosed higher kinase activity in respect to the wild-type protein both basally and after EGF stimulation, while the latter was kinase impaired [12]. Razzaque et al. created a morpholino antisense oligonucleotide knockdown zebrafish model of Raf1 gene, and showed that the Raf1 morphant had enlarged heart tube, especially in the atrial region, which also was unbent compared to the wild type, further confirming the causative role of RAF1 mutations in HCM [13]. Two adult individuals with RAF1 mutations were diagnosed as full-blown LS. One patient carried the S257L mutation, identified also in 11 NS patients, while the other had the L613V

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Table 3. Comparison of clinical features between patients with RAF1 gene mutation, the general NS population and cohorts of patients with PTPN11 or SOS1 gene-mutations, respectively
Features Polyhydramnios Fetal macrosomia Short stature (<3rd) Macrocephaly Downslanting palpebral fissures Ptosis Low-set ears with thickened helices Thick lips/macrostomia Pterygium colli Short neck Short/webbed neck Thorax anomalies Cardiac defects Pulmonary valve stenosis Septal defect RAF1+ (%)a 6/19 (32) 6/20 (30) 28/31 (90) 16/21 (76) 19/21 (90) 19/21 (90) 18/21 (86) All NS (%)b p-Value RAF1+ (%)a 6/19 (32) 6/20 (30) 28/31 (90) 16/21 (76) 19/21 (90) 19/21 (90) 18/21 (86) PTPN11+ (%)c p-Value no no 169/223 (76) 9/23 (39) 39/57 (68) 31/58 (53) 61/72 (85) 0.0173 0.0031 RAF1+ (%)a 6/19 (32) 6/20 (30) 28/31 (90) 16/21 (76) 19/21 (90) 19/21 (90) 18/21 (86) SOS1+ (%)d 8/15 (53) 9/15 (60) 19/53 (36) 9/16 (56) 15/16 (94) 16/16 (100) 16/16 (100) p-Value <0.0001

43/130 (33) no 84/115 (73) 0.0547 19/151 (12) <0.0001 no no no

9/21 (43) no no 25/30 (83) 20/28 (71) 30/32 (93) 4/32 (13) 10/32 (31)

no no no no 144/151 (95) 0.0004 132/151 (87) 93/151 (62) <0.0001 29/151 (19) 30/151 (20) <0.0001 no 21/151 (14) 44/151 (29 64/83 (77) 0.0032

9/21 (43) no no 25/30 (83) 20/28 (71) 30/32 (93) 4/32 (13) 10/32 (31) 25/32 (78) no 2/21 (10) 7/32 (22) 7/18 (39) 17/31 (55) 1/21 (5) 11/11 (100)

no 40/130 (31) 16/37 (43) 23/44 (52) 139/215 (65) 236/285 (83) 247/362 (68) 81/340 (24) 31/362 (9) 4/127 (3) 6% 34% 82/104 (79) 76/185 (41) 62/142 (44) 100/113 (88) 0.0010 0.0005 0.0069 <0.0001

9/21 (43) no no 25/30 (83) 20/28 (71) 30/32 (93) 4/32 (13) 10/32 (31)

14/16 (88) no no 15/16 (94) 33/40 (83) 23/28 (82) 41/56 (73) 10/56 (18) 6/56 (11) no 8/16 (50) 14/16 (88) 16/30 (53) 10/51(19) 13/56 (23) no

0.0073

<0.0001 <0.0001

Hypertrophic cardiomyopathy 25/32 (78) Atrioventricular septal defect Facial keratosis pilaris Curly hair Cryptorchidism Mental retardation Bleeding diathesis Distinct facial features
a

<0.0001 25/32 (78) no 2/21 (10) 7/32 (22) 7/18 (39) 17/31 (55) 1/21 (5) 11/11 (100)

no 2/21 (10) 7/32 (22) 7/18 (39) 17/31 (55) 1/21 (5) 11/11 (100)

0.0095 <0.0001 0.0015

32/105 (30) 0.0187 37/151 (25) 0.0482 no

See references 12 and 13. See reference 31. c See references 1523. d See references 9, 10 and 28.
b

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change, identified in two NS patients aged 3 and 5 years [12, 13]. Accordingly, there is no apparent allele specificity for LS associated with RAF1 mutations. However, the NS patients with S257L reported by Pandit et al. were all young (aged 38 years), showing high prevalence of skin features (5/7, 71%) [12]. Accordingly, a long term followup of patients with S257L and L613V mutations is warranted. Functional studies performed in the P261S and L613V RAF1 proteins, representative of the HCM-associated mutation clusters occurring in NS and LS patients, have disclosed higher kinase activity compared to the wild-type protein both basally and after EGF stimulation [12]. When P261S and L613V RAF1 were expressed, ERK activation was constitutive and higher compared to the wild type, with L613V having a seemingly stronger effect. The increased activation of P261S can be attributed to the loss of 1433-mediated inactivation, whereas that mechanism does not account for the gain of function in L613V. These data indicate that both NS- and LS-related RAF1 mutations are gain-of-function mutations, and probably other mechanisms are responsible for the phenotypic diversity associated with these mutations [12].

6 years. Both patients carried the novel D67N change. Interestingly, even though the same mutation has been reported concurrently in a single CFCS patient as well, clinical feature of these NS patients are overlapping only in part those of CFCS [11].

Conclusions

NS and the MEK1 Gene

MEK1 gene mutations have been reported only in two NS patients so far [11]. Accordingly, no genotype-phenotype correlation can be drawn. The first subject, aged 12 years, had typical NS features including short stature, triangular face without temporal constriction, non-curly hair, ptosis, almost absent eyebrows, borderline intelligence, a hyperactivity-attention deficit disorder, and was following normal schooling with extra help. The second case, diagnosed as a mild NS, had short stature, hypertelorism, wide face without temporal constriction, normal brows and non-curly hair, no failure to thrive, PVS, and normal psychomotor development at age of

A wide variation of phenotypes and natural histories is apparent among the NS patients heterozygous for PTPN11, KRAS, SOS1, RAF1 and MEK1 gene mutations. The cumulative data point to some genotype-phenotype correlations. In particular, PTPN11 gene mutations, which are responsible for almost half of these patients, result in a wide spectrum of anomalies, including a high prevalence of PVS, typical facial features, cryptorchidism and bleeding diathesis. Mutations in SOS1, the second most common anomaly, are associated with a low frequency of short stature and mental retardation, and a high frequency of ptosis, macrocephaly and CFCS-like skin features. RAF1 gene mutations are strongly associated with HCM, mental retardation, short stature, and LS skin features. Patients with KRAS mutations are sporadic and manifest variable mental retardation with some features mimicking CS and CFCS. MEK1 mutations have been reported so far only in two unrelated patients with a clinical diagnosis of NS. Homozygous or compound heterozygous mutations may be associated with severe prenatal outcomes, and need to be further addressed especially with regard to the prenatal diagnosis. The clinical heterogeneity within NS families is still an unresolved question. However, despite large inter-individual and inter-familial clinical variability, NS patients heterozygous for the same mutations or with changes in the same gene or residue, tend to show some recognizable phenotype, in agreement with the specific functional role of the mutant.

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The results of genotype-phenotype correlations could change the protocols for testing the patients with a suspected or proved diagnosis of NS. Due to the high prevalence of PTPN11 gene mutations and its related wide clinical spectrum, this gene should be screened first in these patients, independently from their clinical characteristics. The PTPN11-negative patients should be then differentiated according to their clinical specificities, to speed up the process of mutation detection. The SOS1 gene screening should be performed in patients with PVS, CFCS ectodermal features, normal growth and development, while the RAF1 gene screening should be done in individuals with HCM and short stature. The KRAS gene testing should be advocated in the sporadic patients presenting with mental retardation and

clinical overlap with CFCS or CS. Otherwise, the testing strategy in PTPN11 mutation-negative patients should follow the mutation prevalence of the RAS gene cascade, including first SOS1 and then RAF1, KRAS and, finally, MEK1, at least until other genes will prove to have a major role in this disease.

Acknowledgements
Research in the authors laboratories is supported in part by grants from the Italian Ministry of Health (Ricerca Corrente, to A.S. and B.D.; Progetto Programma ItaliaUsa, Malattie Rare to A.S.) and Italian Ministry of University and Research (Progetto Ateneo 2007, to B.D.; Idea Progettuale 2006 to B.D.).

References
1 Allanson J: Noonan syndrome; in Cassidy SB, Allanson JE (eds): Management of Genetic Syndromes (WileyLiss, NY 2005). 2 Sharland M, Burch M, McKenna WM, Patton MA: A clinical study of Noonan syndrome. Arch Dis Child 1992;67: 178183. 3 Allanson JE: Noonan syndrome. J Med Genet 1987;24:913. 4 Allanson JE, Hall JG, Hughes HE, Preus M, Witt RD: Noonan syndrome: the changing phenotype. Am J Med Genet 1985;21:507514. 5 Bentires-Alj M, Kontaridis MI, Neel BG: Stops along the RAS pathway in human genetic disease. Nat Med 2006;12:283285. 6 Tartaglia M, Mehler EL, Goldberg R, Zampino G, Brunner HG, et al: Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 2001;29:465468. 7 Schubbert S, Zenker M, Rowe SL, Boll S, Klein C, et al: Germline KRAS mutations cause Noonan syndrome. Nat Genet 2006;38:331336. 8 Carta C, Pantaleoni F, Bocchinfuso G, Stella L, Vasta I, et al: Germline missense mutations affecting KRAS Isoform B are associated with a severe Noonan syndrome phenotype. Am J Hum Genet 2006;79:129135. 9 Tartaglia, Pennacchio LA, Zhao C, Yadav KK, Fodale V, et al: Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet 2007;39:7579. 10 Roberts AE, Araki T, Swanson KD, Montgomery KT, Schiripo TA, et al: Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet 2007;39:7074. 11 Nava C, Hanna N, Michot C, Pereira S, Pouvreau N, et al: Cardio-facio-cutaneous and Noonan syndromes due to mutations in the RAS/MAPK signalling pathway: genotype phenotype relationships and overlap with Costello syndrome. J Med Genet 2007;44:763 771. 12 Pandit B, Sarkozy A, Pennacchio LA, Carta C, Oishi K, et al: Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nat Genet 2007;39:10071012. 13 Razzaque MA, Nishizawa T, Komoike Y, Yagi H, Furutani M, et al: Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet 2007;39:10131017. 14 Tartaglia M, Gelb BD: Noonan syndrome and related disorders: genetics and pathogenesis. Annu Rev Genomics Hum Genet 2005;6:4568. 15 Tartaglia M, Kalidas K, Shaw A, Song X, Musat DL, et al: PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity. Am J Hum Genet 2002;70:15551563. 16 Musante L, Kehl HG, Majewski F, Meinecke P, Schweiger S, et al: Spectrum of mutations in PTPN11 and genotype-phenotype correlation in 96 patients with Noonan syndrome and five patients with cardio-facio-cutaneous syndrome. Eur J Hum Genet 2003;11:201206. 17 Zenker M, Buheitel G, Rauch R, Koenig R, Bosse K, et al: Genotype-phenotype correlations in Noonan syndrome. J Pediatr 2004;144:368374.

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18 Maheshwari M, Belmont J, Fernbach S, Ho T, Molinari L, et al: PTPN11 mutations in Noonan syndrome type I: detection of recurrent mutations in exons 3 and 13. Hum Mutat 2002;20:298304. 19 Sarkozy A, Conti E, Seripa D, Digilio MC, Grifone N, et al: Correlation between PTPN11 gene mutations and congenital heart defects in Noonan and LEOPARD syndromes. J Med Genet 2003;40:704708. 20 Jongmans M, Sistermans EA, Rikken A, Nillisen WM, Tamminga R, et al: Genotypic and phenotypic characterization of Noonan syndrome: new data and review of the literature. Am J Med Genet A 2005;134:165170. 21 Shaw AC, Kalidas K, Crosby AH, Jeffery S, Patton MA: The natural history of Noonan syndrome: a long-term follow-up study. Arch Dis Child 2007;92:128132. 22 Bertola DR, Pereira AC, Albano LM, De Oliveira PS, Kim CA, Krieger JE: PTPN11 gene analysis in 74 Brazilian patients with Noonan syndrome or Noonan-like phenotype. Genet Test 2006;10:186191. 23 Sznajer Y, Keren B, Baumann C, Pereira S, Alberti C, et al: The spectrum of cardiac anomalies in Noonan syndrome as a result of mutations in the PTPN11 gene. Pediatrics 2007;119: e1325e1331. 24 Tartaglia M, Martinelli S, Stella L, Bocchinfuso G, Flex E, et al: Diversity and functional consequences of germline and somatic PTPN11 mutations in human disease. Am J Hum Genet 2006;78:279290.

25 Digilio MC, Conti E, Sarkozy A, Mingarelli R, Dottorini T, et al: Grouping of multiple-lentigines/LEOPARD and Noonan syndromes on the PTPN11 gene. Am J Hum Genet 2002;71:389 394. 26 Sarkozy A, Conti E, Digilio MC, Marino B, Morini E, et al: Clinical and molecular analysis of 30 patients with multiple lentigines LEOPARD syndrome. J Med Genet 2004;41:e68. 27 Sarkozy A, Obregon MG, Conti E, Esposito G, Mingarelli R, Pizzuti A, Dallapiccola B: A novel PTPN11 gene mutation bridges Noonan syndrome, multiple lentigines/LEOPARD syndrome and Noonan-like/multiple giant cell lesion syndrome. Eur J Hum Genet 2004;12:10691072. 28 Zenker M, Horn D, Wieczorek D, Allanson J, Pauli S, et al: SOS1 is the second most common Noonan gene but plays no major role in cardio-facio-cutaneous syndrome. J Med Genet 2007;44:651656. 29 Ferreira LV, Souza SA, Arnhold IJ, Mendonca BB, Jorge AA: PTPN11 (protein tyrosine phosphatase, nonreceptor type 11) mutations and response to growth hormone therapy in children with Noonan syndrome. J Clin Endocrinol Metab 2005;90:5156 5160. 30 Limal J-M, Parfait B, Carbol S, Bonnet D, Leheup B, et al: Noonan syndrome: relationships between genotype, growth, and growth factors. J Clin Endocrinol Metab 2006;91:300306.

31 Sharland M, Burch M, McKenna WM, Paton MA: A clinical study of Noonan syndrome. Arch Dis Child 1992;67: 178183. 32 Bertola DR, Pereira AC, de Oliveira PS, Kim CA, Krieger JE: Clinical variability in a Noonan syndrome family with a new PTPN11 gene mutation. Am J Med Genet A 2004;130:378383. 33 Zenker M, Voss E, Reis A: Mild variable Noonan syndrome in a family with a novel PTPN11 mutation. Eur J Med Genet 2007;50:4347. 34 Becker K, Hughes H, Howard K, Armstrong M, Roberts D, et al: Early fetal death associated with compound heterozygosity for Noonan syndromecausative PTPN11 mutations. Am J Med Genet A 2007;143:12491252. 35 Digilio MC, Sarkozy A, de Zorzi A, Pacileo G, Limongelli G, et al: LEOPARD syndrome: clinical diagnosis in the first year of life. Am J Med Genet A 2006;140:740746. 36 Zenker M, Lehmann K, Schulz AL, Barth H, Hansmann D, et al: Expansion of the genotypic and phenotypic spectrum in patients with KRAS germline mutations. J Med Genet 2007;44:131135. 37 Araki T, Mohi MG, Ismat FA, Bronson RT, Williams IR, et al: Mouse model of Noonan syndrome reveals cell typeand gene dosage-dependent effects of Ptpn11 mutation. Nat Med 2004;10:849857.

Anna Sarkozy CSS-Mendel Institute Viale Regina Elena 261 IT00198 Rome (Italy) Tel. +39 0644160536, Fax +39 0644160548, E-Mail a.sarkozy@css-mendel.it

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Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 5565

LEOPARD Syndrome: Clinical Aspects and Molecular Pathogenesis


A. Sarkozya M.C. Digiliob G. Zampinoc B. Dallapiccolaa M. Tartagliad B.D. Gelbe
San Giovanni Rotondo, and Dipartimento di Medicina Sperimentale e Patologia, Universit La Sapienza and IRCCS-CSS, San Giovanni Rotondo and CSS-Mendel Institute IRCCS-CSS, San Giovanni Rotondo and CSS-Mendel Institute, Rome, bGenetica Medica, Ospedale Bambino Ges, Rome, cIstituto di Clinica Pediatrica, Universit Cattolica del Sacro Cuore, Rome, and dDipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanit, Rome, Italy; eCenter for Molecular Cardiology, Departments of Pediatrics and Genetics & Genomic Sciences, Mount Sinai School of Medicine, New York, N.Y., USA
aIRCCS-CSS,

Abstract
LEOPARD syndrome (LS) is an autosomal dominant disorder for which the acronymic name denotes major clinical characteristics including lentigines, facial dysmorphism, heart defects, cryptorchidism, short stature and sensorineural deafness. Caf-au-lait spots are common in LS and tend to appear earlier in life than the lentigines. The features of LS overlap closely with those observed in Noonan syndrome (NS) and distinguishing the two in infants and young children before lentigines emerge can be challenging. LS, like NS, arises from dysregulated RAS/mitogenactivated protein kinase (MAPK) signaling. Mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase SHP-2, are found in roughly 90% of LS patients. Ten missense mutations have been reported, two (Y279C and T468M) being most common. LS-associated SHP-2 mutant proteins have impaired phosphatase activity, contrasting notably with the gain-of-function SHP-2 mutants associated with NS. Mutations in a second LS gene, RAF1, account for an additional 3% of cases. The RAF1 protein is a serine/ threonine kinase that is part of the RAS-MAPK cascade. RAF1 is basally inactive and, when activated, activates the MAPK kinases, MEK1 and 2. The two LS-associated RAF1 mutations engender gain-of-function effects and one of the alleles has also been observed in patients with NS as well. Considering both disorders, RAF1 mutations associate strongly with the development of HCM. In sum, LS, like the

phenotypically similar NS, arises from dysregulated RASMAPK signaling. While biochemical differences between their alleles exist, a fuller explanation of their respective disease pathogenesis awaits elucidation.
Copyright 2009 S. Karger AG, Basel

Historical Background, Definition and Epidemiology

LEOPARD syndrome (LS; OMIM 151100) is a rare genetic disorder, first reported in 1936 in a 24-year-old woman, who presented with multiple lentigines, increasing in number from birth to puberty, pectus carinatum, hypertelorism and mandibular prognathism [1]. In 1962, cardiac abnormalities and short stature were first associated with this condition [2]. Gorlin et al. reviewed all reported patients supporting the concept of a more generalized condition and coined the acronym LEOPARD, a mnemonic for the major features of this disorder: multiple Lentigines, E lectrocardiographic (ECG) conduction abnormalities, Ocular hypertelorism, P ulmonic

stenosis, Abnormal genitalia, Retardation of growth, and sensorineural Deafness [3, 4]. LS is also known as multiple lentigines syndrome, cardiocutaneous syndrome, lentiginosis profusa or progressive cardiomyopathic lentiginosis [58]. The population prevalence of LS is unknown but estimates would depend on the age of assessment because the diagnosis can be challenging in infants and young children. LS is an autosomal dominant, fully penetrant trait with many reported familial cases. It is likely that the percentage of sporadic cases is similar to the 3550% rate observed with other autosomal dominant disorders.

Clinical Description

As suggested by the diseases acronym, affected patients present with a wide clinical spectrum [3, 4, 9, 10], including multiple lentigines, facial dysmorphism, congenital heart defects (CHD), ECG conduction anomalies, short stature, abnormal genitalia and sensorineural deafness. Clinical evolution and prognosis are greatly influenced by CHD, although the long-term prognosis in general is favourable since most LS adults do not require special medical care. Almost all patients show distinct facial features, which change considerably with age (fig. 1). At birth and in the first years of life, affected individuals often manifest hypertelorism (50%), downslanting palpebral fissures (50%), ptosis (50%) and dysmorphic ears (87%), including over-folded helices and large, everted pinnae. The spectrum of facial features, however, may range from full-blown to mildly dysmorphic features [11]. Additional characteristics, such as triangular face, prominent eyes and ptosis, become evident during childhood and adolescence. Adults display hypertelorism, ptosis, low-set ears, deep nasal-labial folds and premature skin wrinkling. Cardiac anomalies are detected in up to 80% of the patients [12 and our unpublished

observation]. The most common defects include ECG anomalies (75%), such as superiorly oriented mean QRS axis in the frontal plan, and atrioventricular or bundle branch conduction defects [9, 10, 12, 13]. Other ECG changes, often secondary to hypertrophic cardiomyopathy (HCM), include left or biventricular hypertrophy (46%), with q waves (19%), prolonged QTc (23%) and repolarization abnormalities (42%) [12]. Conduction defects, found in 23% of the patients, and p wave abnormalities (19%) need annual monitoring and treatment as in the general population. HCM is very common, being detected in 80% of the patients with cardiac involvement (about 60% of all LS cases) [11, 12, 14 and our unpublished data] and represents the major potentially life-threatening aspect of this condition. The left ventricular hypertrophy is typically asymmetric and is associated with significant obstruction of the left ventricular outflow tract in about 40% of cases [12]. HCM can be detected prenatally or at birth, but usually manifests in infancy and precedes the onset of multiple lentigines. On occasion, HCM is diagnosed or worsens as the lentigines appear [12, 13]. Since the cardiac involvement evolves with age, a complete cardiac assessment should be performed annually and particularly at the appearance of multiple lentigines. The clinical characteristics of HCM associated with LS are similar to those occurring in familial HCM so the typical HCM algorithms can be followed [12, 15]. Therapeutic choices for HCM in the presence of a significant left ventricular-aortic gradient include beta-blockade and calcium channel blockers, while surgical removal of the left ventricular outflow obstruction is indicated in the absence of any significant improvement following pharmacological treatment. HCM can result in fatal events and, accordingly, a careful risk assessment and prophylaxis against sudden death in patients at risk is recommended [12, 13, 16]. Mitral valve anomalies are often diagnosed in association with HCM. Morphological mitral valve abnormalities, such as mitral valve prolapse or

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Sarkozy Digilio Zampino Dallapiccola Tartaglia Gelb

Fig. 1. Clinical features of LEOPARD syndrome. Typical facial dysmorphism (a and b) and lentigines (c and d).

valve clefting, have been found in 42% of cases, and mitral regurgitation in 57% [12]. Pulmonary valve stenosis (PVS), with or without dysplasia, is the third most common CHD occurring in 1020% of LS cases [12, 14], a figure much lower compared to the earlier suggestions (40%) [9, 10]. Mild PVS has a good prognosis, while severe valvular obstruction would necessitate balloon valvuloplasty or surgical valvulotomy. A subset of patients present with atrial and atrioventricular septal defects, coronary artery abnormalities, apical aneurysm, non-compaction of the left ventricle, multiple ventricular septal defects, isolated left ventricular enlargement and endocardial fibroelastosis [9, 12]. Multiple lentigines are the most distinctive feature of LS, but are not pathognomonic of this condition as they occur in association with other conditions such as the Carney Complex and Peutz-Jeghers syndrome [1, 9]. Lentigines are dispersed flat, black-brown macules, present on the

face, neck, upper part of the trunk, palms and soles, typically sparing the mucosae (fig. 1). With age, lentigines may become extremely numerous and contiguous to each other, and may darken progressively. On histological examination, lentigines are characterized by pigment accumulation in the dermis and deeper epidermal layers and by an increased number of melanocytes per area of skin. Multiple lentigines, however, are usually absent in the young patients, occurring only in about 12% of newborns [11]. In general, lentigines appear at the age of 45 years, increasing into the thousands by puberty, and seem to arise independent of sun exposure. Quite exceptionally, older children may not exhibit multiple lentigines, but no adult without lentigines has been reported. In our cohort of patients, two individuals with the clinical and molecular diagnosis of LS still had no lentigines at age of 11 and 14 years, respectively. Caf-au-lait spots (CLS) occur in about half of LS patients. They normally

LEOPARD Syndrome: Clinical Aspects and Molecular Pathogenesis

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precede the appearance of the lentigines, being found in the first months of life in about 75% of the patients [11]. CLS are similar to those found in neurofibromatosis type 1 (NF1), although much darker and less numerous. Occasionally, LS patients may manifest some hypopigmented skin lesions [6]. Total UVA-UVB protection is recommended for patients with multiple lentigines and CLS. Intense pulsed light technology has been suggested for removal of multiple lentigines [17]. In newborns, the skin may be redundant and hyperelastic (67%) [11]. In LS, birth weight is generally normal or above the average (37%) [11]. Retardation of growth appears subsequently, having been reported in a variable proportion of these patients (1360%) [911, 18]. Adult stature is below the 3rd centile in as many as 25% of patients and is below the 25th centile in 85% of them [10, 19 and our personal observations]. Accordingly, growth parameters should be monitored annually [20, 21]. Females may be affected by delayed puberty. In the presence of a significant growth delay, GH therapy may be indicated until adult height is reached. Cardiac status, however, should be assessed since HCM is a contraindication to the use of GH treatment. LS patients often display thoracic anomalies, including broad chest and pectus carinatum or excavatum (3075% of affected individuals) [11]. These defects are common in newborns (75%) [7]. Other less frequent skeletal abnormalities include mandibular prognathism, winging of the scapulae, scoliosis and joint hyperflexibility [19]. Sensorineural deafness is detected in 1525% of the cases [9, 10, 14]. Deafness is usually diagnosed at birth or during childhood, but may develop also in the adulthood. Accordingly, annual hearing assessment should be performed until adulthood and hearing aids or cochlear implants may be indicated in severe hypoacusia. Hypotonia is common in affected newborns and can result in delayed psychomotor development [11]. Physical and occupational therapies

can be beneficial. Mild learning difficulties are reported in about 30% of the cases, while mental retardation is rare [10, 11 and our unpublished data]. Central nervous abnormalities have been reported in single cases [22, 23]. Genitourinary tract anomalies are common in LS. About 50% of the affected males have bilateral cryptorchidism, and, less frequently, hypospadias and genital hypoplasia. Females may be affected by hypoplastic ovaries. Reduced male fertility has been hypothesized based on the prevalent maternal transmission in the familial cases. Renal anomalies, including horseshoe kidney, are infrequent (12%) [10, 11]. Hematological/oncological complications, such as myelodysplasia, acute myelogenous leukemia and neuroblastoma, have been reported occasionally in LS [14, 24, 25]. Recently, a malignant melanoma was diagnosed in a woman with LS, who had a germline PTPN11 mutation and a somatic BRAF mutation [26]. Bilateral choristomas have been reported in a 5-year-old girl [27]. Other less frequently reported features are vascular anomalies, including recurrent peripheral aneurysms, congenital intrahepatic portosystemic venous shunt, and dental anomalies, such as delayed dental development, possible agenesis of permanent teeth or supernumerary teeth [2832].

Clinical Diagnosis

Voron et al. outlined diagnostic criteria for LS [10]. According to these recommendations, the clinical diagnosis of LS may be suspected in the presence of multiple lentigines and at least two other cardinal features. In the absence of lentiginosis, three features in the patient and presence of an affected close relative are diagnostic. The emerging data about the progressive nature of this condition indicate, however, that these criteria may not permit the diagnosis of LS in young patients manifesting partial phenotypes, particularly

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when their case is sporadic. With the advent of molecular testing, the frequency of misdiagnosis in these challenging cases has become evident [14]. Digilio et al. suggested that the diagnosis of LS should be considered for neonates and infants if three main features, HCM, facial dysmorphism and CLS, are present [11].

Differential Diagnosis

LS shows clinical overlap with Noonan syndrome (NS; OMIM 163950) including Noonanlike/multiple giant cell lesions syndrome (NL/ MGCLS; OMIM 163955), neurofibromatosis type 1 (NF1; OMIM 162200) including neurofibromatosis-Noonan syndrome (NFNS; OMIM 601321), Costello syndrome (CS; OMIM 218040), and cardio-facio-cutaneous syndrome (CFCS; OMIM 115150). All of these conditions have been recently grouped in the neuro-cardio-facio-cutaneous syndrome (NCFCS) family [33]. These disorders share facial anomalies, CHD and growth retardation, as well as skin, skeletal and genitourinary anomalies, and variable degrees of mental retardation. A few distinct features, however, are useful handles for differentiating them. LS overlaps with NS most closely [20, 21]. Unlike LS, NS patients manifest characteristic facial features at birth and during childhood and affected individuals have a higher frequency of PVS. In NS, CLS and sensorineural deafness are rare. Accordingly, the diagnosis of LS is based on the presence of cutaneous manifestations, such as CLS and multiple lentigines, HCM and deafness. Differentiating LS from NS can be extremely difficult in young individuals who have not yet developed lentigines or HCM, among whom only a minority will be deaf. This clinical overlap has raised the question of nosology of NS and LS; specifically, whether they represent distinct disorders or rather are different manifestations of a single condition. The finding that LS and NS result from different mutations affecting the PTPN11 gene (see below) supports

the idea that these traits are allelic conditions. Accordingly, we suggest that subjects without multiple lentigines or CLS should be diagnosed as LS only when an LS-related PTPN11 mutation is detected. The long-term follow up of these patients supports this. NFNS is a rare condition characterized by features of NS and neurofibromatosis type 1, including CLS, neurofibromas, central nervous system and skeletal anomalies [34, 35]. Molecular distinction between these two conditions is helpful in diagnosing patients with borderline clinical manifestations [36].

Genetic Counseling

LS is an autosomal dominant condition and is fully penetrant, so a 50% offspring risk figure should be given to affected individuals. The recurrence risk for siblings of a sporadic case of LS is near population risk, marginally increased by the theoretical possibility of gonadal mosaicism in a parent.

LEOPARD Syndrome-Disease Genes

PTPN11 PTPN11, which encodes SHP-2, was the first gene that was found to cause LS when mutated [37, 38]. SHP-2 is a member of a small subfamily of cytoplasmic Src homology 2 (SH2) domain-containing protein tyrosine phosphatases functioning as intracellular signal transducers. SHP-2s structure comprises two tandemly arranged SH2 domains in its amino terminal half (N-SH2 and C-SH2), a single catalytic domain (PTP) and a carboxy-terminal tail containing two tyrosyl phosphorylation sites and a proline-rich stretch (fig. 2). The N-SH2 and C-SH2 domains selectively bind to short amino acid motifs containing phosphotyrosyl residues and promote SHP-2s association with cell surface receptors, cell adhesion

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Y279S

T468M

N-SH2

C-SH2

PTP

Y279S

A461T G464A R498W,L Q506P Q510E,G

Fig. 2. PTPN11 mutations causing LEOPARD syndrome. Schematic representation of the PTPN11 protein, SHP-2, showing the LEOPARD syndrome-associated mutations with the most common two above and less frequent eight below. SH2 = src homology 2 domain; PTP = protein tyrosine phosphatase.

molecules and scaffolding adapters. The N-SH2 domain also interacts with the PTP domain using a separate site, which functions as an intramolecular switch controlling SHP-2s catalytic activation and translocation [39]. Specifically, the N-SH2 domain interacts with the PTP domain basally, blocking the catalytic site. Binding of the N-SH2 phosphopeptide-binding site to a phosphotyrosyl ligand promotes a conformational change of the domain that weakens the auto-inhibiting intramolecular interaction, making the catalytic site available to substrate, thereby activating the phosphatase [39]. SHP-2 positively modulates signal flow in most circumstances but can also function as a negative regulator depending upon its binding partner and interactions with downstream signaling networks. It has been documented consistently that SHP-2 positively controls the activation of the RAS-MAPK cascade induced by a number of growth factors and cytokines [4046], as well as IL-1 and TNF-dependent NF-B activation [47] and PDGF- and FGF2-induced Ca2+ signaling [48], while it negatively regulates STAT function [4951] and IFN signaling [52]. Similarly,

SHP-2 seems to play either a positive or a negative role in PI3K signaling downstream from activated growth factor receptors [53, 54]. In most cases, SHP-2s function in intracellular signaling appears to be immediately distal to activated receptors and upstream to RAS, but its function downstream of, or parallel to, RAS activation has also been demonstrated [55]. In 2001, Tartaglia and co-workers established that PTPN11 mutations cause NS [56]. Subsequently, they and others documented that PTPN11 mutations underlie approximately 50% of NS cases [5761]. Nearly all molecular lesions in NS are missense mutations. The distribution of the altered amino acid residues in SHP-2 has a non-random pattern. Specifically, most mutations affect residues located in the N-SH2 or PTP domains and involved in the N-SH2/PTP interdomain binding network that stabilizes SHP-2 in its catalytically inactive conformation or are in close spatial proximity to them. One mutation, N308D, constitutes 30% of lesions due to recurrent occurrences. Digilio and co-workers screened eight independent individuals with LS for PTPN11 defects, hypothesizing that NS and LS might be allelic [37]. This notion proved correct as they identified missense mutations in seven. They also found mutations in two young individuals with a phenotype suggestive of NS and CLS. Among these nine independent mutations, three were A-to-G transitions at position 386 (Y279C) and six were C-to-T transitions at position 1403 (T468M). The Y279C mutation had been found previously in an infant diagnosed with NS; for this and the two young patients from the cohort of Digilio et al., re-examination later in life revealed the presence of multiple CLS or lentigines, changing their diagnosis to LS (S. Jeffery, personal communication; our personal observation). Based on subsequent analysis of individuals affected with LS, PTPN11 mutations account for approximately 90% of cases. Y279C and T468M represent the most common defects, although a few additional mutations

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(Y279S, A461T, G464A, R498W/L, Q506P, and Q510E/G) have been documented subsequently (fig. 2) [14, 18, 38, 6266]. These results suggest specificity for these mutations for the development of lentigines, CLS, HCM, conduction abnormalities, and sensorineural deafness. RAF1 Mammalian genomes contain three RAF genes encoding ARAF, BRAF, and RAF1 (previously named CRAF). These RAF proteins are serine/ threonine kinases that are recruited to the plasma membrane by activated RAS, resulting in their activation [67]. Activated RAF kinases phosphorylate the dual specificity kinase MEK, which in turn activates MAPK. Unlike ARAF and BRAF, RAF1 is expressed ubiquitously. The RAF proteins share structural elements including three conserved regions (CR1, 2 and 3). CR1 contains two RAS-binding domains and a cysteine-rich domain, CR2 is rich in serine and threonine residues, and CR3 contains the kinase domain. RAF1 is highly regulated with numerous Ser and Thr residues that can be phosphorylated, resulting in activation or inactivation [68]. Among these, Ser259, which resides in CR2 (fig. 3), is particularly important. In its inactive conformation, the N-terminal portion of RAF1 is thought to interact with and inactivate the kinase domain at the C-terminus. This conformation is stabilized by 1433 protein dimers that bind to phosphorylated Ser259 and Ser621 [69]. Dephosphorylation of Ser259, which is possibly mediated by protein phosphatase-2A (PP2A) or protein phosphatase 1C (PP1C), facilitates binding of RAF1 to RASGTP at the membrane and subsequent propagation of the signal through the RAS-MAPK cascade via RAF1s MEK kinase activity. In 2007, Pandit and colleagues screened six individuals with LS who did not harbor PTPN11 mutations for RAF1 mutations [70]. This was premised on their discovery of RAF1 mutations in approximately 3% of patients with NS. They discovered missense defects in two patients, predicting

S257L CR1 RBD CRD


CR2

L613V CR3 Activation segment S621

S259

Fig. 3. RAF1 mutations causing LEOPARD syndrome. Schematic representation of RAF1 with two mutations indicated above and critical domains and serine residues that can be phosphorylated as part of RAF1s regulation below. CR = Conserved region; RBD = Ras binding domain; CRD = cyteine-rich domain.

S257L and L613V substitutions in RAFs CR2 and C-terminal domains, respectively.

Genotype-Phenotype Correlations

LS patients with PTPN11 mutations show a fullblown phenotype with a high prevalence of HCM [63]. Clinical analyses of a large cohort of LS patients with PTPN11 mutation indicate that patients inheriting the Thr468Met allele show short stature and deafness less frequently compared to those with missense mutations affecting residue Tyr279 (26 vs. 47% and 9 vs. 24%, respectively) [14 and our unpublished observations]. Such a genotype-phenotype correlation was also observed by Zenker and co-workers [61], who noticed a less adverse effect of the Thr468Met change on body growth and cardiac development. Other reports indicate that mutations affecting residue Gln510 are often associated with an important cardiac phenotype, characterized by rapidly progressive severe biventricular obstructive HCM, often with prenatal onset [71, 72 and our personal observation]. LS patients without PTPN11 mutations show a higher prevalence of ECG abnormalities and HCM [12]. Since only two LS patients with RAF1 mutations have been reported [70], no genotype-phenotype inferences can be made.

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Consequences of LEOPARD SyndromeCausing Mutations on RAS Signaling

PTPN11 Three LS-associated SHP-2 mutants, Y279C, T468M and Q510P, have been expressed in bacteria and/or eukaryotic cells, and phosphatase activities assayed from isolated SHP-2 proteins or immunocomplexes [7375]. All studies demonstrated extremely low basal phosphatase activities for the Y279C and T468M mutants and that these proteins could be stimulated with phosphopeptides or phosphoproteins (resulting in roughly 10% of activity observed in stimulated wild type SHP-2). In contrast, the single study of Q510P protein showed that this mutant possessed minimal basal phosphatase activity that did not respond to stimulation [75]. When expressed in eukaryotic cells, the Y279C and T468M mutants decreased activation of MAPK in a dominant negative manner and increased association of those SHP-2 proteins with the docking protein GAB1 [73]. Of note, the biochemical behaviors of these LS mutants contrast sharply with those observed for SHP-2 mutants relevant for NS. NS-associated SHP-2 mutants engender gainof-function effects with increased phosphatase activity and increased activation of RAS-MAPK signaling as measured by MAPK phosphorylation status [74, 76, 77]. This has been confirmed in a mouse model in which an NS-causing mutation, D61G, was introduced into the Ptpn11 gene [78]. The D61G heterozygous mice, which exhibit many of the hallmark features of the disorder, have increased MAPK activation during development. These findings in LS and NS have generated an enigma: how do mutations with seemingly opposite effects on SHP-2 engender phenotypes that are closely similar? Of note, mice engineered to be hemizygous for Shp-2 have no phenotype [79] and no lesion that would be expected to eliminate SHP-2 (e.g., nonsense or frame-shift

mutation near the N-terminus) has been observed in LS [74]. Thus, loss of SHP-2 activity per se seems unlikely to explain the pathogenesis of this disorder. Rather, mechanisms such as the dominant negative effects on MAPK activation and increased binding to docking partners [73] are being explored in ongoing animal modeling studies. RAF1 The two RAF1 mutants observed in LS, S257L and L613V, have been expressed in eukaryotic cells [70, 80]. The S257L RAF1 had normal phosphorylation at Ser259, but failed to bind 1433 and had increased kinase activity [80]. This mutant protein was more readily recruited from the cytosol to the plasma membrane by activated RAS and resulted in increased downstream signaling to MAPK. The mechanism for this gain of function relates to loss of 1433 binding at Ser259, which normally serves to inactivate RAF1. The L613V RAF1 mutant also possessed increased kinase activity and induced increased MAPK activation [70]. Binding of 1433 at the nearby Ser621 was preserved. Since a role for Leu613 in the regulation of RAF1 has not been identified previously, the precise mechanism through which the L613V substitution results in RAF1 activation remains to be established. Of note, the RAF1 mutants causing LS behaved similarly to several observed in patients with NS [70]. In fact, the S257L allele was found in patients with NS. Thus, the congruity between gain-of-function effects on RAF1 causing these disorders contrasts strikingly with the disparity of effects noted among the SHP-2 mutants associated with LS and NS. Aside from providing additional weight to the argument that the LS-associated PTPN11 mutations are not merely loss-of-function alleles, the overlap among the RAF1 alleles raises further issues about what determines whether an individual manifests LS versus NS.

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Acknowledgements
Research in the authors laboratories is supported in part by grants from the Italian Ministry of Health (Ricerca Corrente 2007, to A.S.; Progetto Programma Italia-

Usa, Malattie Rare to A.S. and M.T.), Telethon-Italy (GGP07115, to M.T.), Italian Ministry of University and Research (Progetto Ateneo 2007, to B.D.), and National Institutes of Health (HD001294, HL071207 and HL074728, to B.D.G.).

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Bruce D. Gelb Center for Molecular Cardiology, Departments of Pediatrics and Genetics & Genomic Sciences Mount Sinai School of Medicine New York, NY 10029 (USA) Tel. +1 212 241 3302, Fax +1 212 241 3310, E-Mail bruce.gelb@mssm.edu

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Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 6672

The Clinical Phenotype of Cardiofaciocutaneous Syndrome (CFC)


A.E. Roberts
Department of Cardiology and Division of Genetics, Childrens Hospital Boston, and Harvard Medical School Partners HealthCare Center for Genetics and Genomics, Boston, Mass., USA

Abstract
The first publication describing eight patients with cardiofaciocutaneous syndrome (CFC) focused on the findings of growth and psychomotor retardation, characteristic facial appearance, congenital heart defect, and ectodermal dysplasia [1]. Twenty years later, these findings have held up as those that are most commonly found in CFC. Many features overlap with Noonan syndrome and the two disorders are often confused. Efforts have been made to identify distinguishing features. The neurological and ectodermal findings appear to be the most specific and sensitive for CFC.
Copyright 2009 S. Karger AG, Basel

Clinical Diagnosis

In evaluating 56 published cases of CFC syndrome, Grebe and Clericuzio identified a subset with a specific, severe phenotype distinct from that of Noonan syndrome and proposed that the more mildly affected cases may actually be Noonan syndrome cases or a different diagnosis [2]. They proposed stringent diagnostic criteria; patients had to have at least seven of ten findings including macrocephaly, characteristic facial features, growth retardation, cardiac defect, sparse/ curly hair, neurological impairment/developmental delay, gastrointestinal dysfunction, ocular

abnormalities/dysfunction, history of polyhydramnios, and hyperkeratotic skin lesions. Because none of the traits associated with CFC are either obligatory or specific, Kavamura et al. [3], using data available from 54 published CFC cases, developed the CFC Index for the clinical diagnosis of CFC. This method is also thought to clinically differentiate CFC from Noonan syndrome and from Costello syndrome. The Index is based upon 82 clinical traits and their frequencies in the population with CFC. For a given patient in whom the diagnosis is being considered, the frequency of each feature present is added and the score compared to the CFC Index distribution (95% of the CFC population has a score between 9.5 and 19.9). Patients with Noonan syndrome and Costello syndrome had CFC Index scores below two standard deviations from the mean [3]. With the recent discovery of the molecular genetic causes of CFC, it will be important to assess the positive predictive values of the Grebe and Clericuzio criteria and of the CFC Index as tools to be used in the clinic to determine the likelihood of finding a gene mutation. Narumi et al. [4] calculated an average CFC Index for three KRAS patients (16.7), 16 BRAF patients (16.0) and six MEK1/2 patients (16.8), all consistent with the diagnosis.

Prenatal History

Three of the original eight cases of CFC reported had a prenatal history of polyhydramnios [1]. A retrospective analysis of 53 cases found ten (18%) had a history of polyhydramnios [5]. No other prenatal findings have been reported to be characteristic of CFC.

Growth

The birth weight is most often within the average range and about a third fall above the 75th centile [5]. However, postnatal growth retardation is found in a majority of children. Eight of the first ten cases reported had growth delay [1, 6]. Though feeding issues can be part of the problem, nutrition is not the only factor. Fifteen of fifty-eight children (26%) with CFC were reported to have a feeding problem [7], lower than the estimated 75% incidence of growth failure [8, 5]. The growth delay can persist and short stature is found in almost 80% of CFC cases [9]. There have not been published reports as to the prevalence of growth hormone deficiency or the efficacy of growth hormone replacement therapy.

with molecular genetic confirmation (a pathogenic BRAF, KRAS, MEK1 or MEK2 mutation) found that 95% had speech delay and 100% had MR [11]. Another 25 cases with a CFC gene mutation were reported and also had a 100% prevalence of MR [4]. Ward et al. [10] reported a family with features of CFC but also with features thought to be distinctive of Noonan syndrome including normal development and cognition, bleeding diathesis and ocular abnormalities. Manoukian et al. [12] reported an adult with a phenotype of CFC (valvular and infundibular pulmonic stenosis, brittle and wooly hair with patchy alopecia, dry and hypohydrotic skin, and characteristic facial traits) who did not have mental retardation. In retrospect it is possible that these are misclassified cases of CFC and are actually SOS1 positive Noonan syndrome cases. Noonan syndrome caused by mutations in the SOS1 gene often have accompanying hair and skin findings seen in CFC and mental retardation is not found in a majority of patients [13].

Facial Features

Development

Generalized hypotonia is common. When children are followed over time, there is significant psychomotor delay in infancy and early childhood which improves with age [10]. The cognitive potential is thought to be limited though this in part may be due to the fact that mental retardation (MR) is considered one of the main diagnostic criteria. In the literature, MR (usually mild to moderate) has been described in most cases of CFC with few exceptions. Wieczoreks retrospective analysis of 53 cases in the literature documented a prevalence of mental retardation of 94% [5]. A recent report of 23 cases of CFC

The typical facial features as described in the first cases published, include high forehead with bitemporal constriction, posteriorly rotated ears with thick helix, high boxy appearance of the cranial vault, hypoplasia of supraorbital ridge, hypertelorism, downslanting palpebral fissures, epicanthal folds, ptosis, depressed nasal bridge with anteverted nares, and highly arched palate (fig. 1) [1]. Before age 5 or 6, it can be difficult to discriminate Noonan syndrome from CFC though even in this age group the facial features of CFC are thought to be more coarse and dolichocephaly is more often present [14]. In a retrospective analysis of 53 cases, 92.5% were found to have typical facial features and 70% to have relative macrocephaly [5]. Narumi et al. [4] reported on the facial features of 25 cases of CFC syndrome with a BRAF,

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KRAS, MEK1 or MEK2 mutation and found each feature (hypertelorism, low and posteriorly rotated ears, thick ears, anteverted nostrils, depressed nasal bridge, relative macrocephaly, bitemporal constriction, high cranial vault, and hypoplasia of the supraorbital ridges) to have a frequency of 68 92%. Based upon long term follow-up of several individuals, it is thought that the face becomes less typical with age [14]. Anthropometric analysis of 35 children with CFC showed increased facial widths, with facial depths and circumference closer to normal, a broad nose and mouth, and widely spaced eyes [15].

Cardiovascular Features

The majority of children with CFC syndrome have a congenital heart malformation though there are many children with structurally normal hearts (79% in an analysis of 53 cases [5] and 84% in an analysis of 25 CFC gene mutation positive cases [4] had a congenital heart malformation). The most common findings are the same as those found in Noonan and Costello syndrome, namely pulmonary stenosis and hypertrophic cardiomyopathy though the relative prevalence differs somewhat by report. Wieczorek et al. [5] found 38% with pulmonary stenosis (75% was mild or trivial PS), 29% with atrial septal defect, and 24% with hypertrophic cardiomyopathy. In contrast, among 25 CFC gene mutation positive cases, 52% had cardiomyopathy, 43% pulmonic stenosis, and 9% atrial septal defect [4]. A second analysis of 23 CFC gene mutation positive cases reported a prevalence of 77% congenital heart disease, 40% with pulmonic valve stenosis, 35% with hypertrophic cardiomyopathy, and 23% with atrial septal defect [11]. Ventricular septal defect and partial atrioventricular canal, thickened mitral valve and mitral valve prolapse have also been reported [5]. It appears that adults without previous evidence of cardiomyopathy may be at risk for late

Fig. 1. Almost 2-year-old girl with BRAF positive CFC and hydrocephalus. Note the high forehead with bitemporal constriction, posteriorly rotated ears with thick helix, high boxy appearance of the cranial vault, hypoplasia of supraorbital ridge, hypertelorism, downslanting palpebral fissues, and depressed nasal bridge with anteverted nares.

onset. Two young adults with severe hypertrophic cardiomyopathy died, one at age 21 and one at 22 years of age. The former also had grade three pulmonary hypertension. Neither was diagnosed with hypertrophic cardiomyopathy until their early 20s. A 52-year-old woman with CFC was not diagnosed with hypertrophic cardiomyopathy until her late 40s [16]. This argues strongly for lifelong cardiac follow-up for all people with CFC.

Neurological Features

In the original series of eight patients with CFC, two had hydrocephalus, one had frontal lobe hypoplasia, and three had an abnormal EEG [1]. Subsequent reports have added cortical atrophy

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[8], seizure disorder [8], hypoplasia of the corpus callosum [5], nonspecific leucodystrophy of the right frontal region [17], brain stem atrophy [5, 18] and hypotonia [5] to the list of potential CNS features in CFC syndrome. In a review of the brain MRI results of 32 cases of gene mutation positive CFC, 14 children had ventriculomegaly, eight had hydrocephalus (three requiring shunts), six had prominent Virchow-Robin spaces, four had abnormalities of myelination, two had type I Chiari malformation, two had subependymal grey matter heterotopia, and one had an arachnoid cyst [19]. In the same study, corticospinal tract findings (hyperreflexia, extensor plantar response) were observed in 7 out of 22 participants who were evaluated by detailed neurological exam [19]. Among CFC gene mutation positive cases that have been reported, 1636% [11, 4] have been found to have seizures and 56% hypotonia [4]. More common than clinical seizures are abnormal EEG findings including decreased anterior voltage, spike-wave or polyspike pattern, sharp and slow waves, generalized dysrhythmia grade I and II, irritative waves, or generalized disorganization [1, 8, 17]. The finding of congenital hypertrophy of peripheral nerves with onion bulb formation was reported at the time of autopsy in a 7-year-old boy with CFC who died after cardiac arrest [20]. There is a case report of a 4-year-old girl with BRAF gene mutation positive CFC and a muscular Coenzyme Q10 (CoQ10) deficiency who improved significantly with CoQ10 treatment [21]. CoQ10 deficiency is a rare treatable mitochondrial disorder. This case could implicate a connection between the MAPK pathway and the mitochondria. There has been one case report of moyamoya syndrome in a child with CFC [22].

of CFC [14]. In the original eight cases published, the hair and skin manifestations noted included sparse, friable, curly hair, patchy alopecia, ichthyosis, hyperkeratosis, and absent eyebrows [1]. Ulerythema ophryogenes (absent eyebrows with hyperkeratosis) is also a common finding. In a retrospective analysis of the first 25 cases, caf-au-lait spots, acanthosis nigricans, syringocystadenoma papilliferum, hemangioma, and dysplastic nails or teeth were added to the ectodermal manifestations [23]. Generalized pigmentation of the skin was also noted [24]. A single case report noted facial and body hemihidrosis in a 3-year-old girl [25]. In reviewing 58 cases, Weiss et al. reported that 100% had a cutaneous abnormality; 93% had hyperkeratosis and 21% hemangioma. All cases also had hair abnormalities [26]. A baby with KRAS gene mutation positive CFC was reported to have ulcerating hemangioma [27]. Of 25 CFC gene mutation positive cases, 36% had absent eyebrows, 64% had sparse eyebrows, 68% had each of sparse eyelashes, dry skin, or thin skin, and 96% had curly hair and/or sparse hair [4]. A second report of 23 CFC gene mutation positive cases reported that 63% had deep palmoplantar creases (a feature usually thought to be seen primarily in the phenotypically related Costello syndrome), 67% had sparse or absent eye lashes, 78% had sparse or absent eyebrows, 91% had curly hair, and 95% had sparse hair [11]. Based upon long term follow-up of several individuals, it is thought that with age the hair becomes thicker and more typical in texture and the dryness of the skin and the follicular keratosis improve with age [14].

Ophthalmologic Features Ectodermal Features

Follicular hyperkeratosis of the arms, legs, and face and sparse, slow-growing curly hair are considered to be hallmark ectodermal findings

Half of the original case series had an eye abnormality: esotropia and/or strabismus [1]. Young and colleagues have examined and reported the most patients to date [14, 28, 29]. One third of

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patients have strabismus (exotropia more common then esotropia). Nystagmus is found in 36% and myopia in 20% [28, 29]. There were three cases of optic nerve hypoplasia and one of optic nerve atrophy possibly secondary to increased intracranial pressure [14]. There were single cases of cataract, vertical strabismus, dissociated vertical deviation and inferior oblique muscle overaction, nasolacrimal duct obstruction, and keratoconus [14].

Renal Features

Individuals with CFC syndrome are not thought to be at increased risk of kidney malformation or dysfunction. However, there is a single case report of persistent hypercalciuria, nephrolithiasis, medullary nephrocalcinosis, obstructing ureteral calculi and bladder calculi, and renal cysts [31].

Skeletal Features Gastrointestinal Features

Gastrointestinal problems, both structural and functional, are thought to affect one third of children with CFC syndrome [5]. Early feeding problems are common and can include poor suck, aspiration, gastroesophageal reflux, and dysmotility. Nasogastric tube feeding may be required and some go on to require gastrostomy tube placement. Umbilical hernia was reported in two of the first eight cases [1] and later in seven of the first 58 cases [7]. Overall, 47% of these 58 cases had at least one gastrointestinal abnormality including feeding problems (15 cases), splenomegaly (8 cases), hepatomegaly (4 cases), umbilical hernia (7 cases), or inguinal hernia (3 cases). There was a single case of anal stenosis. Extensive food allergies have also been reported in one child [6]. A 17-yearold woman was reported to have fatty liver with both macro- and microvesicular steatosis of unclear etiology [30]. There was also a case report of antral narrowing on upper GI with delayed emptying and biopsies showing foveolar hyperplasia (though no evidence of infiltration on culture or staining) and functional megacolon from severe constipation [31]. There have been two cases of malrotation reported [7, 31]. One of the originally described cases, now in her early 30s, became critically ill when she had pneumonia complicated by intestinal obstruction [14].

There are very few reports of orthopaedic issues for people with CFC syndrome. This could reflect a lack of study rather than a true absence of orthopaedic problems. Ades et al. [32] reported a single case of bilateral progressive femoral valgus deformity. There was also a case of a 17-yearold with a bone age of 11 years, scoliosis, and osteopenia (the same case described above with hypercalciuria) [31].

Malignancy

Solid tumors have been reported with increased frequency in Costello syndrome and leukemia is of increased prevalence in Noonan syndrome. With only a handful of cases reported, the predisposition for malignancy in CFC syndrome is less clear. In 1999, van den Berg and Hennekam [33] reported a case of a child with CFC syndrome and ALL with the TEL/AML1 fusion who was treated with vincristine, dexamethasone, and E. coli asparaginase and remission was achieved in five weeks. Subsequently, a 9-year-old child with a BRAF mutation who had ALL at 21 months of age has been published [4]. Finally, a 35-monthold MEK1 mutation positive patient with CFC and a history of hypertrophic cardiomyopathy requiring heart transplant presented with metastatic hepatoblastoma [34]. It is possible that the hepatoblastoma was secondary to the immunosuppression required after transplant but since

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hepatoblastoma can be seen in Costello syndrome, it could also be related to the common perturbation of the Ras/MAPK pathway in CFC and Costello syndromes.

at age 47 with chest pain and was diagnosed with hypertrophic cardiomyopathy.

Conclusions Natural History

With so few cases reported and very little long term follow-up, the natural history of CFC remains largely unknown. A 26-year-old man with CFC presented with gait deterioration, intention tremor, distal weakness of the upper limbs with atrophy of the thenar, hypothenar, and interossei muscles, and large fiber sensory loss in all limbs. Nerve conduction studies and electromyography demonstrated a moderately severe axonal neuropathy [35]. The oldest adult reported to date is a 52-year-old woman with CFC [16]. As an adult she had fine, brittle, slow growing hair, dry scaly skin, height less than the 3%, bilateral ptosis, lax skin on her hands, deep creases on her palms (no evidence of nasal papillomata), and osteoporosis. She had a history of learning problems and lived in sheltered accommodations. She went through menopause in her late 40s. Of note, she presented

CFC is a rare genetic disorder with cardiac, ectodermal, growth, and cognitive involvement. Multiple additional systems can also be involved. CFC can be difficult to distinguish in the first few years from Noonan syndrome and none of the features are exclusive to CFC likely owing to the fact that both disorders are caused by genes that are part of the Ras/MAPK pathway. Much of our knowledge of the clinical findings to date is a result of case reports or small cases series. This remains a crucial way of describing the breadth of the phenotype though, until more cases are studied, it will be difficult to determine if these single case report findings are coincidental or truly related to CFC. Recent molecular genetic discoveries have greatly enhanced the ability to diagnose CFC though not all cases are explained by the genes found to date. Therefore, there remains a place for careful clinical diagnosis.

References
1 Reynolds JF, Neri G, Herrmann JP, Blumberg B, Coldwell JG, et al: New multiple congenital anomalies/mental retardation syndrome with cardio-facio-cutaneous involvement the CFC syndrome. Am J Med Genet 1986;25:413427. 2 Grebe TA, Clericuzio C: Neurologic and gastrointestinal dysfunction in cardio-facio-cutaneous syndrome: identification of a severe phenotype. Am J Med Genet 2000;95:135143. 3 Kavamura MI, Peres CA, Alchorne MMA, Brunoni D: CFC index for the diagnosis of cardiofaciocutaneous syndrome. Am J Med Genet 2002;112:1216. 4 Narumi Y, Aoki Y, Niihori T, Neri G, Cave H, et al: Molecular and clinical characterization of cardio-facio-cutaneous (CFC) syndrome. Am J Med Genet A 2007;143:799807. 5 Wieczorek D, Majewski F, GillessenKaesbach G: Cardio-facio-cutaneous (CFC) syndrome a distinct entity? Report of three patients demonstrating the diagnostic difficulties in delineation of the CFC syndrome. Clin Genet 1997;52:3746. 6 Neri G, Sabatino G, Bertini E, Genuardi M: Brief clinical report: the CFC syndrome report of the first two cases outside of the United States. Am J Med Genet 1987;27:767771. 7 McDaniel CH, Fujimoto A: Intestinal malrotation in a child with cardio-facio-cutaneous syndrome. Am J Med Genet 1997;70:284286. 8 Raymond G, Holmes LB: Cardio-faciocutaneous syndrome: neurological features in two children. Dev Med Child Neurol 1993;35:727741. 9 Kavamura MI, Pomponi MG, Zollino M, Lecce R, Murdola M, et al: PTPN11 mutations are not responsible for the cardiofaciocutaneous syndrome. Eur J Med Genet 2003;11:6468. 10 Ward KA, Moss C, McKeown C: The cardio-facio-cutaneous syndrome: a manifestation of the Noonan syndrome? Br J Dermatol 1994;131: 270274.

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11 Nava C, Hanna N, Michot C, Pereira S, Pouvreau N, et al: CFC and Noonan syndrome due to mutations in RAS/ MAPK signaling pathway: genotype/ phenotype relationships and overlap with Costello syndrome. J Med Genet 2007;44:763771. 12 Manoukian S, Lalatta F, Selicorni A, Tadini G, Cavalli R, Neri G: Cardiofacio-cutaneous (CFC) syndrome: report of an adult without mental retardation. Am J Med Genet 1996;63:382385. 13 Tartaglia M, Pennacchio LA, Zhao C, Yadav KK, Fodale V, et al: Gain-offunction SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet 2007;39:7579. 14 Roberts A, Allanson J, Jadico SK, Kavamura MI, Noonan J, et al: The cardiofaciocutaneous syndrome. J Med Genet 2006;43:833842. 15 Allanson J, Opitz JM, Carey JC, Viskochil D, Noonan J, et al: Cardiofacio-cutaneous syndrome: a distinct entity. Proc Greenwood Genet Ctr 2002;21:67. 16 McGaughran J: Cardio-facio-cutaneous syndrome: first presentation in a 52-year old woman. Am J Med Genet A 2003;116:210212. 17 Sabatino G, Verrotti A, Domizio S, Angelozzi B, Chiarelli F, Neri G: The cardio-facio-cutaneous syndrome: a longterm follow-up of two patients, with special reference to the neurologic features. Child Nerv Syst 1997;13: 238241. 18 Ion A, Tartaglia M, Song X, Kalidas K, van der Burgt I, et al: Absence of PTPN11 mutations in 28 cases of cardiofaciocutaneous (CFC) syndrome. Hum Genet 2002;111:421427.

19 Yoon G, Rosenberg J, Blaser S, Rauen KA: Neurological complications of cardio-facio-cutaneous syndrome. Dev Med Child Neurol 2007;49:894899. 20 Manci EA, Martinez JE, Horenstein MG, Gardner TM, Ahmed A, et al: Cardiofaciocutaneous syndrome (CFC) with congenital peripheral neuropathy and nonorganic malnutrition: an autopsy study. Am J Med Genet A 2005;137:18. 21 Aeby A, Snajer Y, Cave H, Rebuffat E, Van Coster R, et al: Cardiofaciocutaneous (CFC) syndrome associated with mucular coenzyme Q10 deficiency. J Inherit Metab Dis 2007;30:827. 22 Mathews CA, George P, Hood AF: Cardiofaciocutaneous syndrome. Arch Dermatol 1993;129:4647. 23 Ishiguro Y, Kubota T, Takenaka J, Maruyama K, Okumura A, et al: Cardio-facio-cutaneous syndrome and moyamoya syndrome. Brain Dev 2002;24:245249. 24 Turnpenny PD, Dean JCS, Auchterlonie IA, Johnston AW: Cardiofaciocutaneous syndrome with new ectodermal manifestations. J Med Genet 1992;29:428429. 25 Leal-Ugarte E, Macias-Gomez NM, Gutierrez-Angulo M, Barros-Nunez P: Cardio-facio-cutaneous syndrome with hemihidrosis: ectodermal dysplasias spectrum? Int J Dermatol 2006;45:14811482. 26 Weiss G, Confino Y, Shemer A, Traut H: Cutaneous manifestations in the cardiofaciocutaneous syndrome, a variant of the classical Noonan syndrome. Report of a case and review of the literature. JEADV 2004;18:324327.

27 Tang B, Reardon W, Black GC, Kerr BA: Congenital ulcerating hemangioma in a baby with KRAS mutation and cardio-facio-cutaneous syndrome. Clin Dysmorphol 2007;16:203206. 28 Young TL, Ziylan S, Schaffer DB: The ophthalmologic manifestations of the cardio-facio-cutaneous syndrome. J Pediatr Ophthalmol Strabismus 1993;30:4852. 29 Young TL: Cardio-facio-cutaneous syndrome conference ophthalmologic findings summary. Rockville Maryland, June 2003;http://www.cfcsyndrome.org/conference-summary.htm. 30 Nanda S, Rajpal M, Reddy BSN: Cardio-facio-cutaneous syndrome: report of a case with a review of the literature. Int Soc Dermatol 2004;43:447450. 31 Herman TE, McAlister WH: Gastrointestinal and renal abnormalities in cardio-facio-cutaneous syndrome. Pediatr Radiol 2005;35:202205. 32 Ades LC, Sillence DO, Rogers M: Cardiofaciocutaneous syndrome. Clin Dysmorphol 1992;1:145150. 33 Van Den Berg H, Hennekam RCM: Acute lymphoblastic leukemia in a patient with cardiofaciocutaneous syndrome. J Med Genet 1999;36:799800. 34 Al-Rahawan MM, Chute DJ, SolChurch K, Gripp KW, Stabley DL, et al: Hepatoblastoma and heart transplantation in a patient with cardio-faciocutaneous syndrome. Am J Med Genet A 2007;143:14811488. 35 DeRoos ST, Ryan MM, Ouvrier RA: Peripheral neuropathy in cardiofaciocutaneous syndrome. Pediatr Neurol 2007;36:250252.

Amy E. Roberts, MD Cardiovascular Genetics, Department of Cardiology, Farley 2, Childrens Hospital Boston 300 Longwood Ave Boston, MA 02115 (USA) Tel. +1 617 355 6529, Fax +1 617 713 3808, E-Mail amy.roberts@childrens.harvard.edu

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Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 7382

Molecular Causes of the CardioFacio-Cutaneous Syndrome


W.E. Tidymana K.A. Rauenb,c
aDepartment of Anatomy, and bDepartment of Pediatrics, Division of Medical Genetics, University of California, and cUCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, Calif., USA

Abstract
Cardio-facio-cutaneous (CFC) syndrome is a rare multiple congenital anomaly disorder in which individuals have characteristic dysmorphic craniofacial features, cardiac defects, ectodermal anomalies, developmental delay and hypotonia. CFC is caused by alteration of activity through the mitogen-activated protein kinase (MAPK) pathway due to heterozygous de novo mutations in protein kinases BRaf, MEK1 or MEK2. Mutations in K-Ras, a small GTPase, have also been implicated as causing CFC syndrome and Noonan syndrome, however its role has yet to be well defined. In those individuals who are found to have a mutation, the majority occur in BRAF, whereas, mutations in MEK1 or MEK2 comprise about 27%. Functional studies of these novel CFC mutant proteins demonstrate that B-Raf may be activated or kinase impaired, whereas all the MEK mutant proteins studied to date demonstrate increased activity. Since CFC syndrome may have a progressive, evolving phenotype, the possible use of systemic therapies to reduce MAPK activity may be of great benefit to this population of patients and certainly warrants investigation. However, animal studies on the effects of MAPK inhibitors will be essential because of the critical role this pathway plays during development.
Copyright 2009 S. Karger AG, Basel

Blumberg and colleagues at the March of Dimes Birth Defects Conference [1]. The three reported patients had characteristic facial features, ichthyosis with abnormal hair, ocular and cardiac abnormalities, postnatal growth failure and mental retardation. These three patients, in addition to five others, were subsequently reported by Reynolds and colleagues [2] who designated this new entity cardio-facio-cutaneous (CFC) syndrome based on their common phenotypic features. Since then, more than one hundred patients have been reported in the literature. Although the exact incidence of this rare syndrome is unknown, a conservative estimate is approximately three to four hundred individuals worldwide.

Overview of the Clinical Diagnosis of CFC Syndrome

In 1979, a mental retardation syndrome with distinctive craniofacial dysmorphology, ectodermal anomalies and cardiac defects was reported by

The clinical diagnosis of CFC syndrome is made by examining individuals for phenotypic features that are characteristic of the syndrome. However at the present time, no routine diagnostic criteria have been established. Individuals with CFC syndrome display phenotypic variability and,

c
Fig. 1. Individual with CFC syndrome who has a p.G596V missense substitution in B-Raf resulting from a nt 1787G T transversion mutation in exon 15 [20]. This individual is shown at various ages: (a) at birth, (b) age 2.5 years, (c) age 10.5 years and (d) at 25 years.

therefore, each affected individual may not possess all the characteristic features. Although CFC syndrome has a distinct phenotype, it shares many overlapping features with Noonan syndrome (NS) and Costello syndrome (CS). Craniofacial findings in CFC syndrome are reminiscent of those described in Noonan syndrome and include macrocephaly, broad forehead, bitemporal narrowing, hypoplasia of the supraorbital ridges, down-slanting palpebral

fissures with ptosis, short nose with depressed nasal bridge and anteverted nares, low-set, posteriorly rotated ears with prominent helices and a high-arched palate (fig. 1). Ectodermal findings typically consist of sparse, curly hair with sparse eyebrows and eyelashes, hyperkeratosis, keratosis pilaris, hemangioma, nevi and ichthyosis [3, 4]. Cardiac anomalies vary with the most prevalent being pulmonic stenosis, atrial septal defects, and hypertrophic cardiomyopathy [5].

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Musculoskeletal abnormalities are common, as are ocular abnormalities including strabismus, nystagmus, myopia, hyperopia and astigmatism [6]. Failure to thrive is common in infancy, as is gastrointestinal dysfunction such as reflux, vomiting, oral aversion and constipation [7, 8]. Intestinal malrotation and renal anomalies are not uncommon [9]. Also, some instances of chylothoraces and lymphedema have been reported at birth [10]. Neurologic abnormalities are universally present in CFC. In a recent study of 39 mutation-positive individuals, features that were present in this cohort included hypotonia, motor delay, speech delay and learning disability [11]. In addition, macrocephaly, ptosis, strabismus and nystagmus were present in more than 50% of individuals, and corticospinal tract findings present in 32%. Ventriculomegaly or hydrocephalus was present in 66% of participants. Other findings on MRI included prominent Virchow-Robin (perivascular) spaces (19%), abnormal myelination (13%), and structural anomalies (16%). Seizures were present in 38% of individuals. However, no specific genotype-phenotype correlations could be drawn. Neoplasia, such as benign papillomas or malignancies observed in CS, NS or neurofibromatosis type 1, has not been reported in CFC syndrome. Although it is unclear if individuals with CFC are at an increased risk to develop cancer, two individuals with CFC syndrome harboring BRAF mutations have been reported with acute lymphoblastic leukemia [12, 13] and one individual with a MEK1 mutation developed hepatoblastoma after a heart transplant while on immunosuppressive therapy [14].

Molecular Clues to the Genetic Etiology

Prior to understanding the molecular etiology of the Ras/Mitogen-activated protein kinase (MAPK) pathway syndromes, two camps of

thought existed as to whether NS, CFC syndrome and CS were distinct genetic disorders, or allelic with variable expressivity. Identification of the first gene, PTPN11, as causative for NS was the initial clue that these disorders were genetically distinct [15]. Since CFC had long been considered by some to be a more severe manifestation of NS, a cohort of CFC patients was screened for mutations in the PTPN11 gene [16]. Molecular investigations revealed no alterations in this gene, suggesting that NS and CFC are indeed distinct genetic entities. A subsequent study examined a well-characterized cohort of patients with Costello syndrome for PTPN11 mutations, and again no mutations were identified providing further evidence that NS, CS and probably CFC were genetically distinct [17]. Subsequently, a second gene in the Ras/MAPK pathway, HRAS, was identified as being causative for the CS [18]. Rauen and colleagues then confirmed that CFC syndrome was genetically distinct from CS, by screening a cohort of patients with the clinical diagnosis of CFC and finding no mutations in HRAS [19]. Because of phenotypic overlap among CS, NS and CFC syndrome, they went on to hypothesize that the molecular basis of pathogenesis may, therefore, be similar. Since NS and CS involve a Ras pathway perturbation affecting both development and predisposition to malignancy, it was reasonable to assume that the Ras pathway or a downstream effector was also causative of CFC syndrome, albeit without the oncogenic consequence that is observed in NS and CS [19]. They went on to identify three genes within the Ras/MAPK pathway, BRAF, MEK1 and MEK2, which were individually responsible for CFC syndrome [20]. From this, it became apparent that the overlapping phenotypes of NS, CS and CFC syndromes were due to the fact that all were caused by mutations within genes of the Ras/MAPK pathway. The Raf-mediated MAPK signaling cascade is one of the most studied downstream pathways

Molecular Causes of the Cardio-Facio-Cutaneous Syndrome

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BRAF

ATG 1 2 3 4

Q262R Q257K Q257R A246P T244P T241P 5 CR1 Ras binding domain
Cysteine-rich domain

T470 K499N G469Q K499E G469E V487G F468S E501V S467A L485S G466R L485F E501K E501G G464R G464V G534R 462469 7 8 CR2 9 10 11 12 G-loop 13

N580D N581K N581D F595L G596V D638E T599R 14 CR3 15 16 17 18

TGA

Activation segment

Protein kinase domain

Fig. 2. Schematic diagram of BRAF indicating causative mutations identified in CFC syndrome. The start and stop codons are indicated. Thirty-one novel CFC BRAF mutations affecting 23 different codons have been identified to date in CFC syndrome (see text). Mutations include heterozygous missense substitutions and in-frame deletions. The amino acid positions in red indicate that these codons have also been found altered in cancer (www.sanger.ac.uk/ genetics/CGP/cosmic).

of Ras and is highly conserved among eukaryotic organisms. It is critically involved in cell proliferation, differentiation, motility, apoptosis, and senescence, and serves various functions during development in a cell-specific fashion. Extracellular stimuli lead to the activation of Ras, which in turn activates Raf, a serine/threonine kinase (A-Raf, B-Raf, and/or C-Raf). Raf then phosphorylates and activates MEK1 and/ or MEK2 (MAPK kinase). MEK1 and MEK2 are threonine/tyrosine kinases with both isoforms having the ability to phosphorylate and activate ERK1 and ERK2 (MAPK). ERK, once activated by MEK, has numerous cytosolic and nuclear substrates [21]. Aberrant signaling, by overexpression or constitutive activation of this pathway, plays a key role in the pathogenesis and progression of many cancers. Hyperactivated ERK is found in approximately 30% of human cancers with pancreas, colon, lung, ovary and kidney demonstrating the highest levels of ERK activation [22]. Altered signaling through the MAPK pathway in cancer results from somatic mutations in upstream modulators of ERK, including K-Ras, N-Ras, H-Ras, B-Raf and C-Raf (Raf-1). Germline alteration of activation through this critical cancer pathway causes CFC syndrome [20, 23].

Molecular Pathology of CFC Syndrome

BRAF Mutations Two research groups independently identified mutations in BRAF as causing CFC syndrome [20, 23]. Rauen and colleagues [20] examined 23 unrelated individuals with the clinical diagnosis of CFC who were HRAS (the causal gene for CS) and PTPN11 (one causal gene for NS) mutationnegative. Heterogeneous missense mutations in BRAF were identified in 18/23 (78%) of individuals with CFC syndrome. Eleven distinct missense mutations clustered in two regions: in the cysteine-rich domain (CRD) of the conserved region 1 (CR1) and in the protein kinase domain (fig. 2). Niihori and colleagues [23] examined 43 CFC individuals and identified eight unique mutations among 16 individuals (37%). Functional analysis of the proteins resulting from BRAF harboring these mutations revealed that the type of BRAF mutations identified in CFC syndrome is similar to the different types of mutations identified in cancer [24]. Specifically, these mutations resulted in proteins with either high kinase, or kinase-impaired activities [20, 23]. B-Raf is a member of the Raf protein family which also includes C-Raf and the X-linked

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A-Raf. BRAF is located on chromosome 7q34, contains 18 exons with intervening sequences and spans approximately 190 kb. The protein product of BRAF is a serine/threonine protein kinase and is one of the many direct downstream effectors of Ras. There are three conserved regions in B-Raf. Conserved region 1 (CR1), part of the regulatory amino-terminal, contains the Ras binding domain and the cysteine-rich domain both of which are required for recruitment of B-Raf to the cell membrane. CR2, also part of the regulatory amino-terminal, is the smallest of the conserved regions. CR3 encompasses the kinase domain and contains a glycine rich loop (exon 11) and the activation segment (exon 15) of the catalytic domain. B-Rafs only known downstream effectors are MEK1 and MEK2. Somatic mutations in BRAF have been reported at a high frequency in numerous cancers including melanoma, thyroid, colorectal and ovarian cancer. Approximately 70 missense mutations affecting 34 codons have been reported (www.sanger.ac.uk/genetics/CGP/cosmic). The majority of BRAF mutations are missense substitutions found in, but not limited to, exon 11 (the glycine-rich loop) and exon 15 (the activation segment) in the kinase domain [25]. The crystal structure of B-Raf shows that the activation segment is held in an inactive conformation by association with the G-loop. Mutations in these two regions are believed to disrupt this interaction, converting B-Raf into its active conformation [24]. One mutation, B-Raf p.V600E, results from a TA transversion at nt1796 substituting glutamic acid for valine at position 600. B-Raf p.V600E, which has increased kinase activity, accounts for over 90% of BRAF mutations identified in human cancer. This mutation B-Raf protein may exert its oncogenic effect in a similar way to the oncogenic effect of an activated Ras. Some tumors do have mutual exclusivity of B-Raf p.V600E and K-Ras mutations, implying that mutation in either gene reflects redundant function in the activation of MAPK [2629].

Interestingly, somatic B-Raf p.V600E mutations are also found in benign skin lesions and premalignant colon polyps [2830]. The majority of benign nevi, as well as primary and metastatic melanoma, have the B-Raf p.V600E mutation. This suggests that MAPK activation is important in melanocytic neoplasia, but in isolation, is insufficient for tumorigenesis [30]. Although the majority of B-Raf mutations are kinase activating, inactivating mutations in B-Raf also play a causal role in human cancer. This is thought to occur through an indirect mechanism that in turn activates C-Raf and, therefore, also results in the activation of the MAPK cascade [24]. The majority of mutations that cause CFC occur in BRAF. Unlike the mutation spectrum seen in cancer, the BRAF mutations in CFC individuals are more widely distributed (fig. 2). Of the 152 non-overlapping CFC individuals that have been published to date, mutations in BRAF comprise 73% (111/152) [14, 20, 23, 3134; Rauen, unpublished data]. Thirty-one novel CFC BRAF mutations affecting 23 different codons have been identified. Of the 18 exons in BRAF, CFC mutations have been found in exon 6 and in exons 1116. The majority of missense mutations are in exon 6 (41%) and exon 12 (21%). The most common BRAF mutations occur in exon 6 and consist of the missense substitution Q257R (29%), in exon 12 at amino acid position E501 (12%) and in exon 11 consisting of the missense substitution G469E (6%). In contrast, virtually all BRAF mutations reported in cancer are found exclusively in exon 11 and 15; whereas, only 18% of CFC mutations are found in exon 11 and 7% in exon 15. Only rare exon 15 in-frame deletions have been reported in cancer [35, 36] and, similarly, rare inframe deletions in exon 11 have been identified in two CFC individuals [11]. MEK1 and MEK2 Mutations Missense mutations in MEK1 and MEK2, which encode downstream effectors of B-Raf, also cause CFC syndrome [20]. Missense MEK mutations

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were initially identified in three of the five CFC individuals who were BRAF-mutation negative (fig. 3). What is of particular interest is that no somatic or constitutional mutations had ever been described in MEK genes. Functional studies of the proteins encoded by these novel MEK1/2 mutations have shown that all of the CFC mutant proteins studied are more active than wildtype MEK in stimulating ERK phosphorylation, but they are not as active as an artificially generated constitutively active MEK mutant [20; Rauen, unpublished data]. MEK, like Raf, exists as a multigene family [37]. The MEK1 (MAP2K1) gene is located on chromosome 15q22.31 and spans approximately 104 kb. MEK2 (MAP2K2) is located on chromosome 19p13.3 and spans approximately 34 kb. Each gene contains 11 exons with intervening sequences. The MEK1 gene encodes the mitogen-activated protein kinase kinase 1 (MEK1), likewise MEK2 encodes the protein MEK2. MEK1 and MEK2 are threonine/tyrosine kinases with both isoforms having the ability to activate ERK1 and ERK2. The MEK1/2 proteins have about 85% amino acid identity [38] but do not serve redundant purposes [39, 40]. Unlike B-Raf, prior to the discovery of germline mutations in MEK, no naturally occurring mutations had ever been identified in either MEK1 or MEK2. MEK1 and MEK2 mutations comprise 27% (41/152) of mutations in CFC individuals in which a gene mutation has been identified [14, 20, 23, 3134; Rauen, unpublished data]. Mutations in MEK1 and MEK2 are seen in roughly equal frequency (fig. 3). The vast majority are missense substitutions and located in exons 2 and 3. The most common mutation is MEK1 Y130C comprising 41% of all the MEK mutations. Rare in-frame deletions have also been identified [11, 33]. MEK, like Raf, has been well studied in the context of cancer. Cellular transformation due to activation of the MAPK cascade ultimately is the result of MEK activation [41]. Constitutively active

MEK mutants, produced in the laboratory by deleting the N-terminus of the protein, or by altering key regulatory residues, promote transformation of mammalian cells both in vitro and in vivo [42]. Several primary human tumor cell lines have increased MEK1 and MEK2 activities [22, 43], but no somatic kinase domain MEK1 and MEK2 mutations have been reported in cancer [4447; www. sanger.ac.uk/genetics/CGP/cosmic]. Recently, the first functional MEK1 mutation was identified in an ovarian cancer cell line and functional studies determined that this mutant protein has increased activity as measured by an increase in ERK phosphorylation [48]. KRAS Mutations Two studies were simultaneously published that implicate KRAS mutations in a small percentage of individuals diagnosed with Noonan and CFC syndromes [23, 49]. Functional studies of these K-Ras mutant proteins reveal altered intrinsic GTPase activity, response to neurofibromin and response to p120 GAP when compared to the wildtype protein [49, 50]. This functional variability observed in the various K-Ras mutant proteins is reflected in the broad phenotypic spectrum of patients with KRAS mutations [51]. These studies confirm that clinically distinguishing between Noonan syndrome and CFC can be difficult and may partly explain the nature of overlapping phenotype between the two syndromes. Continued evaluation of the patient populations and functional studies will resolve these issues.

Making the Molecular Diagnosis of CFC Syndrome

CFC syndrome is one of many syndromes including NS, LEOPARD syndrome, CS and neurofibromatosis type 1 that are caused by alteration of signaling through the Ras/MAPK pathway [52]. As these syndromes share the same molecular developmental pathway, there are overlapping

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D67N P124L K59 G128V T55P Y130N F53S Y130C ATG E44G MEK1 1 2 3 4 5 6 7

TAA 8 9 10 11

Protein kinase domain A62P K61T K61E F57I F57C ATG 4655 MEK2 1 2

P128R G132V Y134C 3 4 5 6

K273R 7

TGA 8 9 10 11

Protein kinase domain

Fig. 3. Schematic diagram of MEK1 and MEK2 genes (not drawn to scale) indicating causative mutations identified in CFC syndrome (see text). The start and stop codons are indicated. The vast majority of mutations occur in exon 2 or 3. Like in BRAF, rare in-frame deletions have been identified. The MEK1 p.D67N mutation which has been identified in CFC [33] has also been identified in an ovarian cancer cell line (indicated in red) [48].

phenotypic features that are seen among individuals who have these disorders. Although CFC syndrome has a distinctive phenotype, there are many features in common with the other related Ras pathway syndromes [34]. Because of this developmental overlap, making the clinical diagnosis of CFC syndrome can be challenging, particularly during the newborn period. Currently, there are no specific criteria that are routinely used to establish the clinical diagnosis of CFC syndrome. Once a clinical diagnosis is considered, molecular testing can definitively confirm the clinical diagnosis. Obtaining a molecular diagnosis is important for many reasons, including appropriate management for the affected individual, cancer predisposition, recurrence risks for the family and emotional well-being of the parents. As CFC is caused by mutations in genes within the MAPK pathway and has a variable phenotype, a stepwise approach in the sequencing of these genes may be prudent. Identification of mutations in BRAF, MEK1 or MEK2 by direct gene sequencing

establishes the diagnosis of CFC syndrome. The initial step would be direct sequencing of the seven BRAF exons in which causative mutations have been identified (exons 6, 1116). If no causal mutation is identified, then direct sequencing of MEK1 (exons 2 and 3) and MEK2 (exons 2, 3 and 7) should be performed. If no causal mutation is identified, consider sequencing the remaining BRAF exons and remaining MEK1 and MEK2 exons in which causal mutations have not yet been reported. If no causal mutation is identified in BRAF, MEK1, and MEK2, then direct sequencing of the Noonan syndrome genes KRAS [23, 49, 51, 53] followed by SOS1 [5456; Rauen, unpublished data] should be considered whereby additional causal mutations have been demonstrated in individuals with a phenotype that overlaps CFC syndrome. Finally, if no causal mutation is identified, consider direct sequencing of HRAS (all exons) [18, 19, 57]. Individuals who have an HRAS mutation are considered to have a diagnosis of Costello syndrome [34, 58, 59].

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Future Directions and Possible Therapies for CFC

Hyperactivated ERK is found in approximately 30% of human cancers with pancreas, colon, lung, ovary and kidney demonstrating the highest levels of ERK activation [22]. One significant aspect of the identification of germline mutations in BRAF and MEK1/2 as causative for CFC syndrome was the finding that activating germline mutations within the MAPK pathway could be compatible with human development. The development of potential treatment for the group of Ras/MAPK syndromes, including CFC, woefully lags behind the ability to diagnose each at the molecular level. Since CFC syndrome may have a progressive, evolving phenotype, the possible use of systemic therapies to reduce MAPK activity may be of great benefit to this population of patients and certainly warrants investigation. However, animal studies on the effects of MAPK inhibitors will be essential because of the critical role this pathway plays during development. Initial functional studies of B-Raf CFC mutant proteins demonstrate that most have an increased kinase activity, but a few appear to be kinase impaired [20, 23]. In contrast, all MEK1/2 mutants

characterized in vitro confer an increase of kinase activity [20; Rauen, unpublished data]. As the majority of the CFC BRAF mutations are novel and no MEK mutations have ever been identified prior to their discovery in CFC syndrome, the biochemical aspects of the novel mutants, as well as, the roles of these MEK mutants in the MAPK signaling cascade requires further examination. Recent in vitro analysis of select MEK1 and MEK2 CFC mutant proteins have shown that these CFC MEK variants are sensitive to the MEK inhibitor U0126 [60]. This, combined with the observation that active somatic B-Raf mutations which have been identified in cancer appear to have enhanced, selective sensitivity to MEK inhibitors [61], makes MEK inhibitors an attractive potential therapeutic for this population of patients.

Acknowledgements
The authors thank the families, CFC International and the Costello Syndrome Family Network for their ongoing support of research in genetic medicine. The authors apologize for not citing all relevant references due to space limitations. This work was supported in part by NIH grant HD048502 (K.A.R.).

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32 Narumi Y, Aoki Y, Niihori T, Neri G, Cave H, et al: Molecular and clinical characterization of cardio-facio-cutaneous (CFC) syndrome: Overlapping clinical manifestations with Costello syndrome. Am J Med Genet A 2007;143:799807. 33 Nava C, Hanna N, Michot C, Pereira S, Pouvreau N, et al: CFC and Noonan syndromes due to mutations in RAS/ MAPK signaling pathway: genotype/ phenotype relationships and overlap with Costello syndrome. J Med Genet 2007;44:763771. 34 Rauen KA: Distinguishing Costello versus cardio-facio-cutaneous syndrome: BRAF mutations in patients with a Costello phenotype. Am J Med Genet A 2006;140:16811683. 35 Cruz F, 3rd, Rubin BP, Wilson D, Town A, Schroeder A, et al: Absence of BRAF and NRAS mutations in uveal melanoma. Cancer Res 2003;63: 57615766. 36 Trovisco V, Soares P, Soares R, Magalhaes J, Sa-Couto P, Sobrinho-Simoes M: A new BRAF gene mutation detected in a case of a solid variant of papillary thyroid carcinoma. Hum Pathol 2005;36:694697. 37 Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, Cobb MH: Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 2001;22:153183. 38 Wu J, Harrison JK, Dent P, Lynch KR, Weber MJ, Sturgill TW: Identification and characterization of a new mammalian mitogen-activated protein kinase kinase, MKK2. Mol Cell Biol 1993;13:45394548. 39 Brott BK, Alessandrini A, Largaespada DA, Copeland NG, Jenkins NA, Crews CM, Erikson RL: MEK2 is a kinase related to MEK1 and is differentially expressed in murine tissues. Cell Growth Differ 1993;4:921929. 40 Alessandrini A, Brott BK, Erikson RL: Differential expression of MEK1 and MEK2 during mouse development. Cell Growth Differ 1997;8:505511. 41 Cowley S, Paterson H, Kemp P, Marshall CJ: Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell 1994;77:841852.

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42 Mansour SJ, Matten WT, Hermann AS, Candia JM, Rong S, et al: Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 1994;265:966970. 43 Sivaraman VS, Wang H, Nuovo GJ, Malbon CC: Hyperexpression of mitogen-activated protein kinase in human breast cancer. J Clin Invest 1997;99: 14781483. 44 Bignell G, Smith R, Hunter C, Stephens P, Davies H, et al: Sequence analysis of the protein kinase gene family in human testicular germ-cell tumors of adolescents and adults. Genes Chromosomes Cancer 2006;45:4246. 45 Davies H, Hunter C, Smith R, Stephens P, Greenman C, et al: Somatic mutations of the protein kinase gene family in human lung cancer. Cancer Res 2005;65:75917595. 46 Hunter C, Smith R, Cahill DP, Stephens P, Stevens C, et al: A hypermutation phenotype and somatic MSH6 mutations in recurrent human malignant gliomas after alkylator chemotherapy. Cancer Res 2006;66: 39873991. 47 Stephens P, Edkins S, Davies H, Greenman C, Cox C, et al: A screen of the complete protein kinase gene family identifies diverse patterns of somatic mutations in human breast cancer. Nat Genet 2005;37:590592. 48 Estep AL, Palmer C, McCormick F, Rauen KA: Mutation Analysis of BRAF, MEK1 and MEK2 in 15 ovarian cancer cell lines: implications for therapy. PLoS ONE 2007;2:e1279.

49 Schubbert S, Zenker M, Rowe SL, Boll S, Klein C, et al: Germline KRAS mutations cause Noonan syndrome. Nat Genet 2006;38:331336. 50 Schubbert S, Bollag G, Lyubynska N, Nguyen H, Kratz CP, et al: Biochemical and functional characterization of germ line KRAS mutations. Mol Cell Biol 2007;27:77657770. 51 Zenker M, Lehmann K, Schulz AL, Barth H, Hansmann D, et al: Expansion of the genotypic and phenotypic spectrum in patients with KRAS germline mutations. J Med Genet 2007;44:131135. 52 Bentires-Alj M, Kontaridis MI, Neel BG: Stops along the RAS pathway in human genetic disease. Nat Med 2006;12:283285. 53 Carta C, Pantaleoni F, Bocchinfuso G, Stella L, Vasta I, et al: Germline missense mutations affecting KRAS isoform B are associated with a severe Noonan syndrome phenotype. Am J Hum Genet 2006;79:129135. 54 Roberts AE, Araki T, Swanson KD, Montgomery KT, Schiripo TA, et al: Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet 2007;39:7074. 55 Tartaglia M, Pennacchio LA, Zhao C, Yadav KK, Fodale V, et al: Gain-offunction SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet 2007;39:7579.

56 Zenker M, Horn D, Wieczorek D, Allanson J, Pauli S, et al: SOS1 is the second most common Noonan gene but plays no major role in cardio-facio-cutaneous syndrome. J Med Genet 2007;44:651656. 57 Gripp KW, Lin AE, Stabley DL, Nicholson L, Scott CI Jr, et al: HRAS mutation analysis in Costello syndrome: Genotype and phenotype correlation. Am J Med Genet A 2006;140:17. 58 Rauen KA: HRAS and the Costello syndrome. Clin Genet 2007;71: 101108. 59 Kerr B, Allanson J, Delrue MA, Gripp KW, Lacomb D, Lin AE, Rauen KA: The diagnosis of Costello syndrome: Nomenclature in Ras/MAPK pathway disorders. Am J Med Genet A 2008;146:12181220. 60 Senawong T, Phuchareon J, Ohara O, McCormick F, Rauen KA, Tetsu O: Germline mutations of MEK in cardiofacio-cutaneous syndrome are sensitive to MEK and RAF inhibition: implications for therapeutic options. Hum Mol Genet 2008;17:419430. 61 Solit DB, Garraway LA, Pratilas CA, Sawai A, Getz G, et al: BRAF mutation predicts sensitivity to MEK inhibition. Nature 2006;439:358362.

Katherine A. Rauen UCSF Helen Diller Family Comprehensive Cancer Center 2340 Sutter Street, Room S429, Box 0128 San Francisco, CA 94115 (USA) Tel. +1 415 514 3513, Fax +1 415 502 3179, E-Mail rauenk@peds.ucsf.edu

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Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 8393

The Clinical Phenotype of Costello Syndrome


B. Kerr
Regional Genetic Service and Medical Genetics Research Group, Central Manchester and Manchester Childrens Hospitals University NHS Trust, Royal Manchester Childrens Hospital, Manchester, UK

Abstract
Costello syndrome (CS) is a rare syndrome associated with developmental disability, prenatal overgrowth, and postnatal failure to thrive and short stature. Polyhydramnios, severe feeding difficulty and congenital heart disease are common features, as are hypertrophic cardiomyopathy and cardiac arrhythmia, predominantly atrial. Skin changes are striking, particularly increased skin over the palms and soles and the development of papilloma at moist body surfaces. The finding of excess palmar skin is part of a striking hand phenotype, comprising in addition, hyperextensibility of the small joints of the hand and a posture of flexion and ulnar deviation at the wrists. There is an increased risk of malignancy, particularly embryonal rhabdomyosarcoma and bladder carcinoma. The demonstration of activating missense mutations in HRAS, a component of the MAPK pathway, as causative in CS has provided a diagnostic test, and confirmation of the phenotype in classical and suspected cases. Although in most cases of CS the phenotype remains relatively homogenous and distinctive, a diagnostic test has demonstrated clinical overlap with cardio-facio-cutaneous syndrome (CFC). A severe neonatal phenotype has emerged, consisting in some cases of a multi-system disease and in others of profound hypotonia and myopathy associated on biopsy with excess muscle spindles (congenital myopathy with excess of muscle spindles, CMEMS). It is likely that further variability in the phenotype will be identified as further diagnostic testing is undertaken.
Copyright 2009 S. Karger AG, Basel

Original Description

In 1977, Dr Jack Costello, a New Zealand paediatrician, published in the Australian Paediatric Journal a report of two children with similar physical characteristics (table 1) and mild intellectual handicap [1]. He had published a brief description in a New Zealand medical journal several years before [2]. The first case was born after a pregnancy complicated by polyhydramnios, with a birth weight of 3.8 kg and head circumference at birth of 38.1 cm. The face was described as unusual with a prominent upper lip. The baby fed poorly and had to be tube fed frequently until 5 weeks, and did not regain birth weight until 6 weeks. He remained in hospital for 11 weeks, a cardiac murmur was noted. Despite the large size at birth, the height and weight were below the third centiles from around three months, with the head circumference one standard deviation above the mean. Formal IQ testing demonstrated scores within the mild range of impaired cognitive functioning, with higher verbal than performance scores. He developed nasal warts at age 6, and

Table 1. Clinical characteristics of first descriptions of patients with Costello syndrome [1, 3] Polyhydramnios High birth weight Large head circumference at birth Poor feeding Small size Hiatus hernia Gastro oesophageal reflux Relative macrocephaly Mild intellectual handicap Tight Achilles Pes cavus Nasal, face and leg papillomata Squint Keratoconus Cryptorchidism Delayed bone age Short neck Curly hair Low set ears Large ear lobes Depressed nasal bridge Epicanthic folds Thick lips High arched palate Systolic murmur Barrel chest Increased carrying angle Short flat hyperextensible fingers Thin nails Ulnar deviation of fingers Transient hepatosplenomegaly Dark or olive skin

later similar lesions appeared on both legs and face. He had a divergent squint and keratoconus. He required orchidopexy and herniotomy and surgery for tight Achilles tendons and pes cavus. The second case had a birth weight of 3.430 kg, length of 48.3 cm and head circumference of 34.9 cm at thirty six weeks gestation. Unusual facial features were noted; heavy jowls, very short neck, depressed bridge of nose, epicanthic folds, large ear lobes and third fontanelle. She had a biggish tongue and developed curly hair. Her fingers were noted to be hyperextensible, with ulnar deviation of the little fingers. She had transient hepatosplenomegaly. She also required tube feeding, was slow to regain her birth weight and had poor growth with relative macrocephaly. She was also functioning in the mildly intellectually handicapped range on formal testing. She developed a calcaneovalgus deformity of the feet and clawing of the toes and required surgery for tight Achilles tendons. At age two she had similar nasal lesions resembling warts; on biopsy, these were shown to be papillomata, with acanthosis and hyperkeratosis of the overlying stratified squamous epithelium.

Both children were noted to have loose skin of the hands and feet and thin nails. This article emphasised the importance of the skin findings as a clue to diagnosis.

Early Reports

The next published report of a child with Costello syndrome was not until 1991. Der Kaloustian et al. [3] reported a similar child (table 1) and described this particular pattern of physical and developmental characteristics as Costello syndrome (CS). Paroxysmal atrial tachycardia with a normal echocardiogram was a feature in this third case. The papillomata formed plaque like lesions, and biopsy showed similar but more pronounced features to those seen in the previous report. The key features of the three cases were summarised as: loose skin, especially of hands and feet, short stature and failure to thrive, coarse face, warts around the mouth and nares and mental subnormality. In this report, the similarity to Noonan syndrome and cardiofacio-cutaneous (CFC) syndrome was noted and compared.

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Table 2. The health of adults with Costello syndrome [4, 24] Multiple intraducatal breast papillomata Fibroadenosis Gastro-oesophageal reflux Duodenal ulcer Inguinal hernia Haemorrhoids Osteoporosis Chiari malformation Delayed puberty Short stature Hypertension Hypertrophic cardiomyopathy Supraventricular tachycardia Kyphoscoliosis Flexion contractures at elbows

Further reports followed, and by 1996, when Dr Costello published an update on his original cases, he was able to include a review of 16 literature cases [4], and contribute to the definition of the adult phenotype (see table 2). The only clinical finding found in all the literature cases until then was loose skin of the hands and feet.

Natural History

Several of the early publications recognised the distinctive natural history of Costello syndrome [5, 6]. Polyhydramnios is common in pregnancy, and may be sufficiently severe to warrant one or more amniotic fluid reductions. Increased nuchal thickness and ulnar deviation at the wrists has been seen on routine ultrasound. Increased and decreased fetal movements have been reported. Despite relatively large size at birth, severe feeding difficulty, secondary to poor swallowing, and failure to thrive is invariable. This has been described as the marasmic phase [5], and is not completely corrected by measures such as gastrostomy and calorie supplementation [7]. Difficulty controlling the airway may also occur, with stridor, opisthotonos and excessive secretions being relatively common. After a variable time, usually around 3 years, feeding behaviour improves and growth becomes more normal, with a disproportionate

weight gain in comparison with linear growth [5]. Developmental progress is often far better than expected in early life [6, 7]. As more patients were described, little was added to the earliest clinical descriptions of Costello syndrome. This is not surprising, given that mutation analysis has established that Costello syndrome is a relatively homogeneous phenotype [8, 9]. The relative frequencies of the various features could be delineated [6] (table 3), the importance of cardiovascular anomalies emerged [10] and the differential diagnosis became better understood. Segregation analysis [11] had suggested a sporadic new dominant mutation as the likely cause. Specific aspects of the phenotype have been the subject of study.

Tumour Risk in Costello Syndrome

In 1991, Martin and Jones [12] reported a young woman with Costello syndrome and a calcified epithelioma of the neck and bilateral epithelial paratubal cysts in addition to nasal papillomata, extending the range of benign epithelial tumours. A single case with a ganglioneuroblastoma was reported in 1993 [5]. In 1998, two young children with Costello syndrome and living in the north of England both developed an embryonal rhabdomyosarcoma [13]. Single reports of an acoustic neuroma in an adult [14] and an alveolar rhabdomyosarcoma [15] rapidly followed, with no

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Table 3. Distinctive features in a series of patients with Costello syndrome [6] Seen in 100% Poor feeding Poor growth Developmental delay Coarse facial appearance Low set ears Short neck Loose skin Hyperpigmentation Deep creases on palms and soles Seen in more than 80% BW and Birth OFC >50th% Outgoing personality Epicanthic folds Flat nasal bridge Thick lips Macrostomia Increased A-P chest diameter Extensible fingers Wide phalanges Abnormal foot position Tight Achilles tendon Curly hair Sparse hair Delayed bone age

further reports to date of further cases of these latter two tumours. Other reports [1622] demonstrated that the commonest malignant tumours are embryonal rhabdomyosarcoma, bladder carcinoma and neuroblastoma, with a suggested tumour risk as high as 17% [22]. A single inflammatory fibroid polyp in the stomach in a two-year-old child has been recorded [23]. In adult life, breast fibroadenosis, multiple intraductal papillomata, a parathyroid adenoma and a choroid plexus papilloma have occurred [24]. A suggested tumour screening protocol [22] using abdominal ultrasound 3 to 6 monthly until age 8 to 10 years, regular urine catecholamine assay until age 5, and annual urinalysis for haematuria from age 10 was modified after reports of abnormal urinary catecholamines in the absence of neuroblastoma in patients with CS [25]. The value of this protocol remains unproven.

Congenital heart disease occurred in 30%, with pulmonary stenosis accounting for 46%. The other common malformations were atrial and ventricular septal defects. Cardiac hypertrophy was reported in 34%; this involved the left ventricle in 50% and was usually consistent with classic hypertrophic cardiomyopathy. In 50% of patients, this occurred in the first two years of life, but onset in an adult has been documented [4]. Rhythm disturbance was also found in around one third of patients, chiefly atrial tachycardia, described as supraventricular, chaotic or multi-focal. The rhythm disturbance was an isolated cardiac finding in one third of cases.

Respiratory Involvement

Cardiac Involvement

A review of 94 patients with CS [26] found evidence of cardiac involvement in 63% overall.

Stridor and excessive secretions are common in the young child with CS. Bronchomalacia and tracheomalacia have been reported and occasionally will be severe enough for tracheostomy to be required [27]. On formal study, obstructive sleep apnoea is common, along with a high frequency of upper airway narrowing [28].

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Endocrine Abnormalities

Neurological, Developmental and Behavioural Manifestations

Growth deficiency is a key feature of CS. Three of four children reported in the literature with abnormal growth hormone secretion in response to provocative stimuli have had a good response to growth hormone therapy [29, 30]. Hypoglycaemia has also been reported, and treated with growth hormone and thyroid hormone replacement in one case. In this case, an initial (four year) response to growth hormone was not sustained, with height after seven years of treatment 5 SD below the mean, as it had been at commencement of growth hormone treatment [20]. Hypoglycaemia has also been reported with presumed cortisol deficiency and a partial deficiency of growth hormone [31]. Hypoglycaemia persisted with growth hormone treatment (but resolved with hydrocortisone), and there was no effect on growth over 8 months of growth hormone treatment. Hypoglycaemia has also been reported with hyperinsulinism [32, 33] and at post mortem, a nesidioblastosis-like lesion, with hypertrophy and hyperplasia of the Langerhans islets of the pancreas [32]. Concern has been expressed that growth hormone treatment may pose particular hazard in Costello syndrome because of the risk of malignant tumours and a possible effect on cardiac hypertrophy [34]. Two tumours have developed in patients treated with growth hormone [20, 34], and progression of cardiomyopathy has been documented in one case [34]. Recommendations for growth hormone treatment are that its use be restricted to patients with documented growth hormone deficiency, that regular tumour surveillance is undertaken, that cardiac monitoring occurs and that treatment is titrated to avoid a supraphysiologic range [29]. Delayed puberty is common [24], with the suggested mechanism being central hypogonadism. Precocious puberty has also been described.

Review of the findings on neurological investigation in 38 literature cases of Costello syndrome [35] found normal cerebral imaging in around one quarter of patients. Cerebellar abnormalities, especially Chiari 1 malformation, were present in 26% and, usually mild ventricular dilatation and cerebral atrophy, each occurred in around 40%. Ventriculo-peritoneal shunting has rarely been required. Syringomyelia has been described in a number of cases. EEG changes were present in a third of 49 children, with seizures occurring in only 20%. In a younger group of ten children, there were no structural brain abnormalities, but a 50% incidence of seizures [36]. Developmental milestones are all acquired late [36], with a mean age of sitting of 23 months (10 months to 3 years), and walking alone 4 years and 11 months (26 months to 9 years). Language development is frequently more severely delayed, and in several cases has been noted to develop as feeding difficulties abate. First words were spoken between 2 and 9 years. Although children and adults with Costello syndrome are described as having a warm sociable personality, the early years of life are often characterised by extreme irritability with hypersensitivity to sounds and tactile stimuli, sleep disturbance and excessive shyness with strangers [36]. This was independent of the diagnosis of gastro-oesophageal reflux, and often improved between ages 2 to 4 years. Formal testing in one patient [36] confirmed the observation of others [7] that the expected developmental attainment in infancy is often exceeded, with the DQ at 18 months 45, 55 at 3 years 8 months and 71 at age 6 years. Serial assessment of a cohort of children, all now known to be HRAS mutation positive [37, 38], demonstrated a mean full scale IQ score of 57 (range 3087) within the mild range of

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intellectual handicap for the majority of patients. Intellectual function was stable over two years.

Skeletal and Orthopaedic Abnormalities

Orthopaedic problems were part of the first descriptions of Costello syndrome (table 1), particularly in relationship to foot position. Review of bone and joint manifestations in 16 examined patients [39], aged from 3 to 23 years, demonstrated a characteristic gait in all, broad based and shuffling, and none were able to run. All had however walked by 48 months. Decreased range of movement at the elbow and shoulder was common, and most tended to hold their elbows and wrist flexed at 90, and reported difficulty with overhead activity. Ligamentous laxity and digital hyperextensibility was present in all. Tight heel cords occurred in 72%. Vertical talus occurred in 28%. Other foot differences were planovalgus position, bilateral overriding second toes, plantar flexed great toe and mild metatarsus adductus. Three children had had hip subluxation. Kyphosis and scoliosis both occurred in 17%. Severe pectus excavatum occurred on one patient. One older patient had bilateral radial head subluxation.

The mean height was 139 cm for females (130 for females not treated with growth hormone), (range 122 to 154 cm) and 142 cm for males (range 124 to 153 cm). Adult height was reached at a mean age of 21 years (range 16 to 28 years). Three of the female patients had received growth hormone, one for proven growth hormone deficiency achieved an adult height of 151 cm. The second was treated with growth hormone from ages 5 to 9 and was 154 cm in height. The third treated case has been previously described [20], and the non sustained response to treatment and subsequent bladder tumour mentioned above. The health problems described in adult patients are summarised in table 2. Of particular note is the development of symptomatic Chiari malformations in adult life in 3 patients, in one of whom previous cerebral imaging was normal. Gastro-oesophageal reflux was part of the symptoms in these patients, suggesting that adult onset oesophageal reflux may not always be primary. Bone density was abnormal in the 8 patients in whom it was measured, and 3 of these patients had bone pain, crush fractures and loss of height.

The Cause of Costello Syndrome

The Adult Phenotype

In a review of the clinical features in 17 adult patients, aged 16 to 40 years [24], the distinctive facial features are summarised as progressive facial coarseness, broad forehead, broad nose, large mouth and thick lips. Frontal balding occurred in two patients. Redundant loose skin persisted over the joints and hands and feet, and hyperkeratosis of the palms and soles in association with excess sweating was a major problem in several. The standing posture was stooped, with flexed elbows and wrists and the hands held in ulnar deviation.

The search for clues to the cause of Costello syndrome had demonstrated a variety of metabolic abnormalities, all in a small number of cases [6]. These include non-specific generalised aminoaciduria, low maternal serum -fetoprotein, sialuria and elevation of hexosaminadase B [6]. Several studies focussed on elastin and elastic fibres. Fine disrupted and loosely constructed elastic fibres were demonstrated in skin, tongue, pharynx, larynx and upper oesophagus, with hyperplasia of collagen fibres in skin, and normal elastin mRNA expression in skin fibroblasts [40]. Hinek et al. [41] demonstrated a deficiency in elastic fibre assembly in skin fibroblasts due to a secondary deficiency in EBP, a 67-kDa

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elastin binding protein. The observed excess of chondroitin sulfate bearing proteoglycans in fibroblasts from patients with CS was postulated as the cause, due to an inhibition of EBP recycling. Subsequent demonstration of excess chondroitin-6-sulfate bearing glycosaminoglycans in cardiac myocytes in 3 children with Costello syndrome suggested an imbalance in sulfation of chondroitin sulfate molecules as contributing to cardiac hypertrophy [42]. The high malignancy risk in Costello syndrome led several authors to speculate that a tumour suppressor gene may be involved in the cause of Costello syndrome, and after loss of heterozygosity studies in tumour, that the locus might be 11p. Based on the phenotypic similarities between Costello syndrome and Noonan syndrome, Aoki et al. [43] discovered activating missense mutations in HRAS to be the cause of Costello syndrome by studying genes in the MAPK pathway, that being the pathway regulated by the product of PTPN11, the gene mutated most often in patients with Noonan syndrome. The HRAS mutation spectrum seen in patients with CS is discussed in detail in the next chapter. It is particularly noteworthy that for the less common mutations, some variability in phenotype is observed, with atypical physical features and milder cognitive phenotypes emerging. The availability of a diagnostic test means that the true clinical phenotype of Costello syndrome can be defined and confirmed. Whilst this is an ongoing process, some conclusions can already be drawn, particularly for the two most difficult diagnostic areas, the newborn diagnosis and the differentiation from CFC syndrome.

44, 45]. Choanal atresia has been reported in one case [44], and in two, neonatal osteoporosis with enlargement of anterior ribs [44]. Pyloric stenosis has occurred in two mutation positive cases [27, 44]. Diagnostic testing in the newborn period has confirmed that hypoglycaemia secondary to hyperinsulinism, renal abnormalities, severe early cardiomyopathy, tracheomalacia and bronchomalacia, pleural and pericardial effusion, chylous ascites and pulmonary, hepatic and splenic lymphangiectasia are part of the clinical spectrum seen in CS [27]. A lung pathology resembling alveolar capillary dysplasia has been reported in one case [27], as has a nesidioblastosis-like lesion in the pancreas [32]. A distinct early onset severe phenotype due to HRAS mutations has been recognised in 4 patients with congenital myopathy with excess of muscle spindles (CMEMS), hypertrophic cardiomyopathy and variable features resembling Noonan syndrome [45]. Two patients had novel mutations, p.E63K and p.Q22K, while the other two had previously reported mutations, p.G12V and p.G12S. The longest surviving patient, with p.Q22K, was still alive at fourteen months. Death was due to cardio-respiratory failure in two cases. All cases had generalised hypotonia, variable contractures, absence of spontaneous movement and areflexia. Excess muscle spindles have not been observed in skeletal muscle in other patients with CS [12].

Differentiation from CFC Syndrome and the Specificity of Diagnostic Criteria

Diagnosis in the Newborn

The difficulty of diagnosing CS in the neonatal period, given the age related nature of some of the diagnostic features, has been confirmed [27,

In CS, diagnostic criteria exhibit age dependence and are listed in table 4. Facial and physical features at different ages are illustrated in figure 1. Facial features are most often described as coarse, with a broad flat nasal bridge, epicanthic folds, thick lips and large tongue and low set ears, often with large upturned ear lobes.

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b
Fig. 1. Facial appearance in CS. (a) Patient at ages two months, two years, five and six years. Note splayed fingers in early life, relatively thick lower lip, flat nasal bridge, upturned ear lobes, high forehead in early life, characteristic hand and wrist posture. Photos published with permission. (b) Second patient at ages 18 months, three years, nine and eighteen years. Note pectus excavatum, lax abdominal musculature, prominent heels, flat nasal bridge, persisting full lips, prominent cheeks in early life with facial thinning with age, upturned large ear lobes, persistence of excess palmar skin. Photos published with permission.

HRAS mutation testing in classical cases, and those where the diagnosis of both CFC and CS were considered [46], has permitted comparison of clinical features in HRAS positive cases with a confirmed diagnosis of CS and those with confirmed CFC on the basis of mutations in BRAF or MEK1 and MEK2. Statistical significance was achieved for polyhydramnios, the presence of more than one papilloma and growth hormone deficiency, all being significantly more common in CS.

Although heart disease was present in about three quarters of both CS and CFC cases, the pattern was different. Congenital heart disease, particularly pulmonary stenosis with an atrial septal defect, is commoner in CFC than CS. Atrial tachycardia is commoner in CS than CFC. Chaotic atrial rhythm, or multifocal atrial tachycardia, has only been observed in CS. Abnormal catecholamine metabolites in the absence of a neuroblastoma have also been observed in CFC patients.

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Table 4. Diagnostic criteria (age dependent) Prenatal overgrowth Postnatal failure to thrive Severe feeding difficulty Relative macrocephaly Short stature Characteristic facies Excess palmar and plantar skin Hyperextensibility of the small joints of the hand Ulnar deviation at the wrists Skin papillomata at moist surfaces Developmental disability

Conclusion

The most distinctive physical finding in CS patients, the hand phenotype of ulnar deviation, excess palmar skin, deep creases and small joint hyperextensibility, has been observed in a small number of patients with BRAF and MEK mutations, but remains most characteristic of CS and HRAS mutations. A new hand sign [47, 48], distal phalangeal creases, particularly obvious on the palmar surface of the thumb, has been seen in patients with HRAS, KRAS and BRAF mutations and suggested as a marker for disorders of the MAPK pathway.

Despite the rarity of Costello syndrome, the phenotype has been extensively studied. The recent demonstration of mutations in HRAS as causative provides a diagnostic test and has already permitted confirmation and refinement of the phenotype. While the phenotype that has been described in the literature over three decades is relatively homogeneous, testing has led to the recognition of new features and it is likely that further modifications of the phenotype will emerge. It is hoped that improved understanding of the fundamental biology will lead to effective treatments.

Acknowledgements
The author would like to acknowledge the contribution of the patients and families of the International Costello Syndrome Support Group, the Costello Syndrome Family Network and the Association Franaise du Syndrome de Costello, to increasing knowledge about Costello syndrome, and their generosity in sharing their experiences.

References
1 Costello JM: A new syndrome: mental subnormality and nasal papillomata. Austr J Paediatr 1977;13:114118. 2 Costello JM: A new syndrome. NZ Med J 1971;74:397. 3 Der Kaloustian VM, Moroz B, McIntosh N, Watters AK, Blaichman S: Costello syndrome. Am J Med Genet 1991;41:6973. 4 Costello JM: Costello syndrome: Update on the original cases and commentary. Am J Med Genet 1996;62:199201. 5 Zampino G, Mastroiacovo P, Ricci R, Zollini M, Segni G, Martini-Neri ME, Neri G: Costello syndrome: Further clinical delineation, natural history genetic definition and nosology. Am J Med Genet 1993;47:176183. 6 Johnson JP, Golabi M, Norton ME, Rosenblatt RM, Feldman GM, et al: Costello syndrome: Phenotype, natural history, differential diagnosis and possible cause. J Pediatr 1998;133:441448. 7 Fryns JP, Vogels A, Haegerman J, Eggermont E, Van Den Berghe H: Costello syndrome: a postnatal growth retardation syndrome with distinct phenotype. Genet Counselling 1994;5:337343. 8 Zampino G, Pantaleoni F, Carta C, Cobellos G, Vasta I, et al: Diversity, parental germline origin and phenotypic spectrum of de novo HRAS missense changes in Costello syndrome. Hum Mut 2007;28:265272. 9 Van Steensel MA, Vreeburg M, Van Ravenswaaiji-Arts CM, Biljsma E, Schrander-Stumpel CT, van Geel M: Recurring HRAS mutation G12S in Dutch patients with Costello syndrome. Exp Derm 2006;15:731734. 10 Siwik ES, Zahka KG, Wiesner GL: Cardiac disease in Costello syndrome. Pediatrics 1998;101:706709. 11 Lurie IW: Genetics of the Costello syndrome. Am J Med Genet 1994;52:358359. 12 Martin RA, Jones KL: Delineation of the Costello syndrome. Am J Med Genet 1991;41:346349.

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13 Kerr B, Eden OM, Dandamudi R, Shannon N, Quarrell O, et al: Costello syndrome: two cases with embryonal rhabdomyosarcoma. J Med Genet 1998;35:10361039. 14 Suri M, Garrett C: Costello syndrome with acoustic neuroma and cataract. Clin Dysmorphol 1998;7:149151. 15 Feingold M: Costello syndrome and rhabdomyosarcoma, J Med Genet 1999;36:582583. 16 Franceschini P, Licata D, Di Cara G, Guala A, Bianchi M, Ingrosso G, Franceschini D: Bladder carcinoma in Costello syndrome: report on a patient born to consanguineous parents and review. Am J Med Genet 1999;86: 174179. 17 Flores-Nava G, Canun-Serrano S, Moysen-Ramirez SG, Parraguirre-Martinez S, Escobedo-Chavez E: Costello syndrome associated to a neuroblastoma. Presentation of a case. Gac Med Mex 2000;136:605609. 18 Sigaudy S, Vittu G, David A, Vigeron J, Lacombe D, et al: Costello syndrome: Report of six patients including one with an embryonal rhabdomyosarcoma. Eur J Pediatr 2000;159:139142. 19 Moroni I, Bedeschi F, Luksch R, Casanova M, DIncerti L, Uziel G, Selicorni A: Costello syndrome: a cancer predisposing syndrome? Clin Dysmorphol 2000;9:265268. 20 Gripp KW, Scott CI Jr, Nicholson L, Figueroa TE: Second case of bladder carcinoma in a patient with Costello syndrome. Am J Med Genet 2000;90:256259. 21 Urakami S, Igawa M, Shiina H, Shigeno K, Kikuno N, Yoshino T: Recurrent transitional cell carcinoma in a child with the Costello syndrome. J Urol 2002;168:11331134. 22 Gripp KW, Scott CI, Nicholson L, McDonald-McGinn DM, Ozeran JD, et al: Five additional Costello syndrome patients with rhabdomyosarcoma: proposal for a tumour screening protocol. Am J Med Genet 2002;108:80 87. 23 Di Rocco M, Dodero P: Concerning Five additional Costello syndrome patients with rhabdomyosarcoma: Proposal for a tumour screening protocol. Am J Med Genet A 2003;118:199. 24 White S, Graham JM, Kerr B, Gripp K, Weksberg R, et al: The adult phenotype in Costello syndrome. Am J Med Genet A 2005;136;128135.

25 Bowron A, Scott JG, Brewer C, Weir P: Increased HVA detected on organic acid analysis in a patient with Costello syndrome. J Inher Metab Dis 2005;28;11551156. 26 Lin AE, Grossfeld PD, Hamilton R, Smoot L, Proud V, et al: Further delineation of cardiac anomalies in Costello syndrome. Am J Med Genet 2002;111:115129. 27 Lo I, Brewer C, Shannon N, Shorto J, Tang B, et al: Severe neonatal manifestations of Costello syndrome. J Med Genet 2008;45:167171. 28 Della Marca G, Vasta I, Scarano E, Rigante M, DeFeo E, et al: Obstructive sleep apnoea in Costello syndrome. Am J Med Genet A 2006;140:257262. 29 Stein RL, Legault L, Daneman D, Weksberg R, Hamilton J: Growth hormone deficiency in Costello syndrome. Am J Med Genet A 2004;129:166170. 30 Okamoto N, Chiyo H, Imai K, Otani K, Futagi Y: A Japanese patient with the Costello syndrome. Hum Genet 1994;93:605606. 31 Gregersen N, Viljoen D: Costello syndrome with growth hormone deficiency and hypoglycaemia: A new report and review of the endocrine associations. Am J Med Genet A 2004;129:171175. 32 Kerr B, Delrue M-A, Sigaudy S, Perveen R, Marche M, et al: Genotypephenotype correlation in Costello syndrome; HRAS mutation analysis in 43 cases. J Med Genet 2006;43:401405. 33 Alexander S, Ramadan D, Alkhayyat H, Al-Sharkawi I, Backer KC, El-Sabban F, Hussain K: Costello syndrome and hyperinsulinemic hypoglycaemia. Am J Med Genet A 2005;139:227230. 34 Kerr B, Einaudi MA, Clayton P, Gladman G, Eden T, et al: Is growth hormone treatment beneficial or harmful in Costello syndrome? J Med Genet 2003;40:e74. 35 Delrue M-A, Chateil J-F, Arveiler B, Lacombe D: Costello syndrome and neurological abnormalities. Am J Med Genet A 2003;123:301305. 36 Kawame H, Matsui M, Kurosawa K, Matsuo M, Masuno M, et al: Further delineation of the behavioural and neurological feature in Costello syndrome. Am J Med Genet A 2003;118:8 14.

37 Axelrad ME, Glidden R, Nicholson L, Gripp KW: Adaptive skills, cognitive and behavioral characteristics of Costello syndrome. Am J Med Genet A 2004;128:396400. 38 Axelrad ME, Nicholson L, Stabley DL, Sol-Church K, Gripp KW: Longitudinal assessment of cognitive characteristics in Costello syndrome. Am J Med Genet A 2007;143:31853193. 39 Yassir WK, Grottkau BE, Goldberg MJ: Costello syndrome: Orthopaedic manifestations and functional health. J Pediatr Orthop 2003;23:9498. 40 Mori M, Yamagata T, Mori Y, Nokubi M, Saito K, Fukushima Y, Momoi M: Elastic fiber degeneration in Costello syndrome. Am J Med Genet 1996;61:304309. 41 Hinek A, Rabinovitch M, Keeley F, Okamura-Oho Y, Callahan J: The 67-kD elastin/laminin-binding protein is related to an enzymatically inactive, alternatively spliced form of beta-galactosidase. J Clin Invest 1993;91:11981205. 42 Hinek A, Teitell MA, Schoyer L, Allen W, Gripp K, et al: Myocardial storage of chondroitin sulfate-containing moieties in Costello syndrome patients with severe hypertrophic cardiomyopathy. Am J Med Genet A 2005;133:112. 43 Aoki Y, Niihori T, Kawame H, Kurosawa K, Ohashi H, et al: Germline mutations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 2005;37:10381040. 44 Digilio M, Sarkozy A, Capolino R, Testa M, Esposito G, et al: Costello syndrome: clinical diagnosis in the first year of life. Eur J Pediatr 2008;167:621 628. 45 Van der Burgt I, Kupsky W, Stassou S, Nadroo A, Barroso C, et al: Myopathy caused by HRAS germline mutationsimplications for disturbed myogenic differentiation in the presence of constitutive H-Ras activation. J Med Genet 2007;44:459462. 46 Gripp K, Lin A, Stabley DL, Nicholson L, Allen A, et al: Further delineation of the phenotype resulting from BRAF or MEK1 mutations helps differentiate Cardio-facio-cutaneous syndrome from Costello syndrome. Am J Med Genet A 2007;143:14721480.

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47 rstavik K, Tangeraas T, Molven A, Prescott TE: Distal phalangeal creases a distinctive dysmorphic feature in disorders of the RAS signalling pathway. Eur J Med Genet 2007;50:155158.

48 Allanson J, Kavamura I, Neri G, Noonan J, Poss A, Kerr B: Distal phalangeal creases: More evidence of this feature in disorders of the Ras signaling pathway. Eur J Med Genet 2007;50:482483.

Bronwyn Kerr Regional Genetic Service and Medical Genetics Research Group, Central Manchester and Manchester Childrens Hospitals University NHS Trust, Royal Manchester Childrens Hospital Hospital Rd Manchester M274HA (UK) Tel. +44 1619222335, Fax +44 1619222329, E-Mail bronwyn.kerr@cmmc.nhs.uk

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Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 94103

The Molecular Basis of Costello Syndrome


K. Sol-Churcha K.W. Grippb
aCenter for Pediatric Research and bDivision of Medical Genetics, Alfred I duPont Hospital for Children, Nemours Childrens Clinic, Wilmington, Del., USA

Abstract
Costello syndrome is a rare tumor predisposition syndrome with a distinctive phenotype overlapping with Noonan and cardio-facio-cutaneous syndromes. Based on this information its genetic cause was identified as heterozygous HRAS mutations. HRAS is a well-known oncogene, and aberrant activation of the gene product due to specific point mutations affecting glycines in positions 12 and 13 is often found in sporadic tumors. As the germline mutations in Costello syndrome affect similar codons, a similar effect on the gene product can be inferred. Substitutions of glycine 12 or 13 account for 95% of Costello syndrome mutations. The common changes (G12S, G12A) result in the typical phenotype, whereas the presentation of presumably more strongly activating mutations (G12V) appears to be more severe. Mutations affecting amino acids other than G12 or G13 occurred in one individual each (Q22K, T58I, E63K, K117R, A146T, A146V), and may be associated with a less typical phenotype. The vast majority of mutations arose in the paternal germline, but two were maternally derived. Somatic mosaicism for the G12S mutation was seen in one individual. While germline mosaicism is the likely cause for reported Costello syndrome sibling pairs, this has not been molecularly confirmed.
Copyright 2009 S. Karger AG, Basel

and it was the first in which a disease causing gene mutation was identified [1]. About half of all patients with Noonan syndrome carry a mutation in the gene PTPN11 encoding the protein kinase phosphatase SHP2, a positive regulator of the mitogen activated protein kinases (MAPK) pathway. A study by Tartaglia et al. (2003) did not identify PTPN11 mutations in patients with Costello syndrome [2]. Based on this information, Aoki et al. (2005) analyzed other genes encoding MAPK pathway proteins and discovered mutations in HRAS in patients with Costello syndrome [3].

HRAS Mutations in Costello Syndrome

Costello syndrome has long been known to share physical findings with cardio-facio-cutaneous syndrome and Noonan syndrome. Of these, Noonan syndrome is the most common disorder,

Aoki et al. (2005) identified the genetic cause of Costello syndrome by sequencing the entire coding region of the RAS genes from 13 Japanese and Italian individuals with Costello syndrome [3]. In 12 individuals, they found a heterozygous mutation in the HRAS gene, a key regulator of signal transduction of the MAPK pathway. Examination of genomic DNA from different tissues of affected individuals and from parental samples suggested that Costello syndrome is due to de novo mutations

Table 1. HRAS mutations reported in 139 Costello syndrome patients HRAS point mutation AA change Frequency in percent (number of patients) c.34G>A c.34G>T c.35G>C c.35_36GC>TT c.35_36GC>AA c.37G>T c.38G>A c.64C>A c.173C>T c.187G>A c.350A>G c.436G>A c.437C>T G12S G12C G12A G12V G12E G13C G13D Q22K T58I E63K K117R A146T A146V 81.3 (113) 2 (3) 7.2 (10) 1.4 (2) <1 (1) 1.4 (2) 1.4 (2) <1 (1) <1 (1) <1 (1) <1 (1) <1 (1) <1 (1) 3, 57, 9, 11, 12, this work 7, this work 3, 57, 15 3, 12 7 5, 6, this work 3 12 18 12 7 11 18 References

of germline origin [3]. Strikingly, all mutations affected either glycine 12 or 13 of the Ras protein. In seven patients a germline c.34G>A transition in the HRAS gene was identified, predicting a gly12to-ser (G12S) amino acid substitution. A germline c.35G>C transversion causing a gly12-to-ala (G12A) amino acid substitution was found in two patients, while two others carried a c.38G>A transition resulting in a gly13-to-asp (G13D) amino acid substitution. One individual had a c.35GC>TT nucleotide substitution resulting in a gly12-to-val amino acid change (G12V), which is the most common mutation in human cancers [4]. The genetic etiology of Costello syndrome was soon confirmed in three larger studies of American and European patients [57]. Gripp et al. (2006) performed mutation analysis in 40 patients and detected missense mutations in HRAS in 33 (82.5%). No sequence change was identified

in the available parental DNAs, supporting de novo origin [5]. Seventeen patients [8] reported by Gripp et al. (2006) were also included in Estep et al. (2006), who reported HRAS mutations in 33 of 36 Costello syndrome patients [6]. Kerr et al. (2006) identified HRAS mutations in 37 of 43 individuals with a clinical diagnosis of Costello syndrome. Analysis of parental DNA confirmed the mutations as de novo in 19 probands [7]. Currently, 13 different heterozygous DNA variants of the HRAS gene have been identified in Costello syndrome patients (table 1). All amino acid substitutions affect protein regions directly linked to its function as a regulator of the MAPK pathway. In addition to the common mutations affecting either residue G12 or G13 of the Ras protein, six patients with DNA variants causing changes of amino acids 22, 58, 63, 117 or 146 were identified.

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gDNA E1 E2
Intron 1 Intron 2

E3
Intron 3

E4

E5 (IDX)
Intron 4

E6
Intron6

E7

A
mRNAs

Intron5

Splice variants

ATG

Variant 1

Stop

ATG

Variant 2

Stop

Proteins

p19 p21

G N G12S Q22K G13C

Ef Switch I

G Switch II T58I E63K

G C

K117R

A146T A146V

Fig. 1. HRAS structure. (A) Genomic structure of the HRAS gene with boxes representing exons. Coding exons are shaded. (B) mRNA structures of the two HRAS splice variants. Start and stop codon are as indicated. (C) N-terminal protein domains of p21ras. GTP/GDP binding boxes (G-domains) are represented in solid and switch I and II regions are shaded. Ef is the effector region involved in binding of the GTPase activating proteins (GAPs).

G12S Variants G12S is the most common mutation found in Costello syndrome patients, accounting for about 82% of reported cases [3, 57]. Additional patients carrying this change were reported by Sol-Church et al. (2006), Van Steensel et al. (2006), and Zampino et al. (2007) [911]. Not surprisingly, because the majority of patients carry this particular mutation, the phenotype associated with the G12S mutation encompasses all findings recognized as typical for Costello syndrome prior to the gene identification [3, 57, 911]. In addition to these typical clinically diagnosed Costello syndrome patients, Van der Burgt et al. (2007) identified this mutation in a patient originally reported by Selcen et al. (2001) as having a novel congenital myopathy

with muscle spindle excess [12, 13]. The patient had generalized muscle weakness, areflexia and joint contractures, and he died at age 14 months of cardiorespiratory failure [13]. Twelve additional, previously unpublished patients are also included in table 1, bringing the total number of patients with a G12S substitution to 113 out of 139 unique Costello syndrome patients with an identified HRAS mutation. G12S is located in exon 2 of the HRAS gene (fig. 1A) in a region containing several CpG sites, possibly accounting for the mutational hotspot. When these CpGs are methylated, they become vulnerable to mutations affecting not only the cytosines of either DNA strand, but also the neighboring guanines [14]. Spontaneous mutations can occur at these sites, especially G>A

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transitions resulting in the G12S change seen in the majority of Costello syndrome patients. G12A Variants The second most common HRAS mutation associated with Costello syndrome in 10 unrelated patients is a c.35G>C transversion resulting in a gly12-to-ala (G12A) amino acid substitution. Two cases each were identified by Aoki et al. (2005), Gripp et al. (2006) and Estep et al. (2006), and Kerr et al. (2006) identified three patients carrying this DNA variant [3, 57]. More recently, Svik et al. (2007) reported a fascinating family with two sisters initially clinically diagnosed with Costello syndrome [15]. While one of the sisters carries a germline HRAS mutation (G12A), a germline KRAS mutation (F156L) was identified in her sibling. The G12A mutation is seen in less than 1% of sporadic malignancies with an HRAS mutation, specifically in one chondrosarcoma and one papillary thyroid carcinoma [4]. One Costello syndrome patient with G12A developed a ganglioneuroblastoma [3], one had a transitional cell carcinoma of the bladder [5], and two developed rhabdomyosarcoma [7]. This apparent high tumor frequency led to the speculation that the G12A germline mutation confers a higher malignancy risk than the G12S change [7]; however, this hypothesis is not yet supported by statistically valid data. Other Variants Affecting G12 and G13 Nearly 80% of codon 12 mutations seen in sporadic tumors [4] involve a G>T transversion resulting in amino acid changes G12V or G12C. The G12V mutant of HRAS identified by Aoki et al. (2005) was observed in another Costello syndrome patient with congenital myopathy with excess of muscle spindles [12]. This Ras mutant has a low GTPase activity and high transformation potential [16, 17]. Based on several unpublished cases the G12V change may be associated with a severe, early lethal phenotype. The G12C variant resulting from a c.34G>T mutation was present in three

patients, one of whom developed rhabdomyosarcoma [7, and this work]. In the same cohort Kerr et al. (2006) identified a rare variant G12E in a patient who died at age 6 months [7]. Two patients with a gly13-to-cys (G13C) substitution caused by a c.37G>A mutation have been identified [5, 6 and this work]. The first patient was included in Gripp et al. [5] and Estep et al. [6]; he is the tallest Costello syndrome patient who never received growth hormone, and at age 12 years had not developed papillomata. The second patient was previously unpublished, she had a history of typical feeding difficulties but never required feeding tube placement, and had few medical problems throughout childhood. At her current age of 17 years, she attends a regular school with some special classes. Despite never receiving growth hormone, her height is only 4 to 3 SD below the mean. She has not developed papillomata. These two cases may suggest that G13C harbors a slightly milder phenotype, compared to the common G12S change. Despite the fact that the HRAS G13C mutation has been identified in three bladder cancer samples, its overall rarity in sporadic malignancies may support this hypothesis. Rare HRAS Variants Changes of amino acids other than G12 and G13 were seen in six Costello syndrome patients (table 1). Heterozygous transversions in the HRAS gene resulting in Q22K (c.64C>A) or E63K (c.187G>A) substitutions were present in two patients with congenital myopathy, respectively [12]. This myopathy with an excess of muscle spindle fibers on biopsy was reported in four patients with HRAS mutations. While two changes were novel, two others, G12S and G12V, had been seen before. These patients medical histories were typical for Costello syndrome and their phenotype is best described as Costello syndrome with myopathy, rather than a different disorder. It appears likely that the excess of spindle fibers reflects the hyperactive MAPK pathways effect on striated muscle development, rather than a finding unique to rare HRAS mutations.

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One patient with a de novo paternally derived HRAS mutation affecting amino acid 58 (T58I) has been identified recently [18]. While he had the typical early failure to thrive, his facial findings at his current age of 6 years are not as coarse as those of most Costello syndrome patients. A de novo c.350A>G transition resulting in a lys117to-arg (K117R) substitution was identified in one Costello patient, unusual for less coarse facies and autistic features [7]. Two patients with different substitutions of the alanine in position 146 have been seen. Zampino et al. (2007) reported a girl with a de novo c.436G>A transition in the HRAS gene, resulting in an ala146-to-thr (A146T) substitution. She required a feeding tube until age 6 years, but her growth was less compromised and minor involvement of skin and joints was observed. Findings not typical for Costello syndrome included microcephaly, sparse and thin, but not curly hair, and ears lacking the distinctive fleshy and forward-cocked lobes [11]. One additional patient with a change affecting amino acid 146 (A146V) has been seen [18]. While she showed some findings typical for Costello syndrome, including failure to thrive and severe hypertrophic cardiomyopathy, her facial features are less coarse and her cognitive development is reportedly better than expected for patients with Costello syndrome. With each novel sequence change it is important to demonstrate that it is disease causing, rather than a benign polymorphism. Functional relevance may be implied if the particular amino acid is frequently mutated in sporadic malignancies, or if it is located at a crucial position within the protein (fig. 1C). The identification of additional patients with changes of the same amino acid, as reported for alanine 146, also supports clinical relevance. It is impossible to draw phenotype-genotype conclusions based on single patients with a specific mutation. At this time we do not know if these mutations are truly rare, or if they have been rarely identified because the associated phenotype is not easily recognized as

Costello syndrome and the patients remain undiagnosed. It is noteworthy that several patients with rare mutations lack the strikingly coarse facial features and very deep palmar creases. Once more patients with these mutations are studied it will become clear if their phenotype varies significantly from that associated with the G12S or G12A changes.

Parental Origin of Germline HRAS Mutations

Heterozygous missense mutations causing constitutive activation of the protein product often occur in the paternal germline, as suggested by Penrose (1955) who proposed that mitotic replication errors accumulate in male germ cells [19]. The paternal age effect in Costello syndrome [20], in combination with the nature of the missense mutations, suggested a paternal origin of the mutations. In search of the parental origin of the HRAS germline mutations, Sol-Church et al. (2006) analyzed the flanking genomic region in 42 probands and 59 parents, and identified three single nucleotide polymorphisms (SNPs), proximal to the mutation site, that could be used to trace the parental origin of the germline mutations [9]. Of a total of 24 probands carrying one or more heterozygous markers, 16 informative families were identified and a paternal origin of the germline mutation was found in 14 probands. This paternal bias was confirmed by Zampino et al. [11]. All patients reported carried a de novo mutation (G12S in eight and A146T in one) inherited from the fathers and there was an advanced age at conception in fathers transmitting the mutation. The Costello syndrome sibling studied by Svik et al. (2007) carries a G12A mutation of paternal origin [15]. Since our first report [9], we identified 13 additional patients with a germline mutation of paternal origin. Our current cohort consists of two patients with maternally derived G12S mutations, 24 with paternally inherited G12S, and one each with paternally inherited

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G12A, G12C, or T58I mutation, respectively [9, 18 and this work]. The mechanism underlying the paternal bias has not been determined, and several theories have been proposed. HRAS is part of the MAPK signaling pathway and mutations could confer a similar selective advantage on sperm as those in FGFR and PTPN11. It was proposed that mutations encoding gain-of-function, such as the paternally inherited S252W in FGFR2 causing Apert syndrome, might confer a selective advantage to spermatogonia, leading to clonal expansion of mutant cells [21, 22]. Therefore it is possible that the HRAS gain-of-function mutations arising in the paternal germline confer a distinct selective advantage during fertilization. Similar paternal skewing observed in Neurofibromatosis type 1 patients carrying de novo point mutations in the NF1 gene supports this hypothesis. Although NF1 microdeletions are predominantly maternal in origin, sporadic NF1 point mutations are mostly paternally inherited [23, 24]. Neurofibromin, the NF1 gene product, is a negative regulator of Ras function; therefore mutations causing loss-of-function of neurofibromin will result in excessively active Ras signaling. A higher level of DNA methylation in spermatogonia compared to oogonia may allow for a higher mutation rate at CpG dinucleotides [14]. Alternatively, Ras proteins are found in the acrosomal regions of sperm cells and it is possible that sperm carrying certain HRAS mutations may have a selective advantage.

Somatic Mosaicism in Costello Syndrome

Prior to the identification of HRAS mutations, several reports of siblings affected with Costello syndrome [25, 26] had been thought to suggest an autosomal recessive inheritance pattern. However, germline mosaicism is a well known mechanism in the recurrence of autosomal dominant disorders, and, while not proven, is likely

to have caused the recurrence in these families. Bodkin et al. (1999) postulated somatic mosaicism in the father of a male with typical Costello syndrome. The father had a history of feeding problems, a patchy distribution of skin and hair abnormalities, and nasal papillomata. The authors suggested that this apparent male-to-male transmission was consistent with an autosomal dominant inheritance pattern [27]. A single molecularly confirmed case of somatic mosaicism in Costello syndrome has been reported [28]. This female presented with findings suggestive of Costello syndrome, including developmental delay, short stature, sparse hair, coarse facial features and thickened toenails. She had tight Achilles tendons requiring surgical lengthening. The characteristic nasal papillomata of Costello syndrome developed at age 15 years, leaving no doubt about the clinical diagnosis. Findings not typical for Costello syndrome included irregular skin hyper- and hypopigmentation, often associated with mosaicism for chromosome abnormalities. Assays performed in this patient with standard techniques on white blood cell derived DNA did not show an HRAS mutation. However, testing of multiple samples of buccal cell derived DNA revealed a sequence change qualitatively consistent with the G12S mutation. Allelic quantitation demonstrated the presence of this mutation in ~2530% of buccal cells. In this patient, standard techniques failed to identify the disease causing mutation on blood sample derived DNA, highlighting a potential pitfall in the interpretation of negative mutation analysis results. It is of note that this patient with somatic mosaicism for the HRAS mutation had her menarche at an average age and reportedly has regular periods, whereas most females with Costello syndrome have delayed or incomplete puberty or amenorrhea due to central hypogonadism [29]. Thus, this patient may be considered mildly affected in this respect, but she is at risk for having a child with Costello syndrome. Since the degree

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Fig. 2. Life cycle of Ras. Ras-GTPases function through the use of guanine nucleotide exchange factors (GEFs) to catalyze the conversion of Ras from the inactive to the active GTP-bound state. GTPase activating proteins (GAPs) accelerate the rate of hydrolysis of bound GTP to GDP resulting in inactivation of Ras. The 3D structures were generated using Deep View Swiss Pdb viewer v3.7 available as free download from http://www. Expasy.org/spdbv. Residue 12 (located in the first G-domain) and residue 61 (located in the switch II region) are depicted in green. The amino (N) and carboxyl (C) end of the protein are indicated.

Switch II

Switch I Ras-GEF

Switch II

Switch I

N GTP C

GDP Ras-GAP

Ras-GDP inactive

Ras-GTP active

of mosaicism in her germline is not known, this risk could be as high as 50%.

Effect of Mutations on Ras Function

RAS Structure and Function The HRAS gene is located on chromosome 11p15.5 and consists of 7 exons (fig. 1A). The first exon is non-coding, and intronic sequences around exon 5 (aka IDX) carry information for the transcription of two splice variants as depicted in figure 1B. Splice variant 1, in which IDX is spliced out, encodes a 21-kDa membrane anchored GTPase (p21ras), while splice variant 2 encodes a smaller protein, p19ras, thought to be a negative regulator of p21ras [30, 31]. The p21ras contains G motifs (fig. 1C), spanning residues 1014, 5763, 116119 and 144147, that are motifs conserved amongst members of the Ras family. A C-terminal hypervariable region includes a plasma membrane targeting CAAX motif and secondary signals for palmitoylation, proteolysis, and carboxyl methylation [32]. p21ras binds guanine nucleotides (GDP and GTP) through a nucleotide binding pocket formed by the interacting

G-domains. Crystallographic information indicates that residues 28, 116, 117, 118, 119, 144 and 146 interact specifically with the guanine ring of the nucleotides, while residues 11, 12, 32, 5863 are in close proximity of the -phosphate [33, 34]. Ras functions as a molecular switch by cycling from an active GTP-bound form to an inactive GDP-bound state (fig. 2). In the MAPK pathway guanine nucleotide exchange factors (GEFs), such as son of sevenless (SOS), facilitate the loading of the GTP and activation of Ras. The activated GTP-bound protein associates with downstream effectors (such as BRAF) and cellular signaling is propagated to the nucleus via a phosphorylation cascade. The signal transduction is stopped by conversion of Ras from the GTP- to GDPbound form mediated by the concerted action of Ras intrinsic GTPase activity, and GTPase activating proteins (GAPs) such as NF1. X-Ray crystallography further revealed that Ras-GTP/GDP cycling induces changes in the conformation of two switch regions (fig. 1C), formed by residues 3038 (switch I) and residues 6072 (switch II). Proper positioning of the switch regions, notably switch II, is required for GTP hydrolysis [34, 35]. Figure 2 illustrates the conformational changes

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between the two states with switch II moving away from the nucleotide binding pocket, and repositioning of the side chains of the Q61, thereby facilitating GAP binding and GTP hydrolysis [3335]. Mutations in the nucleotide binding and switch I and II domains have previously been shown to cause constitutive activation of the HRAS gene product, by locking the mutant proteins in the active GTP-bound conformation, thus disrupting the normal biochemical Ras function and causing aberrant downstream signaling [17, 3638]. Proteins containing mutated glycine residues at position 12 or 13 have reduced intrinsic and GAP-mediated GTPase activity leading to sustained MAPK activation [36, 38]. An activating gly12-to-val (G12V) mutation in the HRAS gene present in a bladder carcinoma was the first somatic point mutation described in human cancer [39, 40]. The authors showed that the glycine to valine substitution physically blocked access of GAPs to the active site, thus preventing inactivation of the GTP-bound protein. Substitution of the glutamine (Q) residue at position 61, located in the switch II region, is also an oncogenic event and the mutant protein shows reduced GTPase activity. A Defective off Switch Causes Costello Syndrome As illustrated in figure 1C, the majority of the amino acid substitutions identified in patients with Costello syndrome affect regions of the Ras protein that are important for the intrinsic as well as the GAPs-mediated GTPase activity. Thus, mutations found in these patients likely affect the off switch of p21ras and lead to hyperactivation of the MAPK pathway during development. Several of the heterozygous germline mutations (G12C, G12S, G12V, G13C, G13D, and Q22K) identified in Costello syndrome are also oncogenic somatic variants reported in human cancer [4], thus correlating with the increased incidence of cancer in this syndrome.

Costello Syndrome is Caused Exclusively by HRAS Mutations

Costello syndrome is caused by heterozygous point mutations in HRAS, resulting in a gainof-function of the abnormal protein product and increased activation of the MAPK pathway. While most patients share a paternally derived de novo G12S amino acid change, some rare mutations are likely associated with either a more severe or a milder phenotype. Phenotypic variation amongst patients sharing the common mutation may be accounted for by modifier genes, or rarely by maternal origin of the mutation, or by somatic mosaicism. There is overlap in the physical findings and cognitive abnormalities of infants with Costello syndrome and related disorders, such as Noonan or cardio-facio-cutaneous syndrome and the KRAS-mutation phenotype, but the clinical manifestations of these disorders become more distinct with age. Costello syndrome is unique in its predisposition to certain malignancies including rhabdomyosarcoma and transitional cell carcinoma of the bladder. Therefore it is important to correctly diagnose patients as early as possible, and to be consistent in diagnosing only patients with an HRAS mutation with Costello syndrome. In contrast, patients with a phenotype resembling Costello syndrome, but with a mutation in another MAPK pathway gene, should not be diagnosed with Costello syndrome, but rather with the syndrome more consistent with the respective gene mutation. Such a change from the original clinical diagnosis of Costello syndrome to the more accurate diagnosis of cardio-facio-cutaneous syndrome occurred in several reported patients after molecular testing [41, 42]. Particularly instructive are the sisters reported by Svik et al. (2007): In one the clinical diagnosis of Costello syndrome was confirmed by the identification of an HRAS mutation, in contrast the other was found to have a KRAS mutation [15]. As expected, their clinical course differed as the patients

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grew older and the Costello syndrome individual developed acanthosis nigricans, papillomata and four asymptomatic bladder carcinomas, whereas the individual with the KRAS mutation associated phenotype had severe epilepsy with hippocampal sclerosis and atrophy. These sisters illustrate the overlap of clinical presentation in infancy, and the more distinctive phenotype of the MAPK pathway disorders thereafter. Molecular testing should be helpful in clarifying the diagnosis, and it is in the patients best interest to reconsider a clinical diagnosis if the molecular test result is inconsistent. Consistency of molecular and clinical diagnosis allows for clarity in communication amongst patients and advocates, health care providers, and researchers.

In the future, drug treatment may be tailored to counteract the specific effects of certain mutations, and may vary by gene.

Acknowledgements
We thank all patients and their families for their participation in our research studies, our collaborators and numerous clinicians who referred patients and contributed information. We would also like to thank Deborah Stabley for her technical contribution to this work. KSC is supported by a grant from the NIH National Center for Research Resources, Center of Biomedical Research Excellence (COBRE) program (1 P20 RR020173) and by the Nemours Foundation.

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17 Fasano O, Aldrich T, Tamanoi F, Taparowsky E, Furth M, Wigler M: Analysis of the transforming potential of the human H-ras gene by random mutagenesis. Proc Natl Acad Sci USA 1984;81:40084012. 18 Gripp KW, Innes AM, Axelrad ME, Gillan TL, Parboosingh JS, et al: Costello syndrome associated with novel germline HRAS mutations: An attenuated phenotype? Am J Med Genet A 2008;146:683690. 19 Penrose LS: Parental age and mutation. Lancet 1955;2:312313. 20 Lurie IW: Genetics of the Costello syndrome. Am J Med Genet 1994;52: 358359. 21 Goriely A, McVean GA, Rojmyr M, Ingermarsson B, Wilkie AO: Evidence for selective advantage of pathogenic FGFR2 mutations in the male germ line. Science 2003;301:606607. 22 Goriely A, McVean GA, van Pelt AM, ORourke AW, Wall SA, et al: Gain-offunction amino acid substitutions drive positive selection of FGFR2 mutations in human spermatogonia. Proc Natl Acad Sci USA 2005;102: 60516056. 23 Lazaro C, Gaona A, Ainsworth P, Tenconi R, Vidaud D, et al: Sex differences in mutational rate and mutational mechanism in the NF1 gene in neurofibromatosis type 1 patients. Hum Genet 1996;98:696699. 24 Lopez-Correa C, Dorschner M, Brems H, Lazaro C, Clementi M, et al: Recombination hotspot in NF1 microdeletion patients. Hum Mol Genet 2001;10: 13871392. 25 Zampino G, Mastroiacovo P, Ricci R, Zollino M, Segni G, et al: Costello syndrome: Further clinical delineation, natural history, genetic definition, and nosology. Am J Med Genet 1993;47:176183.

26 Johnson JP, Golabi M, Norton ME, Rosenblatt RM, Feldman GM, et al: Costello syndrome: phenotype, natural history, differential diagnosis, and possible cause. J Pediatr 1998;133:441448. 27 Bodkin NM, Mortimer ES, Demmer LA: Male to male transmission of Costello syndrome consistent with autosomal dominant inheritance. Am J Hum Genet 1999;65(Suppl):A143. 28 Gripp KW, Stabley DL, Nicholson L, Hoffman JD, Sol-Church K: Somatic mosaicism for an HRAS mutation causes Costello syndrome. Am J Med Genet A 2006;140:21632169. 29 White SM, Graham JM Jr, Kerr B, Gripp K, Weksberg R, et al: The adult phenotype in Costello syndrome. Am J Med Genet A 2005;136:128135. 30 Cohen JB, Broz SD, Levinson AD: Expression of the H-ras proto-oncogene is controlled by alternative splicing. Cell 1989;58:461472. 31 Huang MY, Cohen JB: The alternative H-ras protein p19 displays properties of a negative regulator of p21Ras. Oncol Res 1997;9:611621. 32 Bourne HR, Sanders DA, McCormick F: The GTPase superfamily: conserved structure and molecular mechanism. Nature 1991;349:117127. 33 Pai EF, Kabsch W, Krengel U, Holmes KC, John J, Wittinghofer A: Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature 1989;341:209214. 34 Pai EF, Krengel U, Petsko GA, Goody RS, Kabsch W, Wittinghofer A: Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 resolution: implications for the mechanism of GTP hydrolysis. EMBO J 1990;9:23512359.

35 Vetter IR, Wittinghofer A: The guanine nucleotide-binding switch in three dimensions. Science 2001;294:12991304. 36 Oliva JL, Zarich N, Martinez N, Jorge R, Castrillo A, et al: The P34G mutation reduces the transforming activity of K-Ras and N-Ras in NIH 3T3 cells but not of H-Ras. J Bio Chem 2004;279:3348033489. 37 Lowy DR, Willumsen BM: Function and regulation of Ras. Annu Rev Biochem 1993;62:851891. 38 Ahmadian MR, Zor T, Vogt D, Kabsch W, Selinger Z, et al: Guanosine triphosphatase stimulation of oncogenic Ras mutants. Proc Natl Acad Sci USA 1999;96:70657070. 39 Tabin CJ, Bradley SM, Bargmann CI, Weinberg RA, Papageorge AG, et al: Mechanism of activation of a human oncogene. Nature 1982;300:143149. 40 Reddy EP, Reynolds RK, Santos E, Barbacid M: A point mutation is responsible for the acquisition of transforming properties by the T24 human bladder carcinoma oncogene. Nature 1982;300:149152. 41 Gripp KW, Lin AE, Nicholson L, Allen W, Cramer A, et al: Further delineation of the phenotype resulting from BRAF or MEK1 germline mutations helps differentiate cardio-facio-cutaneous syndrome from Costello syndrome. Am J Med Genet A 2007;143:14721480. 42 Rauen KA: Distinguishing Costello versus cardio-facio-cutaneous syndrome: BRAF mutations in patients with a Costello phenotype. Am J Med Genet A 2006;140:16811683.

Dr. Katia Sol-Church, Director, Biomolecular Core Laboratory Biomedical Research Department, Rockland Center 1, room 234, A. I. duPont Hospital for Children 1600 Rockland Rd Wilmington, DE 19803 (USA) Tel. +1 302 651 6705, Fax +1 302 651 6767, E-Mail ksolchur@nemours.org

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Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 104108

Endocrine Regulation of Growth and Short Stature in Noonan Syndrome


G. Binder
University-Childrens Hospital Tuebingen, Pediatric Endocrinology Section, Tbingen, Germany

Abstract
Short stature is a major feature of Noonan syndrome with a final height of around 2 SD below the normal mean. Puberty is delayed and the total duration of growth is prolonged. Low insulin-like growth factor-I (IGF-I) serum levels and a mild increase of spontaneous growth hormone (GH) secretion with high trough GH concentrations were described in small cohorts of children with Noonan syndrome. SHP2 in vitro binds and dephosphorylates signaling molecules that are positive regulators of the cellular response to GH. Gain-of-function mutations of PTPN11, the gene encoding SHP2, in Noonan syndrome were therefore predicted to regulate the cellular response to GH negatively. Growth in children with Noonan syndrome due to mutations of SOS1, whose gene product apparently is not involved in GH signaling, was reported to be less compromised than in those with PTPN11 mutations. Recently, IGF-I and insulin-like growth factor binding protein (IGFBP)-3 levels were found to be significantly lower in children with an activating PTPN11 mutation, while these levels are normal in children with Noonan syndrome and no mutation in PTPN11. Concomitantly, GH serum levels showed a trend to higher values in those children with PTPN11 mutations. First data suggest reduced responsiveness to treatment with high-dose GH in children with Noonan syndrome and very short stature, who are positive for PTPN11 mutations, but data are still to scarce to draw any final conclusions. Apart from disturbed GH signaling, there must be other relevant mechanisms which influence longitudinal growth in Noonan syndrome. These

deleterious mechanisms are likely to affect the intrinsic regulation of the bone development itself.
Copyright 2009 S. Karger AG, Basel

Short Stature

Short stature is a major feature of Noonan syndrome. Adults with Noonan syndrome reach a mean final height around 1.9 SD below the normal mean (1.9 SDS), 166 cm in men and 153 cm in women [13]. As a consequence, 40% of adults with Noonan syndrome have short stature (height below 1.88 SDS) and about 15% have severe short stature (height below 2.5 SDS). Birth length and weight are normal in Noonan syndrome [1]. In the growth of a child three phases can be distinguished: the infancy phase (mainly regulated by nutrition), the childhood phase (mainly regulated by growth hormone (GH)) and the pubertal phase of growth (mainly regulated by sex hormones). Low or reduced growth rate is present in all these phases in Noonan syndrome [13]. As the puberty is delayed, the total duration of growth is prolonged which may explain that adult height in males with Noonan syndrome has been underestimated in the past [3].

GH-IGF-I Axis

The central endocrine regulation of growth in childhood is based on the pulsatile secretion of GH from the pituitary gland and the GHregulated insulin-like growth factor (IGF)-I production in the liver. Both hormones, GH and IGF-I act in concert at the epiphyseal plate according to the dual effector theory. While GH promotes differentiation and sensitivity to IGF-I of the precursor cells, IGF-I causes the clonal proliferation. In the past, IGF-I serum levels were often found to be low in children with Noonan syndrome. GH secretion has been studied by several groups and found to be abnormal in a subgroup of patients, but not deficient. In comparison to normal individuals, a mild increase of spontaneous GH secretion with high trough GH concentrations was described [4, 5]. Such a constellation would be compatible with a mild form of GH insensitivity.

a good safety profile [8]. The efficacy, however, is reduced when used for these indications in comparison to the effect seen in GH deficiency. A randomized study reporting final heights revealed a mean effect of 7 cm height gain in girls with Turner syndrome [9]. As Noonan syndrome shares many dysmorphic features with Turner syndrome, paediatric endocrinologists started to treat severely short children with Noonan syndrome with the same GH dosage as used in Turner syndrome on the basis of individual treatment trials. Effects on height velocity were reported to be either less pronounced than in Turner syndrome [10] or similar [11]. Randomized controlled studies on the efficacy of this treatment are still missing. The interpretation of effects on growth is difficult due to the natural course of prolonged growth period in Noonan syndrome. Calculations should implicate the retardation of bone age and puberty, which may enable spontaneous catch-up growth after the time of normal growth arrest [13].

GH Therapy

GH substitution was started about 50 years ago in children with severe GH deficiency with satisfying results: normalization of height by induction of catch-up growth and normalization of final height. Without therapy, these children would have lost 20 to 30 cm of height. In the meantime and with the availability of recombinant GH, new indications were implemented. Recombinant GH is now used in pharmacological dosage (around 150 to 200% of the substitutional dosage) to promote growth in short children who have a disturbance of their target organ, the epiphyseal plate. This is the case in children with Turner syndrome [6]. It is also used in children with a combination of both, disturbed endocrine regulation and target organ resistance, such as the heterogeneous group of short children who are born small-for-gestational-age and do not show catchup growth [7]. Pharmacological GH therapy has

Role of SHP2 in the GHR Signaling Pathway in vitro

With the discovery of PTPN11 mutations in the majority of children with Noonan syndrome, some of the above mentioned findings seemed to get a plausible explanation at the molecular level of GH signaling. The mechanisms implicated in GH signaling are as follows: after binding of GH, the GH receptor (GHR) dimer initially activates JAK2, which includes autophosphorylation of the kinase domain of JAK2. Activated JAK2 then phosphorylates itself at additional tyrosine residues as well as tyrosines within the cytoplasmic domain of the GHR and many other signaling molecules including STAT5b. This factor is able to induce IGF-I expression of the liver [12]. SHP2, the gene product of PTPN11, has been shown to associate directly with GHR in response to GH in vitro [13, 14]. Other in vitro experiments

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demonstrated that SHP2 acts as a cytosolic phosphatase of STAT5 downregulating its activity [15, 16]. Accordingly, a mutation of the SHP2 binding site in GHR has been shown to cause prolongation of tyrosyl phosphorylation of GHR, JAK2 and STAT5b in response to GH in vitro [17]. In summary, SHP2 binds and dephosphorylates signaling molecules which are positive regulators of the cellular response to GH (fig. 1). Therefore, gain-of-function mutations of PTPN11 may be assumed to negatively regulate the cellular response to GH in children with Noonan syndrome.

GH Cytoplasm P STATs P STATs P P JAK2 P JAK2 P SHP2 JAK2 SHP2 STAT5b P P

SHP2 P STAT5b P STAT5b P STAT5b P

Short Stature in Noonan Syndrome: Genotype-Phenotype Correlation


Nucleus

Short stature is considered to be a general characteristic in children with Noonan syndrome and related disorders, irrespective of the protein of the RAS-MAPK pathway affected, suggesting that dysregulation of the RAS-MAPK signaling itself confers the disturbance of growth found in Noonan syndrome. But there are subtle hints that mutations leading to constitutive activation of SHP2 might indeed have additional specific impacts on growth in patients with Noonan syndrome that are clinically relevant. Notably, the growth in children with Noonan syndrome due to SOS1 mutations is less compromised than in the presence of PTPN11 mutations [18]. SOS1 encodes a RAS-specific guanine nucleotide exchange factor which acts downstream from SHP2 in the RAS-MAPK signaling, but is as far as we know not directly implicated in the GH signaling pathway, unlike SHP2. The obvious genotype-phenotype correlation regarding growth in patients with PTPN11 and SOS1 mutations, respectively, suggests that mutant SHP2 has stronger impacts on growth than mutant SOS1. It is tempting to speculate that the negative effect of mutant SHP2 on longitudinal growth is in part mediated by an interference with GH signaling. However, this suggestion is challenged by the

IGF-I transcription

Fig. 1. Schematic drawing illustrating known interactions of SHP2 with components of the GH signaling pathway.

fact that patients affected by germline mutations of other downstream components of the RASMAPK signaling pathway, such as RAF1 and BRAF, exhibit a degree of growth failure that is not significantly different from patients with PTPN11 mutations. Obviously, dysregulation of the RASMAPK pathway itself plays a major role for growth impairment in patients with Noonan syndrome and related disorders through mechanisms that are incompletely understood. It may also be possible that aside from SHP2 other interconnections exist between the RAS-MAPK and GH pathways.

PTPN11 Mutations and Mild GH Insensitivity in Children with Noonan Syndrome

Low IGF-I serum levels have been frequently found in short children with Noonan syndrome in the past. Recently, three groups independently

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observed that IGF-I and IGFBP-3 levels were significantly lower in patients with a PTPN11 mutation, while these levels were normal in children with Noonan syndrome but no mutation in PTPN11 [1921]. Concomitantly, serum GH measurements showed a tendency towards higher levels in those children with PTPN11 mutations [19]. This constellation would be well compatible with mild GH insensitivity. Unfortunately, no such studies are currently available that include patients with proven mutations in other components of the RAS-MAPK pathway. In the presence of GH resistance due to PTPN11 mutations, responsiveness to a pharmacological GH therapy would be suspected to be low. The above mentioned studies have addressed this issue as well: the design was prospective in two studies and retrospective in one. Overall, there was a trend to a better firstyear growth velocity of Noonan children without PTPN11 mutations in comparison to those with a PTPN11 mutation. The difference in the response to GH treatment was significant in one of those studies [19]. However, currently long term data on height development and final height are still too scarce to draw any final conclusions.

Summary

The biological basis of short stature in Noonan syndrome is not yet clear. The detection of PTPN11 mutations in approximately half of all individuals with Noonan syndrome has opened up a new perspective from the endocrinological point of view, since SHP2 is implicated in the downregulation of GH receptor signaling. Current data show decreased IGF-I and IGFBP-3 levels, which are markers of the biological effect of GH, in those children with Noonan syndrome who carry a PTPN11 mutation. Furthermore, spontaneous and stimulated GH secretion tends to be higher in those children. Taken together, these observations suggest a mild form of GH resistance. The GH-IGF-I pathway is of major interest when recombinant GH is administered in high doses to short children to promote growth and improve final height. GH responsiveness seems to be reduced in the presence of PTPN11 mutations, but to date data are too scarce to draw any final conclusions. Children with Noonan syndrome carrying mutations in components of the RAS-MAPK signaling pathway downstream from SHP2 have short stature as well, though less frequently in the case of SOS1 mutations. Therefore, apart from the disturbance of GH signaling, there must be other relevant mechanisms that influence longitudinal growth. These deleterious mechanisms are likely to affect the intrinsic regulation of the bone development itself.

References
1 Ranke MB, Heidemann P, Knupfer C, Enders H, Schmaltz AA, Bierich JR: Noonan syndrome: growth and clinical manifestations in 144 cases. Eur J Pediatr 1988;148:220227. 2 Witt DR, Keena BA, Hall JG, Allanson JE: Growth curves for height in Noonan syndrome. Clin Genet 1986;30:150153. 3 Shaw AC, Kalidas K, Crosby AH, Jeffery S, Patton MA: The natural history of Noonan syndrome: a long-term follow-up study. Arch Dis Child 2007;92:128132. 4 Ahmed ML, Foot AB, Edge JA, Lamkin VA, Savage MO, Dunger DB: Noonans syndrome: abnormalities of the growth hormone/IGF-I axis and the response to treatment with human biosynthetic growth hormone. Acta Paediatr Scand 1991;80:446450. 5 Noordam C, van der Burgt I, Sweep CG, Delemarre-van de Waal HA, Sengers RC, Otten BJ: Growth hormone (GH) secretion in children with Noonan syndrome: frequently abnormal without consequences for growth or response to GH treatment. Clin Endocrinol 2001;54:5359.

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6 Marchini A, Rappold G, Schneider KU: SHOX at a glance: from gene to protein. Arch Physiol Biochem 2007;113:116123. 7 Saenger P, Czernichow P, Hughes I, Reiter EO: Small for gestational age: short stature and beyond. Endocr Rev 2007;28:219251. 8 Cutfield WS, Lindberg A, Rapaport R, Wajnrajch MP, Saenger P: Safety of growth hormone treatment in children born small for gestational age: the US trial and KIGS analysis. Horm Res 2006;65(suppl 3):153159. 9 Stephure DK, 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;90:33602266. 10 Noordam C, van der Burgt I, Sengers RCA, Delemarre-van de Waal HA, Otten BJ: Growth hormone treatment in children with Noonans syndrome: four year results of a partly controlled trial. Acta Paediatr 2001;90:889894.

11 Osio D, Dahlgren J, Albertsson Wikland K, Westphal O: Improved final height with long-term growth hormone treatment in Noonan syndrome. Acta Paediatr 2005;94:12321237. 12 Woelfle J, Chia DJ, Massart-Schlesinger MB, Moyano P, Rotwein P: Molecular physiology, pathology, and regulation of the growth hormone/ insulin-like growth factor-I system. Pediatr Nephrol 2005;20:295302. 13 Argetsinger LS, Carter-Su C: Mechanism of signaling by growth hormone receptor. Physiol Rev 1996;76: 10891107. 14 Kim SO, Jiang J, Yi W, Feng GS, Frank SJ: Involvement of the Src homology 2-containing tyrosine phosphatase SHP2 in growth hormone signaling. J Biol Chem 1998;273:23442354. 15 Yu C-L, Yong-Jiu J, Burakoff SJ: Cytosolic tyrosine dephosphorylation of STAT5. J Biol Chem 2000;275:599604. 16 Chen Y, Wen R, Yang S, Schuman J, Zhang EE, et al: Identification of Shp-2 as a Stat5A phosphatase. J Biol Chem 2003;278:1652016527.

17 Stofega MR, Herrington J, Billestrup N, Carter-Su C: Mutation of the SHP2 binding site in growth hormone (GH) receptor prolongs GH-promoted tyrosyl phosphorylation of GH receptor, JAK2, and STAT5B. Mol Endocrinol 2000;14:13381350. 18 Tartaglia M, Pennacchio LA, Zhao C, Yadav KK, Fodale V, et al: Gain-offunction SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet 2007;39:7579. 19 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;90:53775381. 20 Ferreira LV, Souza SAL, Arnhold IJP, Mendonca BB, Jorge AAL: PTPN11 mutations and response to growth hormone therapy in children with Noonan syndrome. J Clin Endocrinol Metab 2005;90:51565160. 21 Limal J-M, Parfait B, Cabrol S, Bonnet D, Leheup B, et al: Noonan syndrome: relationships between genotype, growth, and growth factors. J Clin Endocrinol Metab 2006;91:300306.

Gerhard Binder University-Childrens Hospital Tuebingen, Pediatric Endocrinology Section Hoppe-Seyler-Strasse 1 DE72076 Tbingen (Germany) Tel. +49 7071 2983781, Fax +49 7071 294157, E-Mail gerhard.binder@med.uni-tuebingen.de

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Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 109118

The Heart in Ras-MAPK Pathway Disorders


M.C. Digilioa B. Marinob A. Sarkozyc P. Versaccib B. Dallapiccolac
aMedical cDepartment

Genetics, Bambino Ges Hospital, Rome, bPediatric Cardiology, La Sapienza University, Rome, and of Experimental Medicine, La Sapienza University and CSS-Mendel Institute, Rome, Italy

Abstract
Noonan syndrome (NS) and related disorders are due to mutations in the genes of the Ras-MAPK pathway. Congenital heart defect (CHD) is diagnosed in 6085% of patients affected by these conditions. Pulmonary valve stenosis (PVS) and hypertrophic cardiomyopathy (HCM) are the most common defects. Nevertheless, the spectrum of cardiac malformations has been progressively widened, including also atrioventricular canal defect (AVCD), mitral valve anomalies (MVA), atrial septal defect (ASD), aortic coarctation (AC), and other defects. The anatomic pattern of CHDs in NS and related disorders is specific. PVS has dysplastic pulmonary valve and fibrous thickening of the annulus and the leaflets, while HCM is characterized by left ventricle hypertrophy with asymmetric septal thickening and frequent systolic anterior motion of the mitral valve. The prevalence of specific CHDs in the different clinical conditions is varying depending on the mutated gene, and at time also on the different allelic mutations. Particularly, PVS is the prevalent CHD in NS due to PTPN11 mutations, while HCM is the predominant cardiac manifestation of NS patients with RAF1 mutations and LS patients with PTPN11 mutation.
Copyright 2009 S. Karger AG, Basel

Noonan syndrome (NS) and related disorders are due to mutations in the genes of the Ras-MAPK pathway. They are the most important cause of Mendelian congenital heart defect (CHD).

NS was described by the cardiologist Jacqueline Noonan in 1963, who reported several syndromic patients with pulmonary valve stenosis (PVS) and remarkably similar facial anomalies [1]. The peculiar cardiac involvement of NS characterized by pulmonary valvular and/or supravalvular stenosis with dysplastic and thickened valves was then confirmed by several other reports [27]. Eccentric left ventricular hypertrophy (asymmetrical septal hypertrophy) was noticed in some NS patients as an additional cardiac defect [8]. In the French literature Pernot et al. [9] and Hoeffel et al. [10] realized that these distinct cardiac defects are not specific of NS, but they also occur in the cardio-cutaneous syndromes (LEOPARD and Watson syndromes) and in neurofibromatosis type 1 (NF1) (also called Noonan-like syndromes). At the same time, Dr. Noonan argued that with further clinical studies the group of patients originally labeled as NS could be separated into several distinct entities [11]. Present clinical and molecular data have confirmed the concept that a specific cardiac anatomy is linking syndromes with similar phenotypes resulting from mutations in the genes

Table 1. Prevalence of congenital heart defects (CHDs) in the different syndromes linked to mutations in genes belonging to the Ras/MAPK pathway Syndrome Noonan syndrome PTPN11 SOS1 RAF1 KRAS LEOPARD syndrome PTPN11 RAF1 Cardio-Facio-Cutaneous syndrome BRAF MEK1/MEK2 Costello syndrome HRAS Neurofibromatosis 1 NF1 26 65 85 45 60 100 80 80 90 65 Prevalence of CHD (%)

belonging to the same pathogenetic pathway [12, 13]. Nevertheless, the prevalence of specific CHDs in the different clinical conditions is varying depending on the mutated gene, and at time also on the different allelic mutations [14].

widened, to include also atrioventricular canal defect (AVCD), mitral valve anomalies (MVA), atrial septal defect (ASD), aortic coarctation (AC), and other defects [16] (table 2). Pulmonary Valve Stenosis (PVS) PVS is the most common defect occurring in NS patients [15, 16]. Therefore, NS and Noonan-like phenotypes are the most common disorders associated with PVS [17]. The anatomic pattern is quite distinct, with dysplastic pulmonary valve and fibrous thickening of the annulus and the leaflets [7, 15, 16, 18]. Stenosis results from the fibrous thickening of the valvular leaflets, which appear deformed, glistening and edematous, eventually without fusion of commissures [19, 20]. In some cases pulmonary

Anatomic Characteristics of Cardiac Defects in Ras/MAPK Pathway

The prevalence of cardiac anomalies in syndromes caused by mutations in the genes belonging to the Ras/MAPK pathway is summarized in table 1. PVS and hypertrophic cardiomyopathy (HCM) have been the first defects linked to NS and NS-like conditions [7, 15]. Nevertheless, the spectrum of cardiac malformations in NS has been progressively

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Table 2. Prevalence (%) of specific types of CHD in syndromes linked to mutations in genes belonging to the Ras/ MAPK pathway, according to the clinical diagnosis and the gene mutated Congenital heart defect Pulmonary valve stenosis Hypertrophic cardiomyopathy Atrial septal defect Atrioventricular canal defect Aortic coarctation Ventricular septal defect Tetralogy of Fallot Mitral anomalies Arrhythmia 5 NS PTPN11 SOS1 70 10 25 3 1 7 73 10 20 7 RAF1 15 75 30 4 40 KRAS 45 20 15 4 40 25 LS PTPN11 RAF1 25 75 4 50 100 100 CFCS BRAF 45 30 20 CS MEK1-2 HRAS 20 20 20 30 30 45 40 7 NF1 NF1 3 1 1 1

CFCS = Cardio-Facio-Cutaneous syndrome; CHD = congenital heart defect; CS = Costello syndrome; LS = LEOPARD syndrome; NF1 = Neurofibromatosis 1; NS = Noonan syndrome. References: NS [14, 25, 37, 38, 4953, 55, 56, 62]; LS [25, 27, 50, 57, 58, 6769]; CFCS [73, 74]; CS [22, 24, 7578]; NF1 [7982].

valve stenosis is supraanular, consisting in the fusion of the valvular cusps with the wall of the pulmonary artery (fig. 1). Severe pulmonary valve dysplasia is a distinct marker of NS, being uncommonly found in nonsyndromic patients with PVS [21]. Histological characteristics of PVS in NS include severe thickening of the spongiosa layer of the leaflets caused by the presence of stellate and fusiform cells resembling embryonic tissue. Due to these peculiar anatomic characteristics, percutaneous balloon valvuloplasty rarely is effective in these patients. A polyvalvular disease has also been described in some patients, including the simultaneous

dysplasia of the pulmonary, aortic and mitral leaflets. Hypertrophic Cardiomyopathy (HCM) HCM occurring in NS and related disorders is characterized by left ventricle hypertrophy, consisting in asymmetric septal thickening, frequent systolic anterior motion of the mitral valve, and decreased left ventricular compliance (fig. 2) [16, 2225]. The association with structural mitral anomalies, as prolapsing, myxomatous, redundant or thick valve leaflets, is common (fig. 2). Moreover, anomalous insertion of the mitral valve causing subaortic stenosis was reported [26]. Severe left ventricular hypertrophy is at risk

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anomalous insertion of the mitral valve on the ventricular septum causing obstruction of the left ventricular outflow tract has been rarely observed in patients [26]. The leaflets of the mitral valve can be dysplastic with short cords and nodular myxomatous tissue. A defect of the cardiac jelly and extracellular matrix has been suggested as a likely pathogenetic mechanism for some CHDs in NS, including MVA associated with HCM and AVCD [3436]. Atrial Septal Defect (ASD) Patients with NS can display the ostium secundum type of ASD, often in association with PVS [14, 16, 37, 38]. Aortic Coarctation (AC) AC is diagnosed in a subgroup of NS patients [3941], mainly without any detectable mutation in some of the known genes [14, 38]. These patients are often males, and a possible relationship with a gene related to the lymphatic hypoplasia has been suggested [39, 40]. Other CHDs Additional anatomic types of CHD occasionally diagnosed in NS patients include tetralogy of Fallot [16, 42], ventricular septal defect [16], and aortic root dilatation [16, 4345].

Fig. 1. Two-dimensional echocardiography: apical 5chamber view shows a dysplastic pulmonary valve with thick leaflets and fusion of the valvular cusps with the wall of the pulmonary artery (arrows). PA: pulmonary artery; PV: pulmonary valve.

for some fatal events during the follow-up, and coronary and myocardial anomalies may also occur in these patients [14, 25, 27]. Compared to children with non-syndromic idiopathic disease, the NS patients show a more severe type of HCM with reduced diastolic function and higher prevalence of left ventricular outflow tract obstruction [25]. It has also been shown that HCM associated with malformation syndromes (78% of these cases are NS) have a worse clinical outcome [28]. Atrioventricular Canal Defect (AVCD) AVCD is a quite common defect in NS individuals [15, 16, 2932]. The partial type is prevailing, and may be associated with subaortic stenosis, as left ventricular outflow obstruction due to anomalous insertion of the mitral valve [16]. Mitral Valve Anomalies (MVA) Structural abnormalities of the mitral valve have been observed in patients with NS and LS, alone or in association with HCM, AVCD or polyvalvular disease [16, 18, 26, 27, 33]. An isolated

Prevalence and Types of Cardiac Defects According to Distinct Phenotypes and Different Mutated Genes (Table 2)

Noonan Syndrome (NS) The frequency of CHD in clinical series of NS patients evaluated before the molecular testing became available was estimated in the range of 50 to 90% [16, 4648]. PVS was the most common defect (2050%), followed by HCM (2030%), AVCD (15%), ASD (10%), and aortic coarctation (10%).

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a
Fig. 2. Two-dimensional echocardiography: (a) Parasternal long-axis view shows an asymmetric HCM involving the interventricular septum (IVS, 16 mm). (b) Apical 4-chamber view shows a significant increase of anterior interventricular septum (IVS) thickness (12 mm). Moreover a mild prolapse of the mitral valve (MV) with redundant leaflets is evident (arrow). RA: right atrium; LA: left atrium, RV: right ventricle; LV: left ventricle; Ao: aorta.

Following the discovery of PTPN11 as the most common affected gene, PVS was confirmed as the prevailing CHD in NS [14, 4954]. It was also proven that HCM was less common in PTPN11-mutated patients, while is the prevailing cardiac defect in the patients with RAF1 mutations [55, 56] and in the LS patients heterozygous for PTPN11 gene mutations [14, 27, 50, 57, 58]. PTPN11: PTPN11 was considered a candidate gene in NS because its protein product, SHP-2, is a component of several signal transduction pathways controlling the protein developmental processes, in particular the cardiac semilunar valvulogenesis and myocardial development [59]. PTPN11 mutations are associated with a wide spectrum of CHDs. In particular, codon 308 mutations are considered a mutational hot spot related to PVS [21, 49]. ASD, alone or in association with PVS, may be somewhat more common with exon 3 mutations [21, 54]. Additional CHDs include HCM, partial AVCD and MVA. This wide spectrum of defects suggests that CHDs in NS could well be related to some anomaly of the cardiac jelly and the extracellular matrix [20, 36, 60]. Accordingly, Krenz et al. [61] modeled the effect of NS-related PTPN11 mutations through the expression in

valve primordia using the chicken explant culture system. These authors have documented a relationship between PTPN11 mutations and the cell proliferation during endocardial cushion development due to an increased signaling via the Ras-MAPK pathway. Aortic coarctation, tetralogy of Fallot and ventricular septal defect are rarely found in the PTPN11 gene heterozygotes. SOS1: Most CHDs in NS patients carrying SOS1 gene mutations are PVS, eventually associated with ASD ostium secundum type [37, 62, 63]. ASD alone can also be found in a proportion of these individuals. RAF1: RAF1 mutations are quite distinctly associated with HCM. In fact, the frequency of HCM in these patients is significantly higher compared to the general NS population [55, 56]. In addition, allelic specificity has been proven, since HCM is clustering around Ser259 and Ser612 [55]. On the other hand, PVS is significantly less common among the RAF1 gene-related NS patients, a minority of which also display tetralogy of Fallot [55, 56]. KRAS: PVS is the prevailing defect among NS patients with KRAS mutations, although HCM and ASD have been found in some cases [6466].

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LEOPARD Syndrome PTPN11: The most common CHD in LS patients with PTPN11 gene mutations is HCM, which is usually manifesting with left ventricular hypertrophy. This anomaly can be associated either with PVS with dysplastic valve leaflets or with polyvalvular dysplasia involving the aortic and mitral valves [50, 58]. In our experience, HCM is found in 75% of LS patients with the PTPN11 mutation, in 80% of LS patients with cardiac defects, and in 87% of patients with a diagnosis of LS in the first year of life [50, 58]. Right ventricular hypertrophy is detectable in about 30% of the patients, in association with left ventricular hypertrophy and PVS. The prevailing PTPN11 mutations in LS affect exons 7 and 12 [50, 57, 6769]. A subset of cases display mutations in exon 13, while the Gln510Glu mutation is associated with a distinct cardiac phenotype, with rapidly progressive severe biventricular obstructive HCM and systolic anterior motion of the mitral valve [27]. The early onset of this severe HCM allows in some instances the antenatal detection of this defect using echocardiography [27]. Heart failure symptoms can manifest in the first years of life, and can even require cardiac surgery (myectomy). Interestingly, PTPN11 gene LS-related mutations appear to dysregulate the Ras-MAPK pathway in a different way in respect to those occurring in NS. In fact, LS mutations seem to have a negative effect onto the catalytic activity, implying a loss-of-function in the pathogenesis of this syndrome, rather than a gain-of-function as proven in NS [70]. Long-term prognosis is usually favorable in LS patients with mild cardiac involvement. However, patients with left ventricular hypertrophy may develop significant symptoms and arrhythmias during the follow-up, and a few fatal events have been documented [25]. ECG anomalies can be detected in 75% of patients, including left or biventricular

hypertrophy in most of the cases, prolonged QTc intervals, repolarization abnormalities and conduction defects [25]. Additional cardiac findings in these patients include coronary artery anomalies [71, 72], noncompaction of left ventricle [25], and AVCD [57]. Of note, patients with AVCD can develop subsequently HCM [50]. RAF1: Up to now, only two patients with LS and RAF1 mutations have been identified. Both had HCM, associated with PVS in one of them [55]. Functional studies of one RAF1 mutation associated with LEOPARD syndrome appear to dysregulate the Ras-MAPK pathway, showing a higher kinase activity compared to the wildtype protein, in a manner similar to those of NSassociated mutations [55]. Accordingly, these data indicate that the pathogenesis of LEOPARD syndrome may not be characterized simply as reduced RAS signal transduction, as previously suggested by functional studies performed on PTPN11 mutants. CFCS (Cardio-Facio-Cutaneous Syndrome) BRAF, MEK12: CHD in CFCS patients include PVS with dysplastic leaflets, HCM and ASD [73, 74]. It has been suspected that the combination of PVS and ASD (with or without HCM) is more common in patients with BRAF and MEK12 mutations, raising the question of whether ASD could represent a physiologic response to the frequent downstream pulmonary flow obstruction, or rather could be related to the impact of BRAF and MEK1 mutations on cardiogenesis [74]. Costello Syndrome (CS) CHDs occur in 65% of patients with CS, and mainly include PVS, HCM, and rhythm disturbances [22, 24, 7578]. Each of these defects can be diagnosed in about one third of the patients, but the combination of two anomalies is not unusual. Rhythm disturbances are often manifesting as atrial tachycardia, also referred to as chaotic, multifocal, and ectopic tachycardia

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[24], possibly secondary to dysplasia of the conduction system. The occurrence of both HCM and atrial tachycardia in a dysmorphic neonate is a striking feature of CS, prompting the molecular search of HRAS gene mutations. The onset of HCM in CS usually occurs in the first two years of life [24]. Also rhythm disturbances are manifesting as a very early symptom [24], and their etiology appears to be related to the genetic defect itself, based on the following observations: (1) rhythm disturbances may occur in the absence of HCM; (2) HCM does not precede arrhythmias in many cases; (3) histological evidence of congenital dysplasia of the conduction system. Neurofibromatosis 1 (NF1) The frequency of CHD in NF1 ranges from 0.4 to 6.4% in published series of patients [79, 80]. The percentage is higher in the selected series of patients with NF1/NS phenotype [81], and in those heterozygous for deletions of the entire NF1 gene and flanking regions [82]. The prevailing CHD is PVS, but also ASD, aortic coarctation and mitral anomalies have been found [80, 81]. PVS is anatomically similar to the defect occurring in other syndromes resulting from mutations in the genes of the Ras/MAPK pathway. Most of the patients with aortic coarctation have a long fusiform type vascular narrowing, which differs from the abrupt segmental constriction often seen in patients without NF1. The involvement of the neurofibromin gene in the cardiac development is supported by Nf1 knockout mouse models. In fact, homozygous Nf1 mutant embryos display CHD, including double outlet right ventricle and associated anomalies of the cardiac outflow tract formation, endocardial cushion development, and myocardial structure [83]. Similarly, the vascular characteristic of NF1 may result from abnormal neurofibromin function, since it has been shown that neurofibro-

min is expressed in the endothelial and smooth muscle cells of blood vessels [84].

Evaluation and Management

All patients should have a cardiologic study at diagnosis, including clinical evaluation, electrocardiography, and two-dimensional color-Doppler echocardiography. Four extremity blood pressure measurements should be controlled, due to possible occurrence of aortic coarctation, particularly in patients with NF1. Cardiac hypertrophy may develop with time. Therefore, an echocardiographic follow-up every 2 years, particularly in the patients with LS and NS with RAF1 mutations, is indicated also when echocardiography is normal at the first evaluation. All patients, particularly those with CS, should be followed also with a 24-hour ECG monitoring, due to the possible occurrence of rhythm disturbances. Patients with a diagnosed CHD should be followed by a pediatric cardiologist, and the timing of follow-up should be programmed based on the results of periodical investigations and clinical course.

Treatment

PVS Correction of pulmonary outflow obstruction caused by valvular dysplasia is indicated. The initial choice can be transcatheter balloon valvuloplasty, although this procedure is frequently ineffective in the patients with NS, due to the peculiar dysplasia of the pulmonary valve leaflets. For this reason, surgical valvotomy is often required. Prophylaxis for bacterial endocarditis is required in all patients with a CHD for dental treatments, surgery, or catheterization.

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HCM Pharmacological treatment of obstructive HCM includes -blockade or calcium channel blockers. The sporadic occurrence of fatal events in patients with HCM suggests careful periodic evaluation

and risk assessment, and consideration for prophylaxis against sudden death with implantable cardioverter defibrillators in some cases. Severe HCM can also be treated by surgical myomectomy or transplantation.

References
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39 Hasegawa T, Ogata T, Hasegawa Y, Honda M, Nagai T, et al: Coarctation of the aorta and renal hypoplasia in a boy with Turner-Noonan surface anomalies and a 46,XY karyotype: A clinical model for the possible impairment of a putative lymphogenic gene(s) for Turner somatic stigmata. Hum Genet 1996;97:564567. 40 Digilio MC, Marino B, Picchio F, Prandstraller D, Toscano A, Giannotti A, Dallapiccola B: Noonan syndrome and aortic coarctation. Am J Med Genet 1998;80:160162. 41 Lemire EG: Noonan syndrome or new autosomal dominant condition with coarctation of the aorta, hypertrophic cardiomyopathy, and minor anomalies. Am J Med Genet 2002;113: 286290. 42 Digilio MC, Marino B, Giannotti A, Dallapiccola B: Exclusion of 22q11 deletion in Noonan syndrome with tetralogy of Fallot. Am J Med Genet 1996;62:413414. 43 Shachter N, Perloff JK, Mulder DG: Aortic dissection in Noonans syndrome (46 XY Turner). Am J Cardiol 1984;54:464465. 44 Lin AE, Garver KL, Allanson J: Aorticroot dilatation in Noonans syndrome. N Engl J Med 1987;317:16681669. 45 Power PD, Lewin MB, Hannibal MC, Glass IA: Aortic root dilatation is a rare complication of Noonan syndrome. Pediatr Cardiol 2006;27:478480. 46 Allanson JE: Noonan syndrome. J Med Genet 1987;24:913. 47 Patton MA: Noonan syndrome: A review. Growth Genet Horm 1994;33:13. 48 Sharland M, Burch M, McKenna WM, Patton MA: A clinical study of Noonan syndrome. Arch Dis Child 1992;67:178183. 49 Tartaglia M, Kalidas K, Shaw A, Song X, Musat DL, et al: PTPN11 mutations in Noonan syndrome: Molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity. Am J Hum Genet 2002;70:15551563. 50 Sarkozy A, Conti E, Digilio MC, Marino B, Morini E, et al: Clinical and molecular analysis of 30 patients with multiple lentigines LEOPARD syndrome. J Med Genet 2004;41:e68. 51 Zenker M, Buheitel G, Rauch R, Koenig R, Bosse K, et al: Genotype-phenotype correlations in Noonan syndrome. J Pediatr 2004;144:368374.

52 Jongmans M, Sistermans EA, Rikken A, Nillessen WM, Tamminga R, et al: Genotypic and phenotypic characterization of Noonan syndrome: New data and review of the literature. Am J Med Genet A 2005;134:165170. 53 Musante L, Kehl HG, Majewski F, Meinecke P, Schweiger S, et al: Spectrum of mutations in PTPN11 and genotype-phenotype correlation in 96 patients with Noonan syndrome and five patients with cardio-facio-cutaneous syndrome. Eur J Hum Genet 2002;11:201206. 54 Kamisago M, Hirayama-Yamad K, Kato T, Imamura S, Joo K, et al: Missense mutations in the PTPN11 as a cause of cardiac defects associated with Noonan syndrome; in Artman M, Benson DW, Srivastava D, Nakazawa M (eds): Cardiovascular Development and Congenital Malformations. Molecular and Genetic Mechanisms, pp 273276 (Blackwell Futura Publishing, Malden, Massachusetts, 2005). 55 Pandit B, Sarkozy A, Pennacchio LA, Carta C, Oishi K, et al: Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nat Genet 2007;39:10071012. 56 Razzaque MA, Nishizawa T, Komoike Y, Yagi H, Furutani M, et al: Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet 2007;39:10131017. 57 Digilio MC, Conti E, Sarkozy A, Mingarelli R, Dottorini T, et al: Grouping of Multiple Lentigines/LEOPARD and Noonan syndrome on the PTPN11 gene. Am J Hum Genet 2002;71:389394. 58 Digilio MC, Sarkozy A, de Zorzi A, Pacileo G, Limongelli G, et al: LEOPARD syndrome: Clinical diagnosis in the first year of life. Am J Med Genet A 2006;140:740746. 59 Chen B, Bronson RT, Klaman RD, Hampton TG, Wang J-F, et al: Mice mutant for Egfr and Shp2 have defective cardiac semilunar valvulogenesis. Nat Genet 2000;24:296299. 60 Marino B, Digilio MC: Congenital heart disease and genetic syndromes: specific correlation between cardiac phenotype and genotype. Cardiovasc Pathol 2000;9:303315.

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61 Krenz M, Yutzey KE, Robbins J: Noonan syndrome mutation Q79R in Shp2 increases proliferation of valve primordia mesenchymal cells via extracellular signal regulated kinase 1/2 signaling. Circ Res 2005;97:813820. 62 Roberts AE, Araki T, Swanson KD, Montgomery KT, Schiripo TA, et al: Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet 2007;39:7074. 63 Zenker M, Horn D, Wieczorek D, Allanson J, Pauli S, et al: SOS1 is the second most common Noonan gene but plays no major role in cardio-facio-cutaneous syndrome. J Med Genet 2007;44:651656. 64 Schubbert S, Zenker M, Rowe SL, Boll S, Klein C, et al: Germline KRAS mutations cause Noonan syndrome. Nat Genet 2006;38:331336. 65 Carta C, Pantaleoni F, Bocchinfuso G, Stella L, Vasta I, et al: Germline missense mutations affecting KRAS isoform B are associated with a severe Noonan syndrome phenotype. Am J Hum Genet 2006;79:129135. 66 Zenker M, Lehmann K, Schulz AL, Barth H, Hansmann D, et al: Expansion of the genotypic and phenotypic spectrum in patients with KRAS germline mutations. J Med Genet 2007;44:131135. 67 Legius E, Schrander-Stumpel C, Schollen E, Pulles-Heintzberger C, Gewillig M, Fryns JP: PTPN11 mutations in LEOPARD syndrome. J Med Genet 2002;39:571574. 68 Keren B, Hadchouel A, Saba S, Sznajer Y, Bonneau D, et al, for the French Collaborative Noonan Study Group: PTPN11 mutations in patients with LEOPARD syndrome: a French multicentric experience. J Med Genet 2004;41:e117.

69 Yoshida R, Nagai T, Hasegawa T, Kinoshita E, Tanaka T, Ogata T: Two novel and one recurrent PTPN11 mutations in LEOPARD syndrome. Am J Med Genet A 2004;130:432434. 70 Tartaglia M, Martinelli S, Stella L, Bocchinfuso G, Flex E, et al: Diversity and functional consequences of germline and somatic PTPN11 mutations in human disease. Am J Hum Genet 2006;78:279290. 71 Yagubyan M, Panneton JM, Lindor NM, Conti E, Sarkozy A, Pizzuti A: LEOPARD syndrome: a new polyaneurism association and an update on the molecular genetics of the disease. J Vasc Surg Apr 2004;39:897900. 72 Pacileo G, Calabr P, Limongelli G, Santoro G, Digilio MC, Sarkozy A, et al: Diffuse coronary artery dilation in a young patient with LEOPARD syndrome. Int J Cardiol 2006;112:e35e37. 73 Roberts A, Allanson J, Jadico SK, Kavamura MI, Noonan J, et al: The cardio-facio-cutaneous (CFC) syndrome: a review. J Med Genet 2006;43: 833842. 74 Gripp KW, Lin AE, Nicholson L, Allen W, Cramer A, et al: Further delineation of the phenotype resulting from BRAF or MEK1 germline mutations helps differentiate Cardio-Facio-Cutaneous syndrome from Costello syndrome. Am J Med Genet A 2007;143:14721480. 75 van Eeghen AM, van Gelderen I, Hennekam RCM: Costello syndrome: Report and review. Am J Med Genet 1999;82:187193. 76 Lin AE, Gripp KW, Kerr B: Costello syndrome; in Cassidy SB, Allanson JE (eds): Management of Genetic Syndromes, ed. 2, pp 151161 (John Wiley & Sons, Inc., Hoboken, New Jersey 2005).

77 Gripp KW, Lin AE, Stabley DL, Nicholson L, Scott CI Jr, et al: HRAS mutation analysis in Costello syndrome: Genotype and phenotype correlation. Am J Med Genet A 2006;140:17. 78 Digilio MC, Sarkozy A, Capolino R, Chiarini Testa MB, Esposito G, et al: Costello syndrome: clinical diagnosis in the first year of life. Eur J Pediatr 2008;167:621628. 79 Lin AE, Birch PH, Korf BR, Tenconi R, Niimura M, et al: Cardiovascular malformations and other cardiac abnormalities in neurofibromatosis 1 (NF1). Am J Med Genet 2000;95:108117. 80 Friedman JM, Arbiser J, Epstein JA, Gutmann DH, Huot SJ, et al: Cardiovascular disease in neurofibromatosis 1: Report of the NF1 Cardiovascular Task Force. Genet Med 2002;4:105111. 81 De Luca A, Bottillo I, Sarkozy A, Carta C, Neri C, et al: NF1 gene mutations represent the major molecular event underlying Neurofibromatosis-Noonan syndrome. Am J Hum Genet 2005;77:10921101. 82 Venturin M, Guarnieri P, Natacci F, Stabile M, Tenconi R, et al: Mental retardation and cardiovascular malformations in NF1 microdeleted patients point to candidate genes in 17q11.2. J Med Genet 2004;41:3541. 83 Brannan CI, Perkins AS, Vogel KS, Ratner N, Nordlund ML, et al: Targeted disruption of the Neurofibromatosis type-1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues. Genes Dev 1994;8:10191029. 84 Hamilton SJ, Friedman JM: Insights into the pathogenesis of neurofibromatosis 1 vasculopathy. Clin Genet 2000;58:341344.

M. Cristina Digilio Medical Genetics, Bambino Ges Hospital Piazza S. Onofrio 4 IT00165 Rome (Italy) Tel. +39 06 68592227, Fax +39 06 68592004, E-Mail digilio@opbg.net

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Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 119127

Myeloproliferative Disease and Cancer in Persons with Noonan Syndrome and Related Disorders
C. Kratz
Division of Pediatric Hematology/Oncology, Department of Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg, Germany

Abstract
The Ras signaling pathway has been extensively studied because of its fundamental role in cancer. This review discusses the occurrence of cancer in persons affected by germline mutations in genes of this pathway. There is a link between Noonan syndrome and myeloproliferative disease (NS/MPD) occurring shortly after birth. Approximately 50% of reported NS/MPD cases are caused by germline mutations of PTPN11 predicting a T73I substitution in SHP2. Persons with Costello syndrome (CS) are at an elevated risk to develop embryonal rhabdomyosarcoma (ERMS), bladder cancer, and ganglioneuroblastoma. The high cancer risk in CS corresponds to the observation that this syndrome is caused by germline mutations of HRAS that typically predict amino acid substitutions at residues Gly12 and Gly13 that are also mutated somatically in cancer. The types of cancer associated with CS suggest that HRAS mutations are major events in the development of these neoplasms. In the future, cancer drugs targeting the Ras pathway may be of potential benefit for patients with these syndromes.
Copyright 2009 S. Karger AG, Basel

(CFC), or LEOPARD syndrome (LS) (table 1) [159]. There is a clear association between NS and MPD during the first months of life [17]. Moreover, several reports link NS and related disorders to acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and other neoplasms, however, the association is weak. There are a number of reports on patients with CS and embryonal rhabdomyosarcoma (ERMS), bladder cancer, or neuroblastoma [13]. This article reviews the occurrence of MPD and cancer in NS and related syndromes and discusses clinical and scientific implications.

Myeloproliferative Disease in Infants with Noonan Syndrome

To date, 88 cases of myeloproliferative disease (MPD) or cancer have been reported in individuals with Noonan syndrome (NS), Costello syndrome (CS), cardio-facio-cutaneous syndrome

Twenty-six cases of NS with infantile MPD (NS/ MPD) have been reported previously [1728, 60] and additional cases of MPD due to germline mutations of PTPN11 implicated in LS (table 1, 2) have also been documented in the literature [22, 26, 29]. This number of reported cases suggests that NS/MPD is not a common complication of

Table 1. Eighty-eight cases of myeloproliferative disease or cancer in patients with Noonan syndrome and related disorders reported by October 2007 Diseasea RMS MPD ALL/NHL AML NBL UC HB CMML TC CLL WT
a

Noonan syndrome 3 [13] 26 [1728] 10 [3037] 3 [34, 41, 42] 4 [42, 4446]

Costello syndrome 20 [416]

CFC syndrome

LEOPARD syndrome

3 [22, 26, 29] 2 [3840] 1 [43] 4 [14, 4750] 4 [5, 16, 5153] 1 [54, 55] 1 [39] 1 [35]

1 [56] 2 [57, 58] 1 [59] 1 [35]

ALL/NHL: Acute lymphoblastic leukemia/non-Hodgkins lymphoma; AML: Acute myeloid leukemia; CLL: Chronic lymphocytic leukemia; CMML: Chronic myelomonocytic leukemia; HB: Hepatoblastoma; MPD: Myeloproliferative disease; NBL: Neuroblastoma; RMS: Rhabdomyosarcoma; TC: Testical cancer; UC: Carcinoma of the urinary tract; WT: Wilms tumor.

NS, which is explained by the observation that NS/MPD is associated with specific PTPN11 mutations (see below). Patients with NS/MPD usually present with thrombocytopenia (which can be present at birth [25, 27]), increased myelomonocytic cells, and splenomegaly in the first months of life [17]. Typically, hematologic abnormalities improve with no or little treatment [17, 20, 25, 27]. In some cases, NS/MPD is aggressive and lethal [17, 19, 28], and requires more aggressive treatment. Interestingly, even in benign cases, splenomegaly and moderate myelomonocytic hyperplasia may persist for years [17]. One patient with NS/MPD developed AML, responded to chemotherapy, and later suffered from myelodysplastic syndrome with additional chromosomal abnormalities [17]. A polyclonal pattern has been documented in one analyzed case of NS/MPD [17]. NS/MPD shares many features with juvenile myelomonocytic leukemia (JMML), a rare,

acquired MPD of early childhood characterized by the proliferation of myelomonocytic cells causing splenomegaly, monocytosis, and thrombocytopenia (reviewed in [61]). The disease is usually rapidly lethal if patients are left untreated, however, approximately 50% of cases can be rescued by allogeneic hematopoietic stem cell transplantation [62]. Hematopoietic cells harbor somatic mutations of PTPN11 or RAS (NRAS and KRAS) genes in ~35 and ~25% of cases, respectively [21, 22], and another 11% of cases of JMML have a clinical diagnosis of neurofibromatosis type 1 (NF1) (reviewed in [63]). Patients with NF1 and JMML show acquired uniparental disomy (UPD) at the NF1 gene locus leading to loss of the wild-type NF1 allele [64, 65]. Together, these data and studies in mice [66, 67] provide strong evidence that JMML is, at least in part, due to aberrant Ras signaling. As illustrated in table 2, the germline PTPN11 mutations

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Table 2. Germline PTPN11 mutations in patients with Noonan syndrome and myeloproliferative disease differ from somatic PTPN11 mutations in juvenile myelomonocytic leukemia PTPN11 mutation [2123, 25, 26, 28, 74] Exon 3 T52S G60R G60V delG60 D61Y D61V D61G D61N Y62D A72T A72V A72G T73I E76K E76G E76V E76A E76M Exon 13 R498Wa S502A S502T G503R G503A G503V Q506Pa
aMutation

Germline: NS/MPD n = 22

Somatic: JMML n = 107

1 2 1 1 1 10

1 2 9

12 7 1

9 7

29 10 2 2 1

1 1 1 1 2 associated with LEOPARD syndrome.

6 4

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in patients with NS/MPD (PTPN11 T73I most common) differ from somatic PTPN11 mutations observed in sporadic JMML (PTPN11 E76K most common) [26]. Although tempting, the mutations identified in cases of NS/MPD currently do not allow for predicting the course of the hematologic disease, because (1) many mutations have been described in only one or two cases (table 2); (2) the PTPN11 T73I mutation has been identified in 10 out of 22 cases of NS/MPD (table 2), however, the phenotype/genotype correlations are hampered by the fact that clinical information is not available for all cases and it is known that PTPN11 T73I is associated with both mild and aggressive disease [23, 28]. Except for rare cases [25], patients with NS/MDP carry de novo mutations and represent sporadic cases of NS [26], indicating that the underlying mutations encode relatively potent gain-of-function SHP2 alleles. While most cases of NS/MPD are due to mutations of PTPN11 affecting exons 3 or 13 (table 2), one patient harbored a KRAS T58I allele [27]. Germline SOS1 mutations have been excluded as a common cause of NS/MPD and somatic SOS1 mutations have been excluded in sporadic JMML [68]. A simplified model of NS/ MPD and non-syndromic JMML is depicted in figure 1.

Cancer in Patients with Costello Syndrome

This topic has recently been comprehensively reviewed by Gripp [13]. The most common malignancy in patients with CS is rhabdomyosarcoma (RMS). The annual incidence of sporadic RMS in children younger than 19 years of age is 45 cases per million children (reviewed in [69]). Twenty cases of RMS have been previously reported in CS [416]. Considering the low incidence of CS, this relatively high number of reports indicates that patients with CS are predisposed to develop RMS [13]. ERMS is the most common histologic subtype in patients with CS [13], however,

alveolar or pleomorphic types also occur [6, 10]. The age at diagnosis of RMS in patients with CS ranges between 6 months and 6 years [13]. The CS gene, HRAS, is located on chromosome 11p15.5, a region showing allelic imbalances in sporadic ERMS and ERMS in association with CS (CS/ ERMS). The critical gene for ERMS development in this region is unknown. Interestingly, somatic HRAS mutations in sporadic ERMS succeed the emergence of UPD [70]. In contrast, HRAS germline mutations are the first step in CS/ERMS. Subsequent development of UPD at 11p15.5 may explain previous observations that CS/ERMS express mutant HRAS only [16, 70]. Figure 2 provides a simplified model illustrating the presumed pathogenesis of CS/ERMS and sporadic ERMS. Neuroblastoma has been reported in four cases of CS [14, 4750]. Ganglioneuroblastoma is the most frequently reported histologic subtype [14, 47, 48, 50]. Considering the association of this tumor in persons with CS, HRAS may be a candidate gene for this histologic subtype of neuroblastoma. Notably, it was shown in one case of CS associated ganglioneuroblastoma that in tumor cells the wild-type HRAS allele is not expressed [14]. CS is the only cancer predisposition syndrome associated with childhood carcinoma of the urinary tract [13] and four cases have been reported [7, 17, 5153]. It is, therefore, not surprising that somatic HRAS mutations are involved in the pathogenesis of sporadic bladder carcinoma [71]. Due to the high tumor risk in persons with CS surveillance is important [10]. In three recent reports on HRAS mutations in patients with CS, the HRAS G12A allele has been identified in a total of seven patients, four of whom developed a malignancy [5, 14, 15], inferring that this particular mutation is potentially associated with a relatively high tumor risk [15]. Nevertheless, there is insufficient data to prove that specific HRAS mutations are associated with a higher tumor risk than other HRAS germline mutations [5].

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Bone marrow and blood

Transient proliferation

Spontaneous remission

T73I

T73I

T73I T73I T73I T73I Fetal period or early infancy

T73I

T73I

Cell of Germline PTPN11 myelomonomutation cytic lineage

Later life JMML E76K E76K E76K Remission post HSCT

Bone marrow and blood

Bone marrow and blood

E76K Cell of myelomonocytic lineage Somatic PTPN11 mutation

E76K

Fig. 1. Model of transient myeloproliferation in association with Noonan syndrome and non-syndromic juvenile myelomonocytic leukemia. (a) In individuals with Noonan syndrome caused by specific germline mutations of PTPN11 (table 2) or KRAS a transient proliferation of myelomonocytic cell is observed. This proliferation may not require additional hits and resolves after the first months of life after affected cells pass a critical developmental stage. (b) Somatic mutations of PTPN11 or KRAS that have stronger biologic effects on Ras signaling than germline mutations and give rise to an aggressive myeloproliferative disorder termed juvenile myelomonocytic leukemia (JMML). Patients can be cured by hematopoietic stem cell transplantation (HSCT).

Neoplasms Occurring at Lower Frequencies in Patients with Noonan Syndrome and Related Disorders

There is considerable overlap in the tumor spectrum observed in patients with NS and related syndromes (table 1). This observation is likely due to the fact that these syndromes share a common pathway. The relatively low number of reported cases of MPD or cancer beside NS/MPD, CS/ERMS, CS/carcinoma of the urinary tract, and CS/neuroblastoma may be due to several reasons: (1) the features of NS can be subtle and overlooked in cancer patients and (2) NS-associated mutants of PTPN11 or RAS are functionally mild in contrast to the cancer-associated mutants, thus requiring several genetic alterations cooperating during transformation. Indeed, for a number of cases second hits such as somatic duplication of

the mutant PTPN11 allele or acquisition of somatic gene fusions have been described [37, 42, 56]. Notably, recent reports describe an individual with CFC due to a germline mutation of MEK1 who developed hepatoblastoma [54, 55]. It remains to be determined if somatic MEK1 mutations are involved in the pathogenesis of nonsyndromic tumors of this kind.

Concluding Remarks

The occurrence of MPD or cancer and the types of cancer arising in individuals with Ras pathway disorders appear to depend mainly on a number of genetic factors: (1) The disease causative gene and the transforming potential of the mutant protein. Patients with HRAS mutations have the highest tumor risk. The respective involved

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Soft tissue

UPD chrom. 11p15.5

Embryonal rhabdomyosarcoma G12S UPD G12S UPD G12S UPD Infancy

G12S Germline HRAS mutation

G12S UPD

a
Soft tissue UPD chrom. 11p15.5 Somatic HRAS mutation

Embryonal rhabdomyosarcoma G12S UPD G12S UPD G12S UPD

UPD

G12S UPD

Fig. 2. Model of embryonal rhabdomyosarcoma in association with Costello syndrome and non-syndromic embryonal rhabdomyosarcoma. (a) Costello syndrome causative HRAS germline mutations are initiating events. To promote tumor development additional hits such as acquired uniparental disomy (UPD) at chromosome 11p15.5 are required. HRAS is located on 11p15.5. Therefore, UPD 11p15.5 leads to duplication of mutant HRAS. (b) In sporadic embryonal rhabdomyosarcoma UPD 11p15.5 is an early event that can be succeeded by somatic HRAS mutations [70].

HRAS proteins are highly potent and the same mutations also occur as somatic mutations in cancer, although the most common cancer associated mutant HRAS G12V is rare in CS [5, 14]. By contrast, there is very little overlap between germline and cancer associated somatic mutations of PTPN11 (table 2), KRAS and BRAF [71] and there are no or rare cases of somatic cancer associated mutations of SOS1, RAF1, MEK1, and MEK2 providing an explanation for the relatively low number of neoplasms in patients with germline mutations in these genes. (2) The role of the disease causative gene in the affected tissues or cells from which cancer arises. Hyperactive Ras appears to have a strong impact on the growth of myelomonocytic cells. Likewise, normal HRAS function appears to be of high relevance in cells that give rise to ERMS, neuroblastoma, and in urothelial cells. (3) The number of genetic events

required for transformation. This may explain the occurrence of NS/MPD in young infants with NS because this kind of MPD may require no additional genetic events. By contrast, cancer types that are caused by several hits such as ALL or AML occur less frequently. (4) Other genetic or epigenetic factors. These modifying factors may explain the observation that there is no strict genotype/phenotype correlation. For example, as discussed above, the PTPN11 T73I allele has been associated with both benign and aggressive NS/ MPD. Efforts to target the Ras pathway in cancer may be of potential benefit to patients with NS and related syndromes. Recently, a small hairpin RNA targeting the dominant mutant form of Fgfr2 prevented Apert-like syndrome in mice. Moreover, treatment of the mutant mice with U0126, an inhibitor of MEK1/2 that blocks phosphorylation

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and activation of ERK1/2, inhibits craniosynostosis [72, 73]. Thus, potentially, it will be possible to prevent tumors in persons with CS in the future. These and related issues will need to be addressed in animal models of syndromes caused by aberrant Ras signaling.

Acknowledgement
I am grateful to Dr. Mwe Mwe Chao for critical reading and discussion of the article.

References
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38 van Den Berg H, Hennekam RC: Acute lymphoblastic leukaemia in a patient with cardiofaciocutaneous syndrome. J Med Genet 1999;36:799800. 39 Niihori T, Aoki Y, Narumi Y, Neri G, Cave H, et al: Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome. Nat Genet 2006;38:294296. 40 Makita Y, Narumi Y, Yoshida M, Niihori T, Kure S, et al: Leukemia in Cardio-facio-cutaneous (CFC) syndrome: a patient with a germline mutation in BRAF proto-oncogene. J Pediatr Hematol Oncol 2007;29:287290. 41 Matsubara K, Yabe H, Ogata T, Yoshida R, Fukaya T: Acute myeloid leukemia in an adult Noonan syndrome patient with PTPN11 mutation. Am J Hematol 2005;79:171172. 42 Chantrain CF, Jijon P, De Raedt T, Vermylen C, Poirel HA, Legius E, Brichard B: Therapy-related acute myeloid leukemia in a child with Noonan syndrome and clonal duplication of the germline PTPN11 mutation. Pediatr Blood Cancer 2007;48:101104. 43 Ucar C, Calyskan U, Martini S, Heinritz W: Acute myelomonocytic leukemia in a boy with LEOPARD syndrome (PTPN11 gene mutation positive). J Pediatr Hematol Oncol 2006;28: 123125. 44 Lopez-Miranda B, Westra SJ, Yazdani S, Boechat MI: Noonan syndrome associated with neuroblastoma: a case report. Pediatr Radiol 1997;27: 324326. 45 Cotton JL, Williams RG: Noonan syndrome and neuroblastoma. Arch Pediatr Adolesc Med 1995;149: 12801281. 46 Ijiri R, Tanaka Y, Keisuke K, Masuno M, Imaizumi K: A case of Noonans syndrome with possible associated neuroblastoma. Pediatr Radiol 2000;30:432433. 47 Zampino G, Mastroiacovo P, Ricci R, Zollino M, Segni G, Martini-Neri ME, Neri G: Costello syndrome: further clinical delineation, natural history, genetic definition, and nosology. Am J Med Genet 1993;47:176183. 48 Moroni I, Bedeschi F, Luksch R, Casanova M, DIncerti L, Uziel G, Selicorni A: Costello syndrome: a cancer predisposing syndrome? Clin Dysmorphol 2000;9:265268.

49 Flores-Nava G, Canun-Serrano S, Moysen-Ramirez SG, Parraguirre-Martinez S, Escobedo-Chavez E: [Costello syndrome associated to a neuroblastoma. Presentation of a case]. Gac Med Mex 2000;136:605609. 50 Zampino G, Pantaleoni F, Carta C, Cobellis G, Vasta I, et al: Diversity, parental germline origin, and phenotypic spectrum of de novo HRAS missense changes in Costello syndrome. Hum Mutat 2007;28:265272. 51 Franceschini P, Licata D, Di Cara G, Guala A, Bianchi M, Ingrosso G, Franceschini D: Bladder carcinoma in Costello syndrome: report on a patient born to consanguineous parents and review. Am J Med Genet 1999;86:174179. 52 Gripp KW, Scott CI Jr, Nicholson L, Figueroa TE: Second case of bladder carcinoma in a patient with Costello syndrome. Am J Med Genet 2000;90:256259. 53 Urakami S, Igawa M, Shiina H, Shigeno K, Kikuno N, Yoshino T: Recurrent transitional cell carcinoma in a child with the Costello syndrome. J Urol 2002;168:11331134. 54 Gripp KW, Lin AE, Nicholson L, Allen W, Cramer A, et al: Further delineation of the phenotype resulting from BRAF or MEK1 germline mutations helps differentiate cardio-facio-cutaneous syndrome from Costello syndrome. Am J Med Genet A 2007;143:14721480. 55 Al-Rahawan MM, Chute DJ, SolChurch K, Gripp KW, Stabley DL, et al: Hepatoblastoma and heart transplantation in a patient with cardio-faciocutaneous syndrome. Am J Med Genet A 2007;143:14811488. 56 La Starza R, Rosati R, Roti G, Gorello P, Bardi A, et al: A new NDE1/PDGFRB fusion transcript underlying chronic myelomonocytic leukaemia in Noonan Syndrome. Leukemia 2007;21:830833. 57 Aggarwal A, Krishnan J, Kwart A, Perry D: Noonans syndrome and seminoma of undescended testicle. South Med J 2001;94:432434. 58 Sriram K, Thomas K, Barnes R: Noonans syndrome. With carcinoma of undescended testis. IMJ Ill Med J 1987;171:294296. 59 Riederer J: [Benign monoclonal gammopathy and chronic lymphatic leukemia in a patient with Noonan syndrome]. Med Klin (Munich) 1998;93:433437.

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60 Wilcox WD: Occurrence of myeloproliferative disorder in patients with Noonan syndrome. J Pediatr 1998;132:189190. 61 Niemeyer CM, Kratz C: Juvenile myelomonocytic leukemia. Curr Oncol Rep 2003;5:510515. 62 Locatelli F, Nollke P, Zecca M, Korthof E, Lanino E, et al: Hematopoietic stem cell transplantation (HSCT) in children with juvenile myelomonocytic leukemia (JMML): results of the EWOG-MDS/EBMT trial. Blood 2005;105:410419. 63 Kratz CP, Niemeyer CM, Zenker M: An unexpected new role of mutant Ras: perturbation of human embryonic development. J Mol Med 2007;85: 223231. 64 Flotho C, Steinemann D, Mullighan CG, Neale G, Mayer K, et al: Genomewide single-nucleotide polymorphism analysis in juvenile myelomonocytic leukemia identifies uniparental disomy surrounding the NF1 locus in cases associated with neurofibromatosis but not in cases with mutant RAS or PTPN11. Oncogene 2007;26: 58165821.

65 Stephens K, Weaver M, Leppig KA, Maruyama K, Emanuel PD, Le Beau MM, Shannon KM: Interstitial uniparental isodisomy at clustered breakpoint intervals is a frequent mechanism of NF1 inactivation in myeloid malignancies. Blood 2006;108: 16841689. 66 Braun BS, Tuveson DA, Kong N, Le DT, Kogan SC, et al: Somatic activation of oncogenic Kras in hematopoietic cells initiates a rapidly fatal myeloproliferative disorder. Proc Natl Acad Sci USA 2004;101:597602. 67 Le DT, Kong N, Zhu Y, Lauchle JO, Aiyigari A, et al: Somatic inactivation of Nf1 in hematopoietic cells results in a progressive myeloproliferative disorder. Blood 2004;103:42434250. 68 Kratz CP, Niemeyer CM, Thomas C, Bauhuber S, Matejas V, et al: Mutation analysis of Son of Sevenless in juvenile myelomonocytic leukemia. Leukemia 2007;21:11081109. 69 Wexler LH, Helman LJ: Pediatric soft tissue sarcomas. CA Cancer J Clin 1994;44:211247.

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Christian Kratz Division of Pediatric Hematology/Oncology, Department of Pediatrics and Adolescent Medicine, University of Freiburg Mathildenstrasse 1 DE79104 Freiburg (Germany) Tel. +49 761 270 4514, Fax +49 761 270 4518, E-Mail christian.kratz@uniklinik-freiburg.de

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Neurofibromatosis Type 1-Noonan Syndrome: Whats the Link?


E. Denayer E. Legius
Department of Human Genetics, Catholic University of Leuven, Leuven, Belgium

Abstract
Neurofibromatosis type 1 is an autosomal dominant condition characterized by caf-au-lait spots, axillary and inguinal freckling, Lisch nodules of the iris, learning difficulties and benign and malign tumours of the peripheral nerves. Noonan syndrome is characterized by short stature, specific heart defects, normal intelligence to mild mental retardation and facial dysmorphism. Watson syndrome and Neurofibromatosis-Noonan syndrome have been described as separate entities combining features of both conditions. During recent years Watson and Neurofibromatosis-Noonan syndrome have been shown to result from mutations in the NF1 gene. Neurofibromatosis type 1 and Noonan syndrome also share some phenotypical features with other syndromes like Costello, cardio-facio-cutaneous, LEOPARD, and the SPRED1 syndrome. These neuro-cardio-facial-cutaneous syndromes are all caused by heterozygous germline mutations in genes of the RAS-MAPK pathway, resulting in deregulation of this important signal transduction cascade. This can explain the partially overlapping phenotypical features of these syndromes.
Copyright 2009 S. Karger AG, Basel

Neurofibromatosis Type 1 (NF1) (OMIM #162200)

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition with a prevalence of 1/3,000 [1]. It is a highly variable disorder with symptoms

and signs evolving during lifetime. Pigmentation abnormalities of the skin occur in almost all patients. Some caf-au-lait spots might be present at birth, but increase in size, number and pigmentation during the first years of life. Axillary or inguinal freckling is commonly present by the age of seven. Lisch nodules are hamartomas of the iris and can best be identified with split-lamp examination. They are present in more than 90% of adults with NF1 and the prevalence increases with age. Typical osseous findings are sphenoid wing dysplasia and thinning of the cortex of long bones (usually tibia and/or fibula) with or without pseudarthrosis. Scoliosis is a more frequent, but less specific skeletal symptom. Although most individuals with NF1 have normal intelligence, learning difficulties [2, 3] and/or attention-deficit disorder [4, 5] occur in about 5075%. The NF1 neuropsychological profile is characterized by deficits in visuo-spatial performance, executive functioning (planning and abstract concept formation), and attention [6]. Cardiovascular complications of NF1 are congenital heart defects (2.1%) [7], most commonly pulmonary valve stenosis [8], and hypertension. The latter can either be essential or secondary to NF1 vasculopathy which can for example produce renal

artery stenosis. Neurofibromas are a key hallmark of NF1. Numerous benign neurofibromas are usually present in adults, but they rarely develop before late childhood. Plexiform neurofibromas are less common. They can cause serious disfigurement and compromise the function of certain organs. Diffuse plexiform neurofibromas are usually obvious in young children, but deep, nodular plexiform neurofibromas can occur later on and may remain asymptomatic until deep in adulthood. Optic pathway glioma usually develops during the first 23 years of life. Other brain tumours, rhabdomyosarcoma and leukaemia, especially juvenile myelomonocytic leukaemia, are also more prevalent in children with NF1 than in the general population. Malignant peripheral nerve sheath tumours (MPNST) are the most frequent malignant neoplasm associated with NF1 with a life time risk of 813% [9]. They usually occur in adolescents and young adults. A variety of other tumours can be seen in adults with NF1 such as gastro-intestinal stromal tumours (GIST) [10] and glomus tumours of the finger tips [11]. A reliable diagnosis of NF1 can be made on a clinical base using the NIH diagnostic criteria [12].

show marked differences in frequency in the two conditions and this appears to provide the major factor differentiating the two conditions. For example, pulmonary valve stenosis, low intelligence and short stature are relatively common in WS, while neurofibromata are relatively rare in WS. Supporting the conclusion that Watson syndrome is allelic to NF1 was the finding by Upadhyaya et al. of an 80-kb deletion in the NF1 gene in a patient with Watson syndrome [15]. Similarly, Tassabehji et al. demonstrated an inframe tandem duplication of 42 bases in exon 28 of the NF1 gene in 3 members of a family with Watson syndrome [16] (table 1).

Neurofibromatosis-Noonan Syndrome (NFNS) (OMIM #601321)

Watson Syndrome (WS) (OMIM #193520)

In the past several reports have pointed to the co-occurrence of features of NF1 and Noonan syndrome. In 1967 Watson described three unrelated families with pulmonary valve stenosis, borderline intelligence, multiple caf-au-lait spots and freckling [13]. Allanson et al. expanded the clinical phenotype of Watson syndrome (WS) to include relative macrocephaly and Lisch nodules in the majority of affected subjects, and neurofibromas in one-third of family members. Molecular linkage studies supported linkage to the NF1-locus [14]. Of the clinical features common to WS and NF1, only axillary freckling and caf-au-lait spots show equal incidence in the two conditions. Other shared clinical features

Concurrently with the reports on WS Allanson et al. [17] reported on yet another distinct entity: Neurofibromatosis-Noonan syndrome (NFNS). They described four patients with NF1 and manifestations of Noonan syndrome including short stature, ptosis, midface hypoplasia, apparently short webbed neck, learning disabilities, and hypotonia. Further reports on the association of NF1 and Noonan-like features followed. Kaplan and Rosenblatt described a distinctive facial appearance resembling Noonan syndrome in children with neurofibromatosis [18]. In 1996 Colley et al. reviewed clinical features of 94 sequentially identified patients with NF1 and found Noonan features in 12, suggesting these features to be more common than previously appreciated [19]. In 1997 Friedman and Birch reported that 3.7% of the individuals in the NF International Database had Noonan syndrome features [7]. At that time it remained unclear whether the co-occurrence of features of NF1 and Noonan syndrome in some patients represented a chance co-incidence, a separate syndrome or a variant of either NF1 or Noonan syndrome. Since both NF1 with a prevalence of about 1/3,000

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Table 1. Overview of clinical and molecular findings in patients with WS or NFNS and a mutation in NF1 Reference Upadhyaya et al. [15] Tassabehji et al. [16] Clinical findings One individual with Watson syndrome Molecular findings 80-kb deletion

3 members of a family with Watson in-frame tandem duplication of 42 bases in syndrome: 26-year-old mother has multiple exon 28 CALM, axillary freckling, learning difficulties (IQ:56), low-set ears, squint; 3.5-year-old dizygous twins have multiple CALM, moderate developmental delay, mild pectus carinatum, hypertelorism, epicanthic folds, low-set ears, squint, one has bilateral cryptorchidy and mild pulmonary valve stenosis 8 family members with NFNS, 2 with NF1 only Family with NFNS Nonsense mutation exon 16 (c.2446C>T; p.R816X) 3-bp deletion in exon 17

Bahuau et al. [22] Carey et al. [23] Baralle et al. [24]

6-year-old boy, more than six CALM, ptosis, 3-bp deletion in exon 25 (c.4312 del GAA) epicanthic folds, low posterior hairline, lowset ears, pulmonic stenosis, severe feeding problems in infancy 20-year-old man, 7 CALM, axillary freckling, 10 neurofibromas, Lisch nodules, vertebral abnormality, downslanting palpebral fissures, ptosis, short/broad neck, widely spaced nipples, ASD, short stature, learning difficulties 2-bp insertion in exon 23-2 (c.4095 ins TG)

De Luca et al. [25]

9-year-old boy, CALM, neurofibromas, axillary freckling, bilateral optic glioma, macrocephaly, hypertelorism, ptosis, low posterior hairline, low-set ears

Missense mutation exon 4b (c.581T>G; p. L194R)

6.5-year-old girl, CALM, 1 neurofibroma, Splice-site mutation exon 11 (c.1721+3A>G) optic glioma, learning difficulties, macrocephaly, hypertelorism, downslanting palpebral fissures, ptosis, epicanthic folds, low-set ears, thoracic anormality 6-year-old boy, CALM, learning difficulties, short stature, hypertelorism, downslanting palpebral fissures, malar hypoplasia, low posterior hairline, webbed neck, low-set ears, hypotonia 2.2-year-old girl, CALM, macrocephaly, PS, hypertelorism, downslanting palpebral fissures, ptosis, epicanthic folds, low posterior hairline, webbed neck, low-set ears 4-bp deletion exon 12a (c.1756 del ACTA)

1-bp deletion exon 12b (c.1862 del C)

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Denayer Legius

Table 1. (continued) Reference De Luca et al. [25] Clinical findings 8-year-old boy, CALM, axillary freckling, mild MR, short stature, macrocephaly, hypertelorism, downslanting palpebral fissures, malar hypoplasia, low posterior hairline, webbed neck, low-set ears, thoracic abnormality, cubitus valgus 3 family members, CALM, axillary freckling, Lisch nodules, scoliosis, learning difficulties in 2, short stature, PS in one, macrocephaly in one, hypertelorism, downslanting palpebral fissures, low posterior hairline, webbed neck, low-set ears, cubitus valgus 3 family members, CALM, neurofibromas in 2, axillary freckling in 2, Lisch nodules, learning difficulties in 2, macrocephaly, mitral prolapse in 1, hypertelorism, low posterior hairline, low-set ears Molecular findings 1-bp deletion exon 13 (c.2153 del A)

3-bp inframe deletion (c.2970 del AAT; p. 991del M)

Nonsense mutation exon 24 (c.4243G>T; p. E1415X)

2.2-year-old boy, CALM, macrocephaly, PS, Missense mutation exon 24 (c. 4267A>G; p. hypertelorism, downslanting palpebral K1423E) fissures, ptosis, epicanthic folds, low posterior hairline, low-set ears, webbed neck 6-year-old girl, CALM, Lisch nodules, short Missense mutation exon 24 (c. 4267A>G; p. stature, hypertelorism, downslanting K1423E) palpebral fissures, ptosis, malar hypoplasia, epicanthic folds, low posterior hairline, webbed neck, low-set ears, thoracic abnormality 4-year-old girl, CALM, macrocephaly, ASD, Missense mutation exon 25 (c.4289A>C; p. hypertelorism, ptosis, epicanthic folds, low- N1430T) set ears 40-year-old female, CALM, neurofibromas, axillary freckling, Lisch nodules, optic glioma, learning difficulties, scoliosis, short stature, hypertelorism, downslanting palpebral fissures, ptosis, malar hypoplasia, epicanthic folds, low posterior hairline, webbed neck, low-set ears, thoracic abnormality, cubitus valgus 9.5-year-old girl, CALM, axillary freckling, scoliosis, short stature, macrocephaly, PS, hypertelorism, downslanting palpebral fissures, ptosis, low posterior hairline, webbed neck, low-set ears, thoracic abnormality, keratosis pilaris, sensorineural deafness Missense mutation exon 25 (c.4294G>C; p. V1432L)

3-bp inframe deletion exon 25 (c.4312 del GAA; p.1438 del E)

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Table 1. (continued) Reference De Luca et al. [25] Clinical findings Molecular findings

38-year-old female, CALM, Nonsense mutation exon 29 (c.5339T>G; p. neurofibromas, 1 plexiform, axillary L1780X) freckling, short stature, mitral valve thickening, downslanting palpebral fissures, epicanthic folds, low posterior hairline, webbed neck, low-set ears, thoracic abnormality 10-year-old boy, CALM, axillary freckling, Lisch nodules, macrocephaly, arrhythmia (right-branch block), ptosis, low-set ears, thoracic abnormality, renal cyst 2 family members, CALM, neurofibromas, axillary freckling, Lisch nodules in 1, learning difficulties, scoliosis, macrocephaly, hypertelorism, downslanting palpebral fissures, ptosis, low-set ears 7-year-old boy, CALM, axillary freckling, Lisch nodules, optic glioma, MR, hypertelorism, downslanting palpebral fissures, ptosis, low-set ears, thoracic abnormality, cubitus valgus Splice-site mutation exon 35 (c.6641+1G>A)

1-bp deletion exon 45 (c.7877del G)

Partial NF1 gene deletion

Hffmeier et al. [26]

4-year-old, 5 CALM, delayed psychomotor development, PS, relative macrocephaly, hypertelorism, downslanting palpebral fissures, ptosis, low posterior hairline, webbed neck, thoracic abnormality 2 family members, CALM, neurofibromas in 1, aortic insufficiency in 1, hypertelorism, downslanting palpebral fissures, low posterior hairline, webbed neck, low-set ears, thoracic abnormality 2 family members, CALM, neurofibromas, axillary freckling, hypertelorism, downslanting palpebral fissures, low posterior hairline, webbed neck, thoracic abnormality 42-year-old, CALM, neurofibromas, axillary freckling, Lisch nodules, short stature, macrocephaly, hypertelorism, downslanting palpebral fissures, ptosis, low posterior hairline, webbed neck, lowset ears, thoracic abnormality, scoliosis

Missense mutation exon 21 (c.3587T>G; p.L1196R)

4-bp deletion exon 21 (c.1756_1759 del 4)

1-bp deletion exon 18 (c.3060 del A)

8-bp deletion exon 6 (c.796 del GTTTGGCC)

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Table 1. (continued) Reference Hffmeier et al. [26] Clinical findings Molecular findings

20-year-old, CALM, neurofibromas, axillary Whole gene deletion freckling, Lisch nodules, learning difficulties, short stature, macrocephaly, hypertelorism, downslanting palpebral fissures, low posterior hairline, webbed neck, low-set ears, thoracic abnormality, scoliosis

and Noonan syndrome with a prevalence of 1/1,000 to 1/2,500 are rather common disorders chance association is a possibility. There has been one report of a female patient with typical findings of NFNS and missense mutations in both the PTPN11 gene on chromosome 12q and the NF1 gene on chromosome 17q [20]. In the study by Colley et al. [19] one of the families showed independent segregation of NF1 and Noonan syndrome whereas in other families half of those affected with NF1 had manifestations of the Noonan syndrome. Bahuau et al. identified a heterozygous truncating mutation in exon 16 of the NF1 gene in a family in which 8 members had NF1/Noonan syndrome, 2 had NF1 only, and 2 had NS only. All 10 patients with features of NF1 carried the mutation, whereas the 2 patients with Noonan syndrome only did not [21, 22]. Carey et al. identified a 3-bp deletion in exon 17 of the NF1 gene in one family with NFNS [23]. More recently several reports confirmed NFNS to be a variant of NF1 caused by mutations in NF1 (table 1). Baralle et al. found 2 mutations in 2 individuals with NFNS, a 3-bp deletion in exon 25 and a 2-bp insertion in exon 232. In four other individuals no mutations were found in NF1 nor in PTPN11 [24]. De Luca et al. identified heterozygous NF1 mutations in 16 of 17 unrelated subjects with NFNS, including nonsense mutations, out-of-frame deletions, missense changes, small in-frame deletions and one large multi-exon deletion. They noted a high prevalence of in-

frame defects affecting exons 24 and 25, which encode a portion of the GAP-related domain of the protein. No defect in PTPN11 was observed. They provided further evidence that NFNS and Noonan syndrome are genetically distinct disorders by excluding mutations in exons 1127 of NF1 in 100 PTPN11-negative Noonan syndrome patients [25]. Hffmeier et al. found heterozygous mutations or deletions of NF1 in seven patients from 5 unrelated families who presented with a variable combination of features of Noonan syndrome and neurofibromatosis type 1 [26].

Some Tumour Types and Leukaemias Occur in Both NF1 and Noonan Syndrome

Haematological malignancies occur with increased frequency in both NF1 and Noonan syndrome. Children with NF1 are predisposed to juvenile myelomonocytic leukaemia (JMML) and other haematological malignancies (ALL, non-Hodgkin lymphoma) [27, 28]. In Noonan syndrome a spectrum of haematological abnormalities has been described including isolated monocytosis, a CMML-like condition that remits spontaneously [29] and JMML [30]. JMML is a rare (annual incidence of 12 per million) myeloproliferative disorder characterized by leukocytosis with tissue infiltration and it has a severe and often lethal course. The incidence of JMML is increased 200- to 500-fold in children with NF1. Loss

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of the normal NF1 allele (LOH, loss of heterozygosity) is common in JMML cells from children with NF1 [31]. Sporadic cases of JMML can be caused by somatic mutations of NRAS or KRAS [32], or PTPN11 [33]. Somatic PTPN11 mutations found in JMML (most frequent mutation: E76K) differ from the PTPN11 mutations in Noonan syndrome. They exhibit stronger biochemical and biological effects than germline PTPN11 mutations. Moreover patients with Noonan syndrome who develop JMML have specific germline PTPN11 mutations, most frequently T73I. Apart from the leukaemias there is also overlap in the type of solid tumours observed in NF1 and Noonan syndrome. Rhabdomyosarcoma [34, 35] and neuroblastoma [3638] have been reported in association with both syndromes. Giant cell tumours of the bone are usually benign, but locally aggressive tumours characterized by the presence of multinucleated giant cells. They have been associated with NF1 [3941], Noonan syndrome [4244] and NFNS [45, 46]. Another example are granular cell tumours which are small, cutaneous or subcutaneous nodules. Immunohistochemistry studies have shown that they stain positive for S100, a protein that is a marker of Schwann cells and other cells of neuro-ectodermal origin. They are now generally accepted to have a Schwann cell origin, comparable to neurofibromas. They have been reported in individuals with NF1 [47] and Noonan syndrome [48].

generation of intracellular cyclic-AMP. However most important is its function as a RAS GTPase Activating Protein (GAP). By means of its GAPrelated domain (exons 2027a) neurofibromin functions as a negative regulator of the RASMAPKinase cascade. It stimulates the hydrolysis of the GTP bound to RAS and thus converts active GTP-bound RAS to inactive GDP-bound RAS. Inactivating mutations in the NF1 gene disturb the GAP activity of the protein resulting in more active RAS and increased signalling through the RAS-MAPK pathway. In 2001 Costa et al. linked the learning difficulties in NF1 individuals to the GAP-activity of neurofibromin by showing that Nf1 mice lacking the alternatively spliced exon 23a exhibited learning difficulties but no apparent developmental abnormalities or tumour predisposition [51]. They proposed that hyperactive RAS resulting from Nf1 haploinsufficiency leads to overactivity of inhibitory GABAergic neurons. The increased inhibition by GABAergic neurons would then lead to the learning disorder phenotype in these animals. To prove this hypothesis they showed that genetically (by crossing Nf1+/ mice with Kras or Nras+/ mice) as well as pharmacologically (by use of the farnesyltransferase inhibitor lovastatin) diminished RAS function rescued the learning difficulties observed in Nf1 heterozygous animals [52]. It can be hypothesized that the learning problems observed in the other NCFC syndromes are also the result of an increased RAS activation in certain brain cells.

The NF1 Gene

The NF1 gene located on chromosome 17q11.2 is a large gene (~350 kilobases) containing 60 exons. It acts as a tumour suppressor gene and NF1 related tumours originate as a result of a somatic inactivation of the normal NF1 copy in an NF1 patient (Knudsons second hit) [10, 49, 50]. The NF1 protein product, neurofibromin, is a large 327 kDa protein which has different functions such as regulation of adenylyl-cyclase activity and

The Neuro-Cardio-Facial-Cutaneous (NCFC) Syndromes: An Expanding Group of Phenotypically Overlapping Disorders

Apart from phenotypic overlap with NF1 Noonan syndrome also shares phenotypical features with Costello, LEOPARD and cardio-facio-cutaneous (CFC) syndrome. Recently the term neurocardio-facial-cutaneous (NCFC) syndromes has

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been coined to group these syndromes. These conditions all share a variable degree of learning disabilities or mental retardation, congenital heart defects, facial dysmorphy and skin abnormalities. In addition they all predispose in some way to malignancy (except for CFC syndrome). During recent years a common genetic and pathophysiologic basis has become obvious. In 2001 gain-of-function mutations in the PTPN11 gene, located on chromosome 12q24.1, have been found to cause about 50% of cases of Noonan syndrome. The PTPN11 gene encodes the non-receptor protein tyrosine phosphatase SHP-2 which relays signals from activated receptor complexes to downstream signalling molecules, like RAS. Noonan-associated PTPN11 mutations result in an enhanced phosphatase activity and activation of the RAS-MAPK pathway [53]. Shortly thereafter specific PTPN11 mutations have also been found in LEOPARD syndrome [54, 55]. Subsequently germline mutations in other components of the RAS-MAPK cascade have been identified in Costello syndrome (HRAS) [56], CFC syndrome (KRAS, BRAF, MEK1/2) [5759] and also in nonPTPN11 associated Noonan syndrome (KRAS mutations in less than 2% [59], SOS1 in 10% [60, 61] and RAF1 in 317% [62, 63]). Functional studies have revealed that most of these mutants result in hyperactivation of the RAS-MAPK cascade. This hyperactive MAPK-signalling is now held responsible as a mechanism for several of the overlapping symptoms in the different NCFC syndromes such as specific facial features, learning difficulties and heart defects. Therefore the presence in NF1 individuals of a pulmonary valve stenosis

and/or facial features typically seen in Noonan syndrome is not surprising. This also explains why differentiation between these syndromes on clinical grounds is not always simple. As an illustration in 1996 a young woman with a prior diagnosis of LEOPARD syndrome and hypertrophic cardiomyopathy who had a de novo missense mutation in exon 18 of the NF1 gene was described [64]. Later on PTPN11 analysis in this individual proved to be negative. Recently a new member of the group of NCFC syndromes has been identified, an autosomal dominant condition caused by germline mutations in the SPRED1 gene [65]. Affected individuals presented with caf-au-lait spots, skinfold freckling and macrocephaly. Most of the individuals fulfilled the NIH diagnostic criteria for NF1. Some typical features of NF1 were systematically absent in the reported patients such as Lisch nodules, neurofibromas and central nervous system tumours. In adults multiple lipomas were observed. A Noonan-like facial morphology has been observed in some patients. One individual had a pulmonary valve stenosis. The SPRED1 gene is, like NF1, a negative regulator of the RASMAPK pathway and acts between RAS and RAF to inhibit the activation of RAF by active RAS. A second hit was found in melanocytes from a caf-au-lait spot of an affected individual, supporting the hypothesis that biallelic inactivation of SPRED1 is responsible for some of the observed features. This new syndrome again confirms that dysregulated RAS-MAPK signalling can be responsible for symptoms seen in either NF1 or Noonan syndrome.

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39 Ardekian L, Manor R, Peled M, Laufer D: Bilateral central giant cell granulomas in a patient with neurofibromatosis: report of a case and review of the literature. J Oral Maxillofac Surg 1999;57:869872. 40 Krammer U, Wimmer K, Wiesbauer P, Rasse M, Lang S, Mullner-Eidenbock A, Frisch H: Neurofibromatosis 1: a novel NF1 mutation in an 11-year-old girl with a giant cell granuloma. J Child Neurol 2003;18:371373. 41 Ruggieri M, Pavone V, Polizzi A, Albanese S, Magro G, Merino M, Duray PH: Unusual form of recurrent giant cell granuloma of the mandible and lower extremities in a patient with neurofibromatosis type 1. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:6772. 42 Bertola DR, Kim CA, Pereira AC, Mota GF, Krieger JE, et al: Are Noonan syndrome and Noonan-like/multiple giant cell lesion syndrome distinct entities? Am J Med Genet 2001;98:230234. 43 Cohen MM Jr, Gorlin RJ: Noonan-like/ multiple giant cell lesion syndrome. Am J Med Genet 1991;40:159166. 44 Ucar B, Okten A, Mocan H, Ercin C: Noonan syndrome associated with central giant cell granuloma. Clin Genet 1998;53:411414. 45 Posligua L, McDonald DJ, Dehner LP: Diffuse-type tenosynovial giant cell tumor in association with neurofibromatosis type 1-Noonan syndrome: possibly more than a chance relationship. Am J Surg Pathol 2006;30: 734738. 46 Yazdizadeh M, Tapia JL, Baharvand M, Radfar L: A case of neurofibromatosisNoonan syndrome with a central giant cell granuloma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:316320. 47 Sahn EE, Dunlavey ES, Parsons JL: Multiple cutaneous granular cell tumors in a child with possible neurofibromatosis. J Am Acad Dermatol 1997;36:327330.

48 Lohmann DR, Gillessen-Kaesbach G: Multiple subcutaneous granular-cell tumours in a patient with Noonan syndrome. Clin Dysmorphol 2000;9:301302. 49 Legius E, Marchuk DA, Collins FS, Glover TW: Somatic deletion of the neurofibromatosis type 1 gene in a neurofibrosarcoma supports a tumour suppressor gene hypothesis. Nat Genet 1993;3:122126. 50 Maertens O, Brems H, Vandesompele J, De Raedt T, Heyns I, et al: Comprehensive NF1 screening on cultured Schwann cells from neurofibromas. Hum Mutat 2006;27:10301040. 51 Costa RM, Yang T, Huynh DP, Pulst SM, Viskochil DH, Silva AJ, Brannan CI: Learning deficits, but normal development and tumor predisposition, in mice lacking exon 23a of Nf1. Nat Genet 2001;27:399405. 52 Costa RM, Federov NB, Kogan JH, Murphy GG, Stern J, et al: Mechanism for the learning deficits in a mouse model of neurofibromatosis type 1. Nature 2002;415:526530. 53 Tartaglia M, Mehler EL, Goldberg R, Zampino G, Brunner HG, et al: Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 2001;29:465468. 54 Digilio MC, Conti E, Sarkozy A, Mingarelli R, Dottorini T, et al: Grouping of multiple-lentigines/LEOPARD and Noonan syndromes on the PTPN11 gene. Am J Hum Genet 2002;71: 389394. 55 Legius E, Schrander-Stumpel C, Schollen E, Pulles-Heintzberger C, Gewillig M, Fryns JP: PTPN11 mutations in LEOPARD syndrome. J Med Genet 2002;39:571574. 56 Aoki Y, Niihori T, Kawame H, Kurosawa K, Ohashi H, et al: Germline mutations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 2005;37:10381040.

57 Niihori T, Aoki Y, Narumi Y, Neri G, Cave H, et al: Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome. Nat Genet 2006;38:294296. 58 Rodriguez-Viciana P, Tetsu O, Tidyman WE, Estep AL, Conger BA, et al: Germline mutations in genes within the MAPK pathway cause cardio-faciocutaneous syndrome. Science 2006;311:12871290. 59 Schubbert S, Zenker M, Rowe SL, Boll S, Klein C, et al: Germline KRAS mutations cause Noonan syndrome. Nat Genet 2006;38:331336. 60 Roberts AE, Araki T, Swanson KD, Montgomery KT, Schiripo TA, et al: Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet 2007;39:7074. 61 Tartaglia M, Pennacchio LA, Zhao C, Yadav KK, Fodale V, et al: Gain-offunction SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet 2007;39:7579. 62 Pandit B, Sarkozy A, Pennacchio LA, Carta C, Oishi K, et al: Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nat Genet 2007;39:10071012. 63 Razzaque MA, Nishizawa T, Komoike Y, Yagi H, Furutani M, et al: Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet 2007;39:10131017. 64 Wu R, Legius E, Robberecht W, Dumoulin M, Cassiman JJ, Fryns JP: Neurofibromatosis type I gene mutation in a patient with features of LEOPARD syndrome. Hum Mutat 1996;8:5156. 65 Brems H, Chmara M, Sahbatou M, Denayer E, Taniguchi K, et al: Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1-like phenotype. Nat Genet 2007;39:11201126.

Eric Legius Department of Human Genetics, Catholic University of Leuven Herestraat 49 BE3000 Leuven (Belgium) Tel. +32 16 345903, Fax +32 16 346051, E-Mail Eric.Legius@uz.kuleuven.ac.be

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Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 138150

Animal Models for Noonan Syndrome and Related Disorders


T. Araki B.G. Neel
Ontario Cancer Institute, University Health Network, TMDT8355, Toronto, Ont., Canada

Abstract
Noonan syndrome (NS) and related disorders, including LEOPARD syndrome (LS), cardio-facial-cutaneous (CFC) syndrome, Costello syndrome (CS) and neurofibromatosis Type-I (NF1) can be grouped together as the neuro-cardio-facio-cutaneous (NCFC) syndromes by virtue of their shared clinical features and molecular pathogenesis. Recent studies have shown that these diseases are caused by germline mutations in key components of the RAS-RAF-MEK-ERK kinase (hereafter, RAS/ERK) cascade. Presumably, the common features of these syndromes reflect abnormal function of this pathway during development and postnatally. Nevertheless, the detailed mechanism by which abnormal ERK activation results in shared syndromic phenotypes remains unclear. Moreover, although clearly related, specific NCFC syndromes are clinically distinguishable. How mutations within the same signaling pathway have such distinct phenotypic consequences is unknown. NCFC syndromes predominantly affect events that occur during embryogenesis and affect complex developmental/morphogenetic pathways. Consequently, it is difficult, if not impossible, to delineate their molecular pathogenesis using cell culture systems or human samples. Also, because the NCFC syndromes are fairly rare, multiple different alleles exist for each disorder, and the human population is extensively outbred, it is difficult to determine whether allele-specific phenotypic differences exist and/or whether there are key genetic modifiers. Animal models provide tools to address these issues, as well as to devise and evaluate potential therapeutic approaches. This review focuses on models of NS and related disorders, with a particular focus on mouse models.
Copyright 2009 S. Karger AG, Basel

It is now clear that germline mutations in members of the RAS/ERK cascade cause NCFC syndromes [1]. Individuals with these syndromes typically display some combination of facial abnormalities, cardiac defects and proportional short stature, although skin, lymphatic and genital abnormalities, as well as cognitive difficulties, ranging from mild to severe mental retardation, also are common. The phenotypic similarities between NCFC syndromes can be explained by effects of this common signaling pathway. Nevertheless, these syndromes are clinically distinguishable. For example, multiple neurofibromas and caf au lait spots are observed in NF1 patients, and severe mental retardation is most often found in patients with CFC syndrome. The NCFC syndromes also differ markedly in their relative predisposition to malignancy. Predisposition to cancer is not a known feature of CFC syndrome, even though somatic mutations in BRAF, the gene mutated in most cases of CFC syndrome [2, 3], occur in ~70% of melanomas [4]. Other NCFC syndromes carry a high risk of cancer development. Brain tumors and hematological malignancies, particularly the rare disorder juvenile myelomonocytic leukemia (JMML), are associated with NF1. JMML, possibly other

hematological (e.g., acute lymphoblastic leukemia (ALL)) malignancies, and potentially neuroblastoma, are associated with NS (caused by PTPN11, KRAS, SOS1 or RAF1 mutations) [510]. Somatic PTPN11 mutations are also common in sporadic JMML [11, 12], and occur less frequently in a variety of other leukemias and myeloproliferative disorders (MPD). CS, caused by HRAS mutations [13], is associated with a high risk of rhabdomyosarcoma and bladder cancer; notably somatic HRAS mutations are also found in sporadic versions of these malignancies [14]. Although much progress has been made in defining the genetic basis for the NCFC syndromes, several fundamental issues remain to be addressed. First, the molecular and cellular mechanisms responsible for the defects seen in these syndromes remain poorly understood. Also unclear is how mutations in components of the same signaling pathway can cause the different features that allow individual NCFC syndromes to be distinguished clinically. Even within the same syndrome, genotype/phenotype relationships (e.g., whether individual mutant alleles contribute differentially to phenotype and if so, how) are poorly defined. For example, biochemical data suggest that different NS mutations in PTPN11 can have substantially different effects on the catalytic activity of its gene product, SHP2 [15], making it possible, if not likely, that such alleles could have distinct phenotypic consequences. But studies of patients with PTPN11 mutations have failed to reveal clear differences, potentially because of the large number of alleles that exist and the relatively small numbers of patients surveyed. Alternatively (or in addition), unknown modifier loci might play critical roles in phenotype determination. Furthermore, some NCFC syndromes can result from mutations in different genes (e.g., mutations in KRAS, SOS1 or RAF1 can also cause NS, whereas mutations in MEK1 or MEK2, instead of BRAF, can cause CFC) [2, 3]. Certain RAF1 mutations may predispose to hypertrophic cardiomyopathy in NS [9, 10]. NS caused by

PTPN11 and SOS1 mutations may also differ in some phenotypes [7, 8], including relative risk of malignancy [16, 17]. In general, though, whether the pathogenic impact of different disease-associated mutations is similar remains largely unknown. Even more confusing, although most mutant alleles associated with NCFC syndromes act as hypermorphic (gain-of-function) mutants in biochemical and/or transfection assays, others either have no effect compared to wild type (WT) or even act as hypomorphic (loss-of-function) or dominant negative mutants. Conceivably, gainand loss-of-function might have similar effects in some developmental pathways. Alternatively, the cellular pathogenesis of these disorders may be quite distinct. Ultimately, of course, one would like to reverse or remediate the defects in the NCFC syndromes (at least the postnatal defects, which are most likely to be treatable). However, the relative rarity of these syndromes limits the patient population likely to be available for any future therapeutic trials. These and other issues can be effectively, if not best, addressed by animal models. Here, we review the studies of the physiological functions of mutants associated with NCFC syndrome, with a particular focus on mouse models of NS.

Properties of Mutant Alleles Associated with NCFC Syndromes

Approximately 50% of NS cases are caused by mutations in PTPN11, which encodes the SH2 domain-containing protein tyrosine phosphatase SHP2. A key component of the RAS/ERK pathway, SHP2 and, in particular, SHP2 catalytic activity, is required for normal RAS activation by most, if not all, growth factors and cytokines [reviewed in 18; 19]. Multiple studies, culminating in the SHP2 crystal structure [20], established that SHP2 is regulated by an elegant molecular switch mechanism that ensures that its catalytic activity is suppressed until it is needed at the

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right time and place. In the basal (closed) state, the amino terminal SH2 (N-SH2) domain interacts with the PTP domain to suppress catalytic activity [21]. Binding of a phosphotyrosyl peptide (e.g., from an appropriate tyrosyl phosphorylated receptor or adapter protein) to the N-SH2 domain disrupts this inhibitory interaction, resulting in opening of the enzyme and potent activation. The physiological relevance of this regulatory mechanism was first demonstrated by the generation of activated mutants, which show enhanced enzymatic and biological activity ([22]; and see below). The discovery of NS- and leukemia-associated PTPN11 mutants provided even more dramatic validation. Almost all such mutants map to the N-SH2 or PTP domain and affect residues that participate in basal inhibition. Not surprisingly, these mutants almost invariably show enhanced catalytic (PTP) activity in vitro [15], indicating a shift towards the open state of the enzyme. Consistent with this interpretation, NS-associated SHP2 mutants show increased binding to the adapter Gab1 and, when co-expressed with Gab1, enhance Erk activation in transfection assays [23]. SOS1, which encodes a major guanine-nucleotide exchange factor (GEF) for RAS proteins, is the second common gene mutated in NS. SOS1, like SHP2, is regulated by an auto-inhibitory module [24, 25]. Remarkably, similar to the effects of NS-associated PTPN11 mutants, SOS1 mutants affect key auto-inhibitory residues and RAS/ERK activation in transfection assays [7, 8]. SOS1 also has RAC-GEF activity [26], but whether NS-associated mutants affect RAC activation remains unclear. Most NS-associated RAF1 mutants also evoke enhanced MEK and ERK activity in 293T and COS7 cells [9, 10]. However, other RAF1 alleles have either the same or even lower activity than WT RAF1 in such assays. Some cancer-associated, somatic BRAF mutants also exhibit decreased activity under similar assay conditions, but when co-expressed with RAF1, show enhanced MEK/

ERK activation. Such findings led to the conclusion that RAF1/BRAF heterodimers comprise the bona fide kinase that activates MEK in vivo [27, 28]. Conceivably, NS RAF1 mutants with decreased activity might show increased activity upon co-expression of BRAF. Alternatively, NS RAF1 mutants may have decreased susceptibility to negative regulators (e.g., SPRED, SPROUTY proteins), whose effects could be obscured by the high levels of expression in transient transfection studies. Intriguingly, in this regard, it was reported recently that loss-of-function SPRED1 mutants cause a variant NF1-like syndrome [29]; conceivably, some NS-associated RAF1 proteins could be resistant to the inhibitory effects of SPRED1. Such complexities and potential complications in interpretation of heterologous expression experiments emphasize the need to assess the effects (both biochemically and biologically) or diseaseassociated mutants expressed at more physiological levels. LS, which shares multiple phenotypic features with NS, also is caused by PTPN11 mutations [5]. Yet surprisingly, LS alleles exhibit markedly decreased catalytic activity and act as dominant negative mutants to inhibit growth factor-evoked ERK activation [30, 31]. Some mutations reportedly cause both NS and LS [5]. It is unclear whether this represents misdiagnosis, similar phenotypes caused by SHP2 gain-of-function and partial deficiency, artifacts of the in vitro and ex vivo SHP2 assays, or as yet unknown PTP activity-independent effects of SHP2. Most CFC syndrome-associated mutants of BRAF and all MEK1/2 alleles cause increased ERK activation of downstream pathway in transient transfection experiments. Again, however, there are some exceptions [2, 3]. These cell line studies have provided a relatively easy way in which to test the effects of human disease-associated mutations on selected signaling pathways, and to provide initial insights into potential biochemical mechanisms of pathogenesis. Yet, as detailed above, some of the

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results obtained have been confusing, as they do not quite fit with the simple model that NCFC syndromes are diseases of RAS/ERK pathway hypermorphism. Even more importantly, heterologous cell lines provide limited insight into the cell biological basis of the type of cell- and tissue-specific phenotypes seen in these disorders.

Primary Cells/ex vivo Models

Primary cells from relevant tissues are potential improvements over heterologous cell systems for evaluating the effects of NCFC mutants. Several groups have studied the effects of disease-associated PTPN11 mutants on primary hematopoietic cells to gain insights into their role in leukemogenesis. Retroviral transduction of somatic leukemia-associated mutants into primary murine bone marrow (BM) cells recapitulates key cellular features of JMML, including the production of monocytic colonies in the absence of exogenous cytokines (factor-independent colony formation) and hypersensitivity of myeloid progenitors to the cytokine granulocyte-macrophage colonystimulating factor (GM-CSF) [3234]. Unlike in human JMML, mouse myeloid progenitors expressing PTPN11 mutants also show increased sensitivity to IL3; such differences likely reflect intrinsic differences between murine and human hematopoiesis. Mutations associated solely with NS are less potent than those found in both NS and leukemia or in leukemia in this myeloid transformation assay [32]. Second site mutations (introduced into the most potent leukemia-associated mutant) indicate that PTP activity, one of the two C-terminal tyrosyl phosphorylation sites, and both SH2 domains are required for maximal transforming activity [32]. The latter finding suggests that even constitutively activated SHP2 must be targeted correctly via its SH2 domains to promote malignant transformation. Finally, transplantation of BM transduced with leukemia-associated mutants causes a fatal

myeloproliferative disorder (MPD) characterized by overproduction of tissue-invading myeloid lineage cells in ~60% of recipients [32]. The remaining mice succumb to T cell leukemia/lymphoma, a type of neoplasm not associated with PTPN11 mutations in humans. Hematological disease may be strain-dependent however, as MPD (or lymphoid malignancy) is not observed following transduction of C57BL6 BM with potent leukemogenic mutants [33]. Such experiments have also provided some insight into the biochemical effects of leukemogenic mutants. Macrophages derived from BM transduced with such mutants display enhanced GM-CSF-evoked ERK activation [33, 34], whereas bone marrow mast cells from mice with SHP2-evoked MPD show increased activation of ERK, AKT and STAT5 [32]. In contrast to earlier transient transfection studies [12, 23], there was no requirement for co-expression of Gab1 (or another Gab protein) in these primary cell systems. These results suggest that differential expression of important SHP2-binding proteins may be one reason for the tissue-specific effects of disease-associated PTPN11 mutants. Taken together, these retroviral gene transduction studies have provided firm evidence for the causal role of PTPN11 mutants in leukemogenesis, and identified key structure/function relationships (most notably, the PTP domain requirement) and abnormal biochemical consequences that might be pertinent for development of novel therapeutics. KRAS mutants, which are found in a small percentage of NS and NS/JMML patients [6], also cause cytokine hypersensitivity and increased activation of RAS, MEK and AKT. For further details about the hematological effects of PTPN11 and KRAS mutants, the reader should consult more comprehensive reviews [35, 36]. The effects of CS-associated HRAS mutations have been assessed using fibroblasts from affected individuals. Compared to normal fibroblasts, these have increased proliferation (as assayed by

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BrdU incorporation) in response to epidermal growth factor (EGF) or fetal calf serum [13]. Although the common features of the NCFC syndromes include short stature, facial abnormalities and cardiac defects, there have been few studies of the effects of NCFC mutations on the cell types relevant to these defects (in part, because in most cases, these have not been defined; see below). One exception has been an attempt to assess the effects of an NS mutant on valve development, using so-called AV cushion explant assays [37]. Valvulogenesis is a complex process [38], involving at least three cell types, which takes place in specialized structures termed cardiac (or endocardial) cushions. There, specialized endothelial cells (termed cushion endothelium or cushion endocardium), which rest upon a specialized extracellular matrix (the cardiac jelly), respond to signals from the subjacent myocardium and undergo an endothelial to mesenchymal transition (EMT). Once transformed, cushion mesenchymal cells invade the cardiac jelly and proliferate. A complex morphogenetic process ensues, which entails cessation of cushion mesenchymal cell proliferation, cell shape changes and substantial apoptosis. The third cell type, the cardiac neural crest (NC), migrates into the developing cushion and is important for proper valve and septum generation [39]. In the mouse, cardiac NC migration occurs at around E10.5 and only involves the outflow tract (OT) valves [40]. The cushion explant assay models the early events of EMT, mesenchymal cell invasion and, to some extent, mesenchymal proliferation. Initially developed for studies of chicken valve development [41], explant assays subsequently were optimized for murine AV and outflow (OT) cushions [42, 43] and can be used to test the effects of various agonists/antagonists on the above processes. Robbins and coworkers found that the PTPN11 mutant Q79R, introduced by adenoviral gene transduction, had no effect on EMT per se, but increased Erk activation and enhanced the

proliferation of transformed mesenchymal cells in chicken AV cushion explants [37]. These results are consistent with the increased cushion mesenchymal cell proliferation (as assayed by BrdU incorporation) and Erk activation (by anti-pErk immunohistochemistry) seen in a mouse model of NS (see below), and could help explain the hypertrophic valves seen in many NS patients. However, these results are somewhat inconsistent with previous studies of Nf1/ mice. Nf1 homozygosity is not compatible with life, and thus Nf1/ mice do not model a specific human syndrome. However NF1 patients have pulmonic stenosis more often than in the general population [44], suggesting that, contingent upon the genetic background, NF1 may be haploinsufficient for vavulogenesis. Consequently, the cardiac phenotype of Nf1/ embryos may represent a more severe version of the consequences of NF1 heterozygosity in humans. Such embryos exhibit pan-valvular stenosis, atrial and ventricular septal defects, and double outlet right ventricle. Studies using a conditional Nf1 allele indicate that these result selectively from the absence of Nf1 in endothelial cells [43, 45]. Similar to the phenotype of NS mice (see below), Nf1/ embryos show increased mesenchymal cell proliferation, but in contrast to the above chicken explant studies, Nf1/ cushion explants reportedly show increased EMT, as do explants from NS mice ([43]; also, see below). This could reflect an intrinsic difference in chicken and murine explant assays (and possibly, differences between the effects of NS mutants in avian and murine systems). Alternatively, and perhaps more likely, the Q79R allele is not expressed early enough to alter EMT in the chicken explant experiments.

Drosophila Models

The strong conservation of the Ras/Erk pathway across evolution allows the study of human disease-associated NCFC mutants in model genetic

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organisms. When such mutants evoke relevant phenotypes, such organisms as Drosophila melanogaster provide the potentially big advantage of rapidly deciphering key pathways using genetic analysis and the large number of hyper- or hypomorphic lines already established. Flies also have some clear limitations: obviously, key syndromic features such as cardiac, facial and stature abnormalities cannot be modeled accurately. Drosophila does, however, have a rudimentary hematopoietic system, and earlier work established that expression of activated RAS mutants causes excessive hemocyte production [46]. Similarly, expression of the leukemia-associated mutant PTPN11 E76K under the control of a hemocyte-selective promoter causes an ~6-fold increase in the number of plasmatocytes, which are myeloid-like cells that comprise the major circulating blood cells in flies [32]. The mutant PTPN11 allele also alters cellular morphology, suggesting an additional effect on myeloid differentiation. These effects are qualitatively similar, although considerably weaker, than those evoked by activated RAS. It is unclear if this reflects a lower leukemogenic potential of mutant SHP2 compared to RAS, or that human SHP2 is less leukemogenic than the cognate mutation in the fly SHP2 ortholog, corkscrew (csw) might be. Notably, though, mutant Ras also is more potently leukemogenic than mutant Ptpn11 in mice. Oishi et al. generated transgenic flies with GAL4-inducible expression of wild type csw or a series of csw mutants, corresponding to PTPN11 E76K, A72S and N308D, which show different degrees of catalytic activation (E76K>A72S>N308D) [47]. Interestingly, ubiquitous expression of the A72S or E76K mutants causes lethality, but the N308D mutant was compatible with viability. Doubling N308D gene dosage also resulted in lethality, suggesting that the extent of catalytic activation or at least the degree to which a mutation causes SHP2 to reside in the open state helps determine the disease phenotype. Similar observations have been made using mouse model

of NS ([48], and T. A., G. C., and B.G. N., manuscript in preparation; see below). Much like the selective effects of NS mutants on human (and mouse; see below) development, the phenotypes evoked by the cognate csw mutants do not reflect universal abnormality of Drosophila tyrosine kinase signaling. For example, the N308D mutant causes a wing vein phenotype similar to that evoked by gain-of-function mutants in the Drosophila EGFR, and this phenotype is rescued by loss-of-function mutants in the EGFR signaling pathway. Epistasis studies identify additional genetic interactions between N308D and the Notch, BMP and Jak/Stat pathways, respectively, which may have important implications for the pathogenesis of key NS phenotypes. For example, EGFR, Notch and BMP signaling are important for valvulogenesis [38], whereas the Jak/Stat pathway mediates the effects of cytokines such as GM-CSF and IL-3, and may therefore be relevant to the pathogenesis of MPD evoked by PTPN11 mutants.

Zebrafish Model

Very recently, Jopling et al. compared the effects of NS and LS mutants of PTPN11 in zebrafish [49]. Injection of either type of mutant results in significantly shorter embryos at 4 dpf without affecting cell specification. These results suggest impaired gastrulation, and indeed, cell tracing experiments indicate that both extension and convergence movements are reduced significantly upon injection of the NS mutant. NS and LS mutant embryos also showed wide-set eyes as well as edematous hearts, although these defects were not characterized further in this report. In general, the phenotypes caused by NS or LS mutants were indistinguishable, which is similar to the human situation. This is, of course, surprising, given that NS and LS alleles have opposite effects on SHP2 catalytic activity. However, the effects of the two mutants were neither additive

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nor synergistic, which the authors interpreted as indicating opposing actions on the same pathway. On the other hand, the two mutants did not rescue each other, so their precise mode of action remains unclear. Lowering endogenous Shp2 levels by means of morpholinos resulted in a gastrulation defect, similar to the effects of dominant negative Shp2 expression in Xenopus embryos [50] or homozygosity of a Ptpn11 allele that results in an N-terminal Shp2 truncation in the mouse [51]. The effects of zebrafish Shp2 deficiency were attributed to defective Src activity and Rho activation. The effects of the NS and LS mutants on these pathways were not reported, however. In unpublished studies, we (R. Stewart, M. Kontaridis, K. Swanson, B.G. N. and A. T. Look) have also characterized the effects of NS and LS mutants, compared to those of zebrafish Ptpn11 morphants. Similar to the above results, embryos injected with NS and LS mutants, as well as morphants, have defective gastrulation. However, we also find distinct effects of these three treatments on NC development, which suggest that SHP2 has PTP-dependent and independent effects on key NC developmental pathways. The differential ability of NS and LS mutants to mediate these pathways may help explain the phenotypic similarities and differences between LS and NS.

Mouse Models

Over the past decade, several groups have developed mouse models of NF1, by means of conventional and conditional Nf1 gene inactivation. As noted above, Nf1/ mice are not viable. In contrast, Nf1+/ mice, which are genetically similar to NF1 patients, are healthy at birth but succumb to leukemia or pheochromocytoma (both of which are NF1 characteristics) by 1518 months of age [52]. Nf1+/ mice also have learning defects, which can be rescued by genetic and pharmacological manipulations that decrease Ras function [53,

54]. Notably, however, these mice develop neither neurofibromas nor astrocytomas, the hallmark features of Type 1 NF. Homozygotic Nf1 inactivation in Schwann cells (peripheral nervous system) or astrocytes (central nervous system) also fails to cause tumors. By contrast, Nf1 deletion in hematopoietic cells results in a progressive myeloproliferative disorder that resembles JMML [55]. Parada and colleagues resolved this paradox by realizing that in NF1 patients, neurofibromas, which are of Schwann cell origin, are generated in the context of NF1 heterozygous tissues. Remarkably, they found that homozygous Nf1 deletion in Schwann cells, in the background of Nf1 heterozygosity results in fusiform paraspinal masses with histological features of plexiform neurofibroma [56]. Nf1+/ mice lacking neurofibromin in astroglial precursors developed fusiform masses of the optic nerve and chiasm, resembling optic nerve gliomas in children with NF1 [57]. These results indicate that NF1+/ cells in these environments cooperate with NF1/ Schwann or astroglia cells to cause neurofibromas or astrocytomas, respectively. Parada and colleagues subsequently found that Nf1/ Schwann cells secrete mast cell chemotactic factors, including Kit Ligand. In response to such factors, Nf1+/ mast cells are recruited to the vicinity of Nf1/ Schwann cells to facilitate neoplastic transformation [58]. Our group has generated and characterized knock-in mice expressing the NS mutant Ptpn11D61G (hereafter, DG) [48]. Homozygous DG mice die at mid-gestation, with a phenotype similar to global Nf1 deletion. At E13.5, DG/DG embryos exhibit severe cardiac defects, including atrial, ventricular, or atrio-ventricular septal defects, double-outlet right ventricle, enlargement of AV and OT endocardial cushions and markedly thinned myocardium. These abnormalities, with the possible exception of myocardial thinning (see below), represent severe versions of cardiac phenotypes seen in NS patients (and in DG/+ mice).

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Mid-gestation DG/DG embryos also are markedly edematous (probably due to their severe cardiac defects), and show evidence of hemorrhage and liver necrosis. Bleeding and clotting abnormalities are seen with variable penetrance in NS patients [59, 60]; hence, the hemorrhage seen in DG/ DG embryos might reflect NS-associated platelet or clotting factor defects. Liver abnormalities are not a known feature of NS, however, and hemorrhage could be an indirect consequence of liver and/or cardiac defects in these embryos. Although we have not yet pursued the molecular basis of the liver defects in DG/DG embryos (mainly because it is not a feature of the human syndrome), midgestation hepatic necrosis is a classical manifestation of defects in the NF-B pathway (which is required to prevent apoptosis in response to TNF produced at that time). There has been a report that SHP2 regulates NFB activation [61]. As TNF family receptors regulate a wide array of physiological functions that could contribute to bona fide NS phenotypes, further exploration of the molecular pathophysiology of hepatic necrosis in DG/DG mice might prove informative. The relevance of the myocardial thinning seen in DG/DG mice to NS pathogenesis also is unclear. Hypertrophic cardiomyopathy (HCM) is one of the features of human NS [62], but in one of the few clear genotype/phenotype correlations reported, HCM was found to be less common in NS caused by PTPN11 mutations [63]; indeed, recent studies show markedly increased incidence of HCM in NS caused by specific RAF1 alleles [9, 10]. In any event, it appears that high levels of SHP2 activation cause the opposite phenotype (myocardial thinning), at least in mice. Notably, however, ventricular noncompaction or hypoplasia has been reported in some NS patients [6466], although the genotypes of these individuals have not been reported. Transgenic expression of an NS mutant causes a similar phenotype [67], although as discussed below, the relevance of that system is unclear. In contrast to the uniform lethality of DG/ DG embryos, ~50% of D61G/+ mice (on 129Sv

C57BL6/J background) die in late gestation or perinatally with multiple cardiac defects. At E13.5, D61G/+ embryos are obtained at the expected Mendelian ratio, but fall into 2 groups: severely affected or mildly affected. Severely affected embryos have ventricular septal defects, double-outlet right ventricle and increased size of all valve primordia. These phenotypes are similar to, although less severe than, those found in DG/DG embryos; also, unlike the latter, DG/+ embryos have normal myocardial thickness (and no edema or hepatic necrosis). BrdU labeling experiments show increased mesenchymal cell proliferation and decreased apoptosis in DG/+, compared to WT embryos. All of these cardiac defects resemble those seen in Nf1/ mice, suggesting that they result from increased activity of the Ras/Erk pathway. Indeed, increased numbers of pErk-positive cells are found in DG/+ [48] and Nf1/ [45] endocardial cushions. Overall, DG/+ mice provide a reasonable model for the cardiac defects in NS. There are, however, some important caveats. First, the most common cardiac defect in human NS patients is pulmonic stenosis, whereas AV valve hyperplasia is more common in the murine model. This could reflect differences in NC function in human and mouse valvulogenesis, and raises the possibility that NS alleles may have effects in human cardiac NC that are not adequately modeled in the mouse (see below). In addition, human patients typically have some, but rarely all, of the cardiac defects seen in DG/+ mice. Furthermore, the cardiac defects in DG/+ mice (as well as more recent models that we have generated; see below), appear in an all-or-none fashion. This could indicate some fundamental property of murine valvuloseptal development that will limit the conclusions that can be drawn from this and other NCFC models. Nevertheless, it seems likely that the fundamental cellular and biochemical abnormalities revealed by these models will be relevant to pathogenesis of the human syndromes. Although enhanced Erk activation (as assessed by whole mount immunohistochemistry) is seen in

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a few other sites in DG/+ embryos most notably those, such as the developing face and limb bud, in which other NS-like defects are observed subsequently (see below) Erk hyper-activation is not uniform. Furthermore, primary mouse embryo fibroblasts fail to show increased Erk activation in response to low or high doses of EGF, FGF, IGF or PDGF. Thus, as in the Drosophila model, NS mutants have a cell/tissue-selective ability to increase Erk activation. The reason for this selectivity remains unknown, although possible explanations include differential levels of SHP2 binding proteins (discussed above) or substrates or differential activity of homeostatic feedback pathways able to diminish the effects of increased SHP2 activity. Severely affected DG/+ embryos survive to at least E18.5 (the last developmental time point analyzed), and probably die perinatally. In contrast, mildly affected DG/+ mice survive to adulthood and manifest other NS features, including facial abnormalities and proportionate short stature. These mice also exhibit an initially mild MPD, characterized by increased splenic size, mild myeloid hyperplasia, and factor-independent colony production by BM and spleen cells. Although this MPD is well-tolerated at first, DG/+ mice eventually die much earlier (at 1215 months) than their WT counterparts (T.A. and B.G.N., unpublished). Notably in this regard, the D61G allele, which initially was reported only in NS patients, was subsequently observed in JMML as well [68]. The effects of D61G on neurogenesis have been analyzed by means of combined ex vivo and in vivo approaches [69]. Over-expression of D61G in cortical precursors promotes neurogenesis and inhibits astrogenesis. There also is a small, but statistically significant, increase in neurogenesis and decrease in astrogenesis in the dorsal cortex and hippocampus of DG/+ mice. These alterations may perturb neural circuit formation to cause the cognitive deficits seen in NS patients. In summary, DG/+ mice recapitulate many of the main features of human NS. In addition to providing avenues for exploring the pathogenesis of

NS phenotypes, these mice also raise several new questions. First, the effects of genetic background on phenotypes are not clear: e.g., it is unclear whether the incomplete penetrance of the DG/+ cardiac phenotype is stochastic or reflects modifier loci segregating in the mixed background used for the initial studies. Second, biochemical analyses show that D61G is one of, if not the, most highly activated PTPN11 alleles associated with NS [15]. Given that increasing gene dosage enhances phenotypic severity caused by activated Shp2 in flies and mice (see above), mutants with varying degrees of catalytic activation might also be expected to have phenotypic differences. Finally, the key cell types in which PTPN11 mutants act to cause NS phenotypes have not been defined. We have begun to address several of these issues using both our initial DG mice and several new mouse models, including knock-in mice expressing Ptpn11N308D and inducible knockin mice expressing Ptpn11D61Y (T.A., G.C., and B.G.N., manuscript in preparation). By crossing the DG allele onto 129S6/SvEv, C57BL6/J or Balb/c, we have found that the genetic background strongly influences the NS phenotype. It is not yet clear, however, if cloneable modifiers exist or if these strain differences are attributable to heterosis. Our data also indicate, however, that, as in the Drosophila studies, the specific Ptpn11 allele (on the same genetic background) can strongly influence the NS phenotype. This, in turn, may depend on the degree of Shp2 hyper-activation; indeed, there appears to be a hierarchy of phenotypes contingent on increasing Shp2 activity, with the lowest levels of Shp2 hyper-activation capable of affecting growth, while increasingly higher levels are required to evoke facial abnormalities, cardiac defects and fatal MPD, respectively. Studies using tissue-specific Cre recombinase lines to activate the Ptpn11D61Y allele selectively show that the facial abnormalities result from mutant expression in NC-derived cells, whereas all cardiac defects are caused by mutant expression in

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endocardium/endothelium, not the NC or myocardium. Notably, tissue-specific deletion of Nf1 using the same Cre lines has similar phenotypic consequences [45]. Studies to elucidate the cell(s) responsible for NS growth defects are ongoing. Finally, using explant assays from DG/+ embryos, we have found that NS mutants extend the normal interval during which EMT occurs, and as a consequence of the increased Erk activation that they evoke. In contrast to the chicken explant studies [37], we do not observe increased mesenchymal proliferation in mouse AV cushion explants. This could reflect differences between the chicken and mouse systems. Given that cushion mesenchymal cell proliferation is enhanced in DG/+ embryos, though, it is perhaps more likely that the mouse explants are inadequate to model increased proliferation for technical reasons. Notably, mesenchymal outgrowths are observed in these explants when growth factors (e.g., PDGF, FGF, Neuregulin) are added exogenously, suggesting that endogenous growth factors may be limiting in mouse explants (but not in chicken). The emerging picture, combining the mouse and chicken studies, is that NS mutants may both extend the normal interval for EMT and cause excess proliferation of cushion mesenchymal cells. Although both of these defects appear to result from enhanced Erk activation, it is unclear if the same upstream (i.e., growth factor, cytokine, integrin) signals mediate both effects. Also unclear is how increased Erk activation translates into enhanced EMT and proliferation. One attractive possibility is that NS alleles hyper-activate the transcription factor Sox9. Sox9 is known to be an immediate-early gene dependent on Erk activation [70], and Sox9-deficient mice have hypoplastic cardiac valves due to defective EMT [71], the converse phenotype to NS mice. Altered Sox9 activity also could explain other NS phenotypes (e.g., facial and stature abnormalities), given that Sox9 is also required for chondrogenesis [72], and that Erk activity reportedly affects Sox9 activity differentially in micromass cultures from chicken facial NC [73].

In contrast to the above observations using Ptpn11 knock-in mice, Nakamura et al. analyzed transgenic mice expressing Ptpn11Q79R in the myocardium under the control of the MHC and MHC promoters, respectively [67]. Because the MHC promoter becomes active in early gestation, whereas MHC turns on postnatally, mutant Shp2 expression occurs during different time windows in each line. Embryonic expression of Q79R resulted in altered cardiomyocyte cell proliferation, ventricular non-compaction, and ventricular septal defects. In contrast, postnatal expression of Q79R mutant had no apparent effect. Erk activation was increased in mutant hearts, and decreasing expression of Erk1 or 2 (by crossing to Erk1+/ or Erk2+/ mice) ablated the cardiac defects caused by embryonic Q79R expression. It is difficult to reconcile these findings with our observations that myocardial expression of the even more potently activated Ptpn11 mutant DY (see above), or myocardial-specific Nf1 deletion [45], has no phenotypic consequences, whereas endothelialdriven expression phenocopies all aspects of the mouse NS phenotype (including ventricular thinning). Conceivably, the total level of myocardial SHP2 activity in the transgenic model (caused by over-expression of the protein plus its increased activity) is greater than that in mice with myocardial-specific DY expression. Indeed, there was a Q79R dosage-dependent difference in penetrance of the myocardial phenotype in different transgenic lines generated by Nakamura et al. Alternatively, owing to position effects, the Q79R allele could have been expressed gratuitously in the developing endocardial cells in the transgenic mice.

Related Animal Models

Jacks group has generated inducible knock-in mice expressing the strongly activated KrasG12D mutant [74]. Although more potently activated than the KRAS alleles found in NS patients, the effects of G12D are likely to be qualitatively

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similar (though more severe). Global G12D expression causes early embryonic lethality due to trophoblast defects. The early lethality can be bypassed by evoking expression in the epiblast only, using Mox2-Cre mice, but mutant embryos then succumb to cardiovascular defects quite similar to those seen in DG/DG (and Nf1/) mice [75]. Mutant embryos also demonstrate hematopoietic abnormalities and a profound defect in lung branching morphogenesis, associated with upregulation of Sprouty-2, a member of the Spry family (Sprouty 14) of poorly understood feedback inhibitors of Ras/Erk pathway. Although defective lung branching morphogenesis is not characteristic of NCFC syndromes, these findings nevertheless suggest that Spry proteins and their relatives, the Spreds (Spred13) may be important modifiers of hyper-activated RAS/ERK pathway components, and thus may help explain phenotypic variation in the NCFC. Consistent with this notion, Spred1 and Spred2 knockout mice exhibit features similar to NS and other NCFC [76]. Indeed, SPRED1 should probably be added to the list of NCFC genes, given the recent finding of SPRED1 mutations in a group of patients with a neurofibromatosis-like syndrome [29].

Conclusions and Perspectives

Existing mouse models should provide fertile ground for future examination of the pathophysiological basis of NS and NF, as they appear to reproduce many important syndromic features. The challenge now is to explore the cellular and molecular basis of these defects in detail by determining the precise upstream signaling pathways affected and how aberrant signaling by these pathways is translated into abnormal morphogenesis. Further analysis of more genetically tractable organisms such as the fly and fish may provide valuable insights into such pathways, and act as hypothesis generators for studies in more complex systems. It will also be important to define the precise temporal windows during which disease-associated mutants act, as these may suggest (or certainly impose limits on) timing for potential therapeutic interventions. Finally, mouse models for other NS genes, as well as for other NCFC syndromes, will be critical if we are to elucidate the molecular basis for the similarities and differences between these fascinating syndromes.

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25 Sondermann H, Soisson SM, Boykevisch S, Yang SS, Bar-Sagi D, Kuriyan J: Structural analysis of autoinhibition in the Ras activator Son of sevenless. Cell 2004;119:393405. 26 Nimnual A, Bar-Sagi D: The two hats of SOS. Sci STKE 2002;PE36. 27 Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, et al: Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004;116:855867. 28 Garnett MJ, Rana S, Paterson H, Barford D, Marais R: Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization. Mol Cell 2005;20:963969. 29 Brems H, Chmara M, Sahbatou M, Denayer E, Taniguchi K, et al: Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1-like phenotype. Nat Genet 2007;39:11201126. 30 Kontaridis MI, Swanson KD, David FS, Barford D, Neel BG: PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects. J Biol Chem 2006;281: 67856792. 31 Hanna N, Montagner A, Lee WH, Miteva M, Vidal M, et al: Reduced phosphatase activity of SHP-2 in LEOPARD syndrome: consequences for PI3K binding on Gab1. FEBS Lett 2006;580:24772482. 32 Mohi MG, Williams IR, Dearolf CR, Chan G, Kutok JL, et al: Prognostic, therapeutic, and mechanistic implications of a mouse model of leukemia evoked by Shp2 (PTPN11) mutations. Cancer Cell 2005;7:179191. 33 Schubbert S, Lieuw K, Rowe SL, Lee CM, Li X, et al: Functional analysis of leukemia-associated PTPN11 mutations in primary hematopoietic cells. Blood 2005;106:311317. 34 Chan RJ, Leedy MB, Munugalavadla V, Voorhorst CS, Li Y, Yu M, Kapur R: Human somatic PTPN11 mutations induce hematopoietic-cell hypersensitivity to granulocyte-macrophage colony-stimulating factor. Blood 2005;105:37373742. 35 Schubbert S, Shannon K, Bollag G: Hyperactive Ras in developmental disorders and cancer. Nat Rev Cancer 2007;7:295308. 36 Mohi MG, Neel BG: The role of Shp2 (PTPN11) in cancer. Curr Opin Genet Dev 2007;17:2330.

37 Krenz M, Yutzey KE, Robbins J: Noonan syndrome mutation Q79R in Shp2 increases proliferation of valve primordia mesenchymal cells via extracellular signal-regulated kinase 1/2 signaling. Circ Res 2005;97:813820. 38 Armstrong EJ, Bischoff J: Heart valve development: endothelial cell signaling and differentiation. Circ Res 2004;95:459470. 39 Kirby ML, Gale TF, Stewart DE: Neural crest cells contribute to normal aorticopulmonary septation. Science 1983;220:10591061. 40 Kirby ML, Waldo KL: Neural crest and cardiovascular patterning. Circ Res 1995;77:211215. 41 Runyan RB, Markwald RR: Invasion of mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue. Dev Biol 1983;95:108114. 42 Nakajima Y, Miyazono K, Kato M, Takase M, Yamagishi T, Nakamura H: Extracellular fibrillar structure of latent TGF beta binding protein-1:role in TGF beta-dependent endothelial-mesenchymal transformation during endocardial cushion tissue formation in mouse embryonic heart. J Cell Biol 1997;136:193204. 43 Lakkis MM, Epstein JA: Neurofibromin modulation of ras activity is required for normal endocardial-mesenchymal transformation in the developing heart. Development 1998;125:43594367. 44 Friedman JM, Arbiser J, Epstein JA, Gutmann DH, Huot SJ, et al: Cardiovascular disease in neurofibromatosis 1: report of the NF1 Cardiovascular Task Force. Genet Med 2002;4:105111. 45 Gitler AD, Zhu Y, Ismat FA, Lu MM, Yamauchi Y, Parada LF, Epstein JA: Nf1 has an essential role in endothelial cells. Nat Genet 2003;33:7579. 46 Dearolf CR: Fruit fly leukemia. Biochim Biophys Acta 1998;1377:M13M23. 47 Oishi K, Gaengel K, Krishnamoorthy S, Kamiya K, Kim IK, et al: Transgenic Drosophila models of Noonan syndrome causing PTPN11 gain-of-function mutations. Hum Mol Genet 2006;15:543553. 48 Araki T, Mohi MG, Ismat FA, Bronson RT, Williams IR, et al: Mouse model of Noonan syndrome reveals cell typeand gene dosage-dependent effects of Ptpn11 mutation. Nat Med 2004;10:849857.

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49 Jopling C, van Geemen D, den Hertog J: Shp2 Knockdown and Noonan/LEOPARD mutant Shp2-induced gastrulation defects. PLoS Genet 2007;3:e225. 50 Tang TL, Freeman RM Jr, OReilly AM, Neel BG, Sokol SY: The SH2-containing protein-tyrosine phosphatase SHPTP2 is required upstream of MAP kinase for early Xenopus development. Cell 1995;80:473483. 51 Saxton TM, Henkemeyer M, Gasca S, Shen R, Rossi DJ, et al: Abnormal mesoderm patterning in mouse embryos mutant for the SH2 tyrosine phosphatase Shp-2. EMBO J 1997;16: 23522364. 52 Jacks T, Shih TS, Schmitt EM, Bronson RT, Bernards A, Weinberg RA: Tumour predisposition in mice heterozygous for a targeted mutation in Nf1. Nat Genet 1994;7:353361. 53 Silva AJ, Frankland PW, Marowitz Z, Friedman E, Laszlo GS, et al: A mouse model for the learning and memory deficits associated with neurofibromatosis type I. Nat Genet 1997;15:281284. 54 Costa RM, Federov NB, Kogan JH, Murphy GG, Stern J, et al: Mechanism for the learning deficits in a mouse model of neurofibromatosis type 1. Nature 2002;415:526530. 55 Le DT, Kong N, Zhu Y, Lauchle JO, Aiyigari A, et al: Somatic inactivation of Nf1 in hematopoietic cells results in a progressive myeloproliferative disorder. Blood 2004;103:42434250. 56 Zhu Y, Ghosh P, Charnay P, Burns DK, Parada LF: Neurofibromas in NF1: Schwann cell origin and role of tumor environment. Science 2002;296: 920922. 57 Bajenaru ML, Zhu Y, Hedrick NM, Donahoe J, Parada LF, Gutmann DH: Astrocyte-specific inactivation of the neurofibromatosis 1 gene (NF1) is insufficient for astrocytoma formation. Mol Cell Biol 2002;22:51005113. 58 Yang FC, Ingram DA, Chen S, Hingtgen CM, Ratner N, et al: Neurofibromin-deficient Schwann cells secrete a potent migratory stimulus for Nf1+/ mast cells. J Clin Invest 2003;112:18511861.

59 de Haan M, vd Kamp JJ, Briet E, Dubbeldam J: Noonan syndrome: partial factor XI deficiency. Am J Med Genet 1988;29:277282. 60 Sharland M, Patton MA, Talbot S, Chitolie A, Bevan DH: Coagulation-factor deficiencies and abnormal bleeding in Noonans syndrome. Lancet 1992;339:1921. 61 You M, Flick LM, Yu D, Feng GS: Modulation of the nuclear factor kappa B pathway by Shp-2 tyrosine phosphatase in mediating the induction of interleukin (IL)-6 by IL-1 or tumor necrosis factor. J Exp Med 2001;193:101110. 62 Marino B, Digilio MC, Toscano A, Giannotti A, Dallapiccola B: Congenital heart diseases in children with Noonan syndrome: An expanded cardiac spectrum with high prevalence of atrioventricular canal. J Pediatr 1999;135:703706. 63 Tartaglia M, Kalidas K, Shaw A, Song X, Musat DL, et al: PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity. Am J Hum Genet 2002;70:15551563. 64 Antonelli D, Antonelli J, Rosenfeld T: Noonans syndrome associated with hypoplastic left heart. Cardiology 1990;77:6265. 65 Amann G, Sherman FS: Myocardial dysgenesis with persistent sinusoids in a neonate with Noonans phenotype. Pediatr Pathol 1992;12:8392. 66 Wilmshurst P, Da Costa P: Probable right ventricular dysplasia and patent foramen ovale presenting with cyanosis and clubbing in a patient with characteristics of Noonan syndrome. Br Heart J 1995;74:471475. 67 Nakamura T, Colbert M, Krenz M, Molkentin JD, Hahn HS, Dorn GW, 2nd, Robbins J: Mediating ERK 1/2 signaling rescues congenital heart defects in a mouse model of Noonan syndrome. J Clin Invest 2007;117: 21232132.

68 Kratz CP, Niemeyer CM, Castleberry RP, Cetin M, Bergstrasser E, et al: The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and Noonan syndrome/myeloproliferative disease. Blood 2005;106: 21832185. 69 Gauthier AS, Furstoss O, Araki T, Chan R, Neel BG, Kaplan DR, Miller FD: Control of CNS cell-fate decisions by SHP-2 and its dysregulation in Noonan syndrome. Neuron 2007;54: 245262. 70 Murakami S, Kan M, McKeehan WL, de Crombrugghe B: Up-regulation of the chondrogenic Sox9 gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway. Proc Natl Acad Sci USA 2000;97:11131118. 71 Akiyama H, Chaboissier MC, Behringer RR, Rowitch DH, Schedl A, Epstein JA, de Crombrugghe B: Essential role of Sox9 in the pathway that controls formation of cardiac valves and septa. Proc Natl Acad Sci USA 2004;101: 65026507. 72 Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B: Sox9 is required for cartilage formation. Nat Genet 1999;22:8589. 73 Kulyk WM, Franklin JL, Hoffman LM: Sox9 expression during chondrogenesis in micromass cultures of embryonic limb mesenchyme. Exp Cell Res 2000;255:327332. 74 Tuveson DA, Shaw AT, Willis NA, Silver DP, Jackson EL, et al: Endogenous oncogenic K-ras(G12D) stimulates proliferation and widespread neoplastic and developmental defects. Cancer Cell 2004;5:375387. 75 Shaw AT, Meissner A, Dowdle JA, Crowley D, Magendantz M, et al: Sprouty-2 regulates oncogenic K-ras in lung development and tumorigenesis. Genes Dev 2007;21:694707. 76 Bundschu K, Walter U, Schuh K: Getting a first clue about SPRED functions. Bioessays 2007;29:897907.

Benjamin G. Neel Ontario Cancer Institute, University Health Network 101 College Street, TMDT8355 Toronto, ON, M5G1L7 (Canada) Tel. +1 416 581 7757, Fax +1 416 581 7698, E-Mail bneel@uhnres.utoronto.ca

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Zenker M (ed): Noonan Syndrome and Related Disorders. Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 151164

Towards a Treatment for RAS-MAPK Pathway Disorders


V.A. Joshia,b A.E. Robertsa,c R. Kucherlapatia
aHarvard

Medical School Partners HealthCare Center for Genetics and Genomics, Boston, Mass., of Pathology, Massachusetts General Hospital, Boston, Mass., cDepartment of Cardiology, Childrens Hospital Boston, Boston, Mass., USA
bDepartment

Abstract
The molecular pathogenesis of Noonan, Costello, and Cardio-facio-cutaneous syndromes has recently been described. All of these disorders result from an abnormal activation of the RAS-MAPK pathway. RAS-MAPK pathway activation is a common occurrence in tumor cells, and much effort has been made to develop inhibitors of this pathway to treat cancer. This chapter will describe several different strategies of RAS-MAPK pathway inhibition that are being evaluated in clinical trials. The potential application of these inhibitors to individuals with RAS-MAPK developmental disorders will also be discussed.
Copyright 2009 S. Karger AG, Basel

Human genetic disorders can be classified into monogenic and complex disorders. Monogenic disorders, in turn, are classified based upon their inheritance patterns: autosomal dominant, autosomal recessive and sex-linked being the most common. A large number of monogenic disorders have been described (OMIM). In most cases of autosomal recessive disorders, both parents are carriers of a recessive allele. In autosomal dominant disorders, one of the parents may be affected and pass on the dominant allele to their offspring. Alternatively, these disorders may also result from spontaneous mutations in the germ

cells. Noonan syndrome (NS; MIM 163950) and LEOPARD syndrome (LS; MIM 151100) are inherited in an autosomal dominant fashion and individuals with a mutation in one of several genes (see below) are affected. Cardio-faciocutaneous syndrome (CFC; MIM 115150) and Costello syndrome (CS; MIM 218040), by contrast, typically occur sporadically in families. Newborns with one of these syndromes can often be diagnosed based on the manifestations of the syndrome. Accurate diagnosis helps with prediction of the course and the severity of the disorders and thus can assist with management of the patients. Although many of the later onset symptoms can be predicted based on the diagnosis and examination of the nature of the mutations in the causal gene, there are no curative therapies for these disorders. The identification of several genes involved in these disorders and an understanding of the pathways in which these genes function now provides a possible approach for developing therapeutics. Because of the relative rarity of these monogenic disorders, pharmaceutical companies, who have the expertise to develop new drug entities and test them in patients, are unlikely to be interested in developing

drug based therapies. However, these syndromes have unique properties that may allow them to take advantage of drugs that are already in development by several major pharmaceutical companies. The majority of NS, LS, CFC and CS result from dominant mutations in components of the RAS-MAPK pathway. RAS is an important upstream member of this pathway and specific activating mutations in the RAS genes are detected in more than 30% of all solid tumors [1]. Activating mutations in the RAS genes lead to a cascade of events, among which is the activation of ERK and MEK. Because of the involvement of RAS mutations in a large proportion of human cancers, many pharmaceutical companies are developing inhibitors that aim to block the activation of the RAS-MAPK signaling pathway with the expectation that such inhibition would halt tumor progression and reverse the growth of the tumor. We will consider the possibility of developing targeted therapies for NS, LS, CFC, and CS and what type of drug development pathway may be undertaken.

Clinical Features and Management of RAS-MAPK Developmental Disorders

Individuals with NS, LS, CFC, and CS share clinical features that result from defects in the RASMAPK signaling pathway. NS is characterized by variable developmental delay, short stature, webbed neck, pectus abnormalities, coagulation defects, and cryptorchidism, with characteristic facial features. Congenital heart defects, primarily pulmonary valve stenosis and hypertrophic cardiomyopathy, which affect 2050% and 2030% of individuals, respectively, are the primary cause of morbidity and mortality. Juvenile myelomonocytic leukemia (JMML) and acute lymphoblastic leukemia (ALL) have been associated with NS [2]. In one longitudinal study of the natural history of NS, only one case of breast cancer was reported,

with no additional cases of any cancer [3]. Based on the current understanding of the molecular pathogenesis of NS, this is somewhat surprising. However, the mean age of the cohort examined was 25.3 years, with a mean follow-up of 12.02 years, and PTPN11 mutations were only identified in 35% of individuals. It is possible, therefore, that this cohort is not completely representative of the adult NS population. Additional longitudinal studies should help clarify the risk of neoplasia associated with NS. NS can be inherited in an autosomal dominant fashion, but sporadic cases are common. It is estimated that as many as 1/1,000 individuals are affected with NS [4, 5]. LS, CFC, and CS share with NS similar facial features, congenital heart defects, growth retardation, and developmental delay or mental retardation. LEOPARD (Lentigines, Electrocardiographic conduction defects, Ocular hypertelorism, Pulmonary stenosis, Abnormalities of the genitals, Retarded growth resulting in short stature, and Deafness) is an allelic disorder of NS. The multiple lentigines and deafness observed in these individuals are characteristic and unique. CFC is distinctive in that individuals typically have hair and skin abnormalities such as sparse curly hair, absent eyelashes, patchy alopecia, ichthyosis, hyperkeratosis, and ulerythema ophryogenes (absent eyebrows with hyperkeratosis). Individuals with CFC and CS typically have more significant cognitive delays than those with LS or NS. One of the distinctive features of CS is the risk of neoplasia. Papillomas, benign tumors, frequently develop around the mouth and anus. The most common malignant tumor is rhabdomyosarcoma, although neuroblastoma and bladder cancer have also been observed in multiple individuals [6]. Because the cancer risk is as high as 17%, a screening protocol has been recommended for individuals with CS consisting of ultrasound examination of the abdomen and pelvis, urine catecholamine metabolite analysis, and urinalysis [7].

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The medical and developmental issues seen in these disorders are treated symptomatically as there is no curative treatment for the underlying molecular perturbation of the RAS-MAPK pathway. The treatment of cardiac manifestations is largely the same as in the general population. The pulmonary valve stenosis varies in severity from mild, requiring no intervention, to severe, requiring surgery. Dysplastic pulmonary valves respond less often to balloon dilation than pulmonary stenosis without valve dysplasia [8]. Hypertrophic cardiomyopathy may be treated pharmacologically with beta blockers or calcium channel blockers, may require myomectomy or, less commonly, progress to the point of requiring heart transplant. Arrhythmias (usually supraventricular or paroxysmal tachycardia, most distinctively ectopic atrial tachycardia) are most common in CS though reported in all three disorders [9]. Anyone with a disorder of the RAS-MAPK pathway should be followed by a cardiologist throughout both childhood and adulthood. Certain congenital heart defects require antibiotic prophylaxis for subacute bacterial endocarditis. Feeding problems are common and a majority of children require treatment for gastroesophageal reflux. Most children with CFC and CS require nasogastric or gastrostomy feeding and Nissen fundoplication may be required. These interventions are less commonly indicated in NS. Short stature is prevalent in these disorders and growth hormone deficiency is documented, though not in all children with short stature. It appears that children with NS and a PTPN11 mutation have relative growth hormone resistance and there is some thought that augmentation of growth hormone replacement with IGF-1 may yield a better linear growth response [10]. Growth velocity appears to increase during the first three years of treatment with the greatest increase in growth velocity in the first year [11]. Growth hormone deficiency may present as hypoglycemic seizure in CS [12]. Hypertrophic cardiomyopathy

is considered a relative contraindication by some to growth hormone therapy though no impact on ventricular wall size has been documented. A variety of skeletal issues have been observed. Scoliosis and kyphoscoliosis most often respond to bracing, though surgical intervention with rod placement may be required. The ulnar deviation of the wrists and fingers in CS is treated with bracing, physical therapy, and occupational therapy. Large joint extension might be limited and requires physical therapy and occasionally surgical tendon lengthening. Only very rarely does the pectus carinatum or excavatum of NS require surgical correction. Seizures can occur in any of the RAS-MAPK disorders and are treated as in the general population though hypoglycemia, low serum cortisone, and hydrocephalus need to be ruled out as potential causes [13]. Symptomatic Arnold Chiari malformation has been reported in NS and often responds to decompression surgery. Hydrocephalus may require shunting. Malignant hyperthermia has been reported in NS, though it is not clear if these cases are coincidental or truly related to the NS diagnosis. It appears that the risk is greatest if there is myopathy or an elevated serum CK level and thus dantrolene prophylaxis is suggested during surgery when CK levels are elevated or if there is a clinical suspicion of malignant hyperthermia or myopathy [14]. Early developmental milestones are often delayed; this may be more profound in CFC and CS. Gross and fine motor delays are often attributable to low muscle tone and respond well to early intervention with occupational and physical therapy. Articulation difficulties respond well to hearing aids when indicated and speech therapy. In general, IQ falls within the normal range for children with NS and in the mild to moderate mental retardation range for children with CFC and CS. As there is no specific cognitive profile recognized in any of these disorders, children are best served by having regular, detailed neurocognitive evaluations with an individualized education plan.

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Maximization of vision and hearing will help children reach their full developmental potential. Visual problems including myopia, hyperopia, strabismus, and astigmatism can be treated as in the general population [15]. Recurrent ear infections can be complicated by middle ear effusion causing conductive hearing loss and may require placement of pressure equalization tubes. The xerosis and pruritis of CFC syndrome can be treated with an increase in ambient humidity and the use of hydrating lotions [16]. The papillomata of CS can cause irritation or inflammation and can be removed surgically or, in the facial region, treated with cryotherapy. Local medications for keratosis pilaris atrophicans faciei is not usually effective [14]. Skin, particularly in areas of lymphedema, can be prone to infection and is treated with antibiotics as indicated. The multiple nevi often seen in NS and CFC are not thought to be at increased risk for malignant transformation though periodic dermatologic evaluation may be indicated until the natural history is more definitively understood. Children with NS are at increased risk for a bleeding disorder. Platelet aggregation abnormalities, factor deficiencies (most commonly factors V, VIII, XI, and XII, and Protein C), von Willebrand disease, and thrombocytopenia have all been reported. Aspirin and aspirin-containing medications should be avoided. The need for surgical pre-treatment should be assessed by a hematologist. Lymphatic abnormalities including peripheral edema, pulmonary lymphangiectasia, and intestinal lymphangiectasia are reported in a minority of cases but when present can cause significant morbidity and mortality. Support stockings and careful foot hygiene are important for lower extremity edema. Chylothorax may require surgical drainage and/or respond to low-fat diet. Treatment with prednisone has also been reported to be effective [14]. Cryptorchidism and genitourinary reflux are treated as in the general population.

Molecular Pathogenesis of RAS-MAPK Developmental Disorders

Over the past several years, the molecular defect in each of these four disorders has been at least partially revealed. Mutations in PTPN11 have been identified in 50% of cases of NS and the majority of LS, but were not found in individuals with CFC or CS [1720]. This discovery set off a flurry of studies with aims to identify other NS genes and the genes responsible for CFC and CS. Using a candidate gene approach, it was shown that mutations in KRAS, SOS1, and RAF1 cause 1, 10, and 317% of cases of NS respectively [2126]. A fraction of cases of LS also have mutations in RAF1 [22]. Mutations in BRAF, KRAS, MEK1, and MEK2 cause 3778, 7, 9, and 4%, respectively, of cases of CFC [27, 28]. Mutations in HRAS cause up to 92% of CS [29]. Knowledge of the underlying molecular pathogenesis of these disorders could help guide the selection and development of therapies that can be used to treat affected individuals. All of these genes encode proteins that are components of the RAS-MAPK signal transduction pathway. This pathway is responsible for the communication of extracellular growth signals to the nucleus through a complex phosphorylation cascade. Activation of this pathway initiates transcription of genes involved in cell proliferation, inhibition of apoptosis, and metastasis. It has been recognized for some time that upregulation of this pathway is a common feature of tumorigenesis. Somatic activating BRAF mutations, for example, are present in 66% of malignant melanomas and a significant fraction of other tumor types. Activating KRAS mutations are present in up to 21% of all human tumors [30, 31]. Mutations in the eight genes implicated in the RAS-MAPK developmental disorders also typically activate the pathway, although often to a lesser extent than observed by somatic changes. NS-associated mutations in PTPN11 typically lie at the interface of the N-SH2 and PTP interacting

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surfaces, an interaction which is critical for the basal inactive state of the protein [32]. Disruption of the autoinhibited state leads to increased phosphatase activity and activation of the pathway. Similarly, NS mutant alleles of SOS1 show increased ERK and RAS activation [23, 24]. The majority of CS causing HRAS mutations thus far reported have also been identified as somatic mutations in tumors and are known to be activating [29]. The MEK mutations identified in individuals with CFC also stimulate ERK phosphorylation [28]. Exceptions to the gain-of-function mechanism of disease pathogenesis have been described. The majority of the mutations in PTPN11 that cause LS do not show an increase in protein tyrosine phosphatase activity in vitro [33]. RAF1 mutations have also been reported to cause LS [22]. Some RAF1 mutations showed an increase in kinase activity with a consummate increase in ERK activation, whereas others had reduced or absent kinase activity and a decrease in ERK activation [21, 22]. LS-associated RAF1 mutations have increased kinase activity as compared to wild type [22]. In addition, unlike LS-associated mutations in PTPN11 which do not show overlap with NS-associated mutations, RAF1 mutations have been associated with both NS and LS. Therefore, strict loss or gain of function as a mechanism of either NS or LS clinical features may not be the case. BRAF mutations in CFC cluster in either the cysteine-rich domain of the conserved region 1 or in the protein kinase domain [28]. This is in contrast to that observed in most tumors, where the majority of mutations affect a small number of codons; the V600E mutation has been identified in up to 19% of tumors examined and is found in up to 12% of tumors of the large intestine [31]. The kinase activity of CFC-associated BRAF mutants was, in some cases, as activating as the V600E mutant, whereas other CFC mutations impaired kinase activity [27, 28]. This is similar to what has been observed with some somatic mutations [34]. Likewise, activated and impaired

kinase activity were observed with two different CFC-associated KRAS mutations [27]. While it is possible that these results can be derived from technical differences in the assays used, it is quite possible that both loss and gain of function result in similar RAS-MAPK pathway signaling defects. This is very important to keep in mind as consideration is made for targeted treatment for these disorders.

RAS-MAPK Pathway Inhibitors in Development

Because of their prominent role in tumorigenesis, components of the RAS-MAPK cascade have become attractive targets for inhibition in the treatment of cancer. Inhibition strategies fall into several different categories, including antibody or small molecule inhibitors that target receptors, inhibitors that block post-translational modifications, and small molecule inhibitors of protein kinases. Antibody and small molecule inhibitors of receptors have been well described and are currently in use or under evaluation for the treatment of many solid tumors. However, the targets of these inhibitors lie upstream of the proteins affected by RAS-MAPK developmental disorders, and may therefore not be the best candidates for the treatment of these disorders. Many different inhibitors and inhibition strategies are in various stages of development; only the most advanced inhibitors will be discussed here (table 1, fig. 1, for a comprehensive review see [35]). RAF proteins are serine/threonine kinases that directly phosphorylate MEK1 and MEK2 kinases. The RAF family is comprised of A-RAF, BRAF, and RAF1 (C-RAF). A-RAF mutations have not been found in individuals with NS, and only rarely in tumor cells [21, 31]. Whereas both RAF1 and BRAF are commonly mutated in individuals with NS or CFC, respectively, in tumor cells, RAF1 is rarely mutated (<1%, n = 210), and BRAF is frequently mutated (20%, n = 23,269, [31]).

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Table 1. Selected RAS-MAPK pathway inhibitors. Five inhibitors that target components of the RAS-MAPK pathway are highlighted. Their generic name, brand name, sponsoring company, mechanism of action and development stage are listed Generic Name Sorafenib Brand Name Nexavar Company Bayer Pharmaceuticals Mechanism small molecule multikinase inhibitor small molecule MEK inhibitor small molecule MEK inhibitor Stage of development FDA-approved for advanced renal cell carcinoma phase I trial in breast, colon cancer, and melanoma phase I/II trials in pancreas, biliary cancer, liver cancer, advanced solid tumors phase I/II trials in breast cancer, AML, CML, lymphoma, brain and CNS cancer, melanoma, NF1 phase I/II/III trials in progeria, brain and CNS cancer, advanced breast cancer, MDS, CMML

PD-325901

Pfizer

AZD6244

Astra Zeneca/Array

Tipifarnib

Zarnestra

Johnson & Johnson

farnesyltransferase inhibitor

Lonafarnib

Sarasar

Schering Plough

farnesyltransferase inhibitor

Sorafenib (BAY 439006, Nexavar, Bayer Pharmaceuticals) is a RAF inhibitor that is FDAapproved for use in advanced renal cell carcinoma. This drug is generally well-tolerated with skin rash and diarrhea being the most common adverse events. Sorafenib was developed as an inhibitor of RAF1; however, inhibition by this drug is not specific and it has proven to be capable of inhibiting both wild-type and mutant BRAF, as well as receptor tyrosine kinases such as VEGFR-2, PDGFR-, Flt-3, c-Kit, and FGFR-1 [36]. As such, the mechanism of anti-tumor activity is not certain. Sorafenib treatment does inhibit mitogenstimulated RAF activity in vivo, as measured by ERK phosphorylation [37]. In addition, RAF1 is activated in up to 55% (6/11) of RCC tumors, supporting the idea that inhibition of this target is involved in the efficacy of the drug in this cell type [38]. However, it is possible that Sorafenib is

functioning either solely through its off-target activities or that co-inhibition of these other targets is equally important in this drugs efficacy. The substrates of the RAF kinases are MEK1 and MEK2, dual specificity kinases that act upon ERK1 and ERK2. Neither activating MEK1 nor MEK2 mutations have been identified in tumors to date (n = 229, [31]). The transforming activity of RAS is dependent on MEK and ERK, and ERK activation is commonly observed in tumor cells. Therefore, MEK inhibitors have been pursued as anti-tumor agents. After phase II trials did not show anti-tumor activity in breast, colon, NSCLC, or pancreatic tumors, the first MEK inhibitor to enter clinical evaluation, CI-1040 (PD184352), halted development [39]. However, a vast amount of in vitro data suggests that MEK inhibition could be an effective treatment strategy, and two second-

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Growth factor or cytokine Receptor


GTP GDP GTP

Cell membrane

GDP

KRAS HRAS SOS1 Grb2

KRAS HRAS

FTase inhibitors

PTPN11

BRAF RAF1 MEK1/2 ERK1/2

RAF inhibitors

MEK inhibitors

Transcription

Fig. 1. RAS-MAPK Pathway. Components of the RAS-MAPK pathway in which germline mutations have been identified in NS, LS, CS, or CFC are highlighted in yellow. The target of inhibition of each of the three inhibitor classes outlined in the text is indicated.

generation MEK inhibitors, PD325901, a derivative of CI-1040, and AZD6244 (ARRY-142886) are in development. PD325901 suppressed the growth of tumor xenografts that carried an activating BRAF mutation; however, xenografts that were ras and raf wild type were insensitive to the drug [40]. In one phase I/II trial of PD325901, of 30 individuals treated, one exhibited partial response (melanoma) and five exhibited stable disease (4 melanoma, 1 NSCLC). Common adverse events included rash, fatigue, diarrhea, nausea, and vomiting [41]. PD325901 is currently in phase I clinical trials of advanced breast, colon and melanoma [42]. AZD6244 inhibited ERK1/2 phosphorylation in several cell lines, including two with activating BRAF and RAS mutations, as well as in xenograft tumors in mouse models [43]. AZD6244 is currently in phase I/II studies in pancreatic cancer or other advanced solid

tumors. MEK inhibitors, unlike RAF inhibitors, are highly selective due to the fact that they do not bind to the ATP-binding domain, but rather bind to another region of the protein. The fact that these inhibitors suppress the growth of cells driven by RAS-MAPK pathway activation specifically supports the idea that inhibition is functioning through this pathway. RAS activity is dependent upon a number of post-translational modifications that target the protein to the inner plasma membrane surface. Farnesylation, the transfer of a lipid moiety to RAS is the first step in the process and is catalyzed by farnesyltransferase (FTase). FTase inhibitors have been the focus of much investigation, the hypothesis being that the retention of RAS in the cytoplasm will prevent RAS-dependent MAPK pathway activation. This strategy has been complicated by the observation that in the presence of

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FTase inhibitors, RAS proteins undergo alternative lipid modification: prenylation by geranylgeranyltransferase I (GGTaseI), which is sufficient to target RAS to the plasma membrane. GGTase inhibitors also exist and may complement the inhibition of FTase. FTase inhibitors (FTI), like RAF inhibitors, are non-specific and inhibit farnesylation of a number of substrates including Rho-B, Rac, Rheb, and nuclear lamins. R115777 (tipifarnib; ZarnestraTM; Johnson & Johnson) and SCH-66336 (lonafarnib, Sarasar; Schering Plough) are two FTIs that are undergoing clinical development. Several others, including BMS-214662, L778123, FTI-277, and L744832, are also being tested. Although tipifarnib has been proven to be ineffective in several different types of advanced solid tumors, it has shown promise in the treatment of leukemias [44]. In a phase II trial of single agent tipifarnib of 158 older adults with previously untreated AML, 14% achieved complete remission, with an overall response rate of 23% [45]. A phase II trial of 82 individuals with myelodysplastic syndrome similarly yielded a complete response rate of 15%, and an overall response rate of 32% [46]. Common treatment-related toxicities included grade 3 or 4 infection, GI disturbances, skin rash, neutropenia, and thrombocytopenia. Both trials demonstrated equivalent or improved survival benefits compared with other standard treatments. Interestingly, although inhibition of farnesylation was observed in responders, a decrease in MAPK phosphorylation did not correlate with response, suggesting that RAS inhibition may not be a major mechanism of response [45]. Lonafarnib monotherapy or combination therapy has been shown to have activity in the treatment of several solid tumors, including NSCLC and advanced breast cancer [4749]. Both lonafarnib monotherapy and imatinib combination therapy have demonstrated activity in individuals with CML [50, 51]. In advanced urothelial cancer, metastatic colorectal cancer, and advanced head and neck squamous cell carcinoma, no tumor responses were observed [44]. The primary

toxicities included diarrhea, neutropenia, nausea, vomiting, and fatigue. Ongoing phase I, II, and III clinical trials of tipifarnib and lonafarnib in various adult cancers are underway. Both tipifarnib and lonafarnib are also currently being evaluated for the treatment of several pediatric disorders. A phase I trial of tipifarnib treatment in children with either refractory solid tumors (n = 23) or NF-1-related neurofibromas (n = 17) revealed that this drug was well-tolerated in children with primary dose-limiting toxicities being myelosuppression, rash, nausea, vomiting, and diarrhea [52]. At the maximum tolerated dose, median residual FTase activity was 30% in treated individuals. No responses were observed. An ongoing randomized, placebo-controlled phase II clinical trial is underway to evaluate tipifarnib treatment in children and young adults with NF1 and progressive plexiform neurofibromas [53]. One phase II clinical trial of tipifarnib in children with JMML has been performed. In this study, 47 newly diagnosed patients were given the option of tipifarnib treatment prior to stem cell transplant. A response rate of 58% was observed. FTPase activity was inhibited in 13/15 cases; however, no correlation between FTPase inhibition and presence of a somatic RAS or PTPN11 mutation was made [54]. One phase I clinical trial of lonafarnib in pediatric cases of advanced CNS tumors has been reported [55]. Of 48 assessable patients, one exhibited partial response and nine had stable disease. Toxicities included grade 4 neutropenia, alterations in electrolytes, respiratory tract complaints, neuropathy, fatigue, and pain. Diarrhea was treatable with loperamide. Four of six assessable individuals analyzed showed evidence of farnesylation inhibition. Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging syndrome caused by a truncating lamin A mutation which interferes with the integrity of the nuclear membrane. FTase activity is required for lamin localization to the nuclear membrane, and FTase treatment of mouse models of HGPS

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improved bone health through a number of parameters [56, 57]. Evaluation of lonafarnib as a treatment for HGPS is underway [58].

The Horizon for the Treatment of Germline and Somatic RAS-MAPK Disorders

A possible drug evaluation strategy is as follows. A preclinical model, such as a mouse model(s) that appropriately recapitulates the clinical features of these diseases, could be identified or generated. The various inhibitors currently available could be evaluated in this model. The possibilities are not limited to those outlined above; however, these may be suitable candidates. If any of the characteristics in the model are positively affected by inhibition, phase I clinical trials in patients can be considered. This would likely be most appropriate in individuals that develop cancer. The design of the phase I trial (starting dose, regimen specifics) will likely be well informed by other trials of these inhibitors. Response rates and outcomes can be assessed, with an assessment of other clinical features (cardiac, cognitive) as secondary endpoints. If phase I trials show promise, an assessment of these therapies in an adjuvant setting could be conducted to determine the benefits and effects associated with chronic admission of these inhibitors. Additional trials of other individuals that do not have cancer can also be considered. Several mouse models of NS have been generated. Mice heterozygous for the D61G mutation in PTPN11 exhibit several of the clinical features of NS [59]. They have short stature (proportional growth failure), craniofacial features, and myeloproliferative disease. In addition, about 50% of mice have ventricular septal defects, doubleoutlet right ventricle, and enlarged valve primordia; however, the mice do not develop cardiac hypertrophy. Mice that express the Q79R mutation in PTPN11 during development similarly showed ventricular non-compaction, ventricular septal defects, and abnormal anatomy of the

interventricular groove [60]. These may serve as preclinical models of NS, but will likely not be sufficiently representative of all the individuals with these three disorders. Drug selection is one of the most critical aspects to the success of this line of investigation. It may be true that a single inhibitor will not be effective in all individuals with any of the RASMAPK disorders, and that even within disease types, a single inhibitor will not be effective in all individuals. Selection of an effective inhibitor may require knowledge of an individuals specific causative mutation. One challenge could be that target inhibition is circumvented by an activated downstream pathway member (fig. 1). In the treatment of NSCLC, for example, small molecule inhibitors of EGFR, a receptor tyrosine kinase, are ineffective in individuals that have somatic activating KRAS mutations [61]. Likewise, treatment of an individual with a RAS-MAPK disorder with a mutation in a pathway component downstream of a particular inhibitor target may not be effective. One preliminary study evaluating the use of RAS-MAPK inhibitors in cultured cells expressing CFC-associated MEK mutations has been reported [62]. In this study, RAF inhibition by SB-590885 did not prevent pathway activation by mutated MEK2. The MEK inhibitor U0126, however, did prevent pathway activation by mutated MEK1 (F53S and Y130C) and MEK2 (F57C). Inhibition of MEK, the most downstream effector, may be a strategy that will be effective in the majority of individuals. An added benefit of MEK inhibitors could be that, unlike RAF inhibition by sorafenib or FTase inhibition by lonafarnib or tipifarnib, inhibition is highly specific. A remaining challenge will be the small but significant subset of individuals with NS, CFC, and CS whose molecular defect is not known. It is possible that these individuals have mutations in downstream or parallel pathways and will not be affected by RAS-MAPK pathway inhibition. The individuals with RAS-MAPK disorders most likely to immediately benefit from the

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development of inhibitor therapy are those who develop cancer, whether due to germline or somatic genetic mutation. These individuals require treatment, and existing options are currently marginally effective with severe side effects. As mentioned earlier, individuals with NS, CFC, and CS have a predisposition to the development of neoplasia. Monocytic proliferation may affect as many as 10% of cases of NS [63]. Individuals with NS can develop JMML, and deregulation of the RAS-MAPK pathway is observed in up to 85% of JMML cases: 35% have somatic mutations in PTPN11, 20% somatic mutations in RAS, 15% somatic mutations in NF1, and 15% germline mutations in NF1. JMML in individuals without NS is often rapidly fatal; left untreated, the mortality rate nears 100% within the first year. In individuals with NS, myeloproliferative features like JMML often resolve spontaneously, but can also follow an aggressive course similar to JMML. Hematopoietic stem cell transplant has been the primary treatment choice for JMML. While this can be curative, identification of an HLAmatched donor can be difficult, and the side effects associated with treatment can be severe in the pediatric patient group. Disease recurrence can be expected in up to 48% of individuals [64]. Individuals with CS have an incidence of tumor formation that is as high as 17%. Tumors observed include rhabdomyosarcomas, neuroblastomas, bladder carcinomas, vestibular schwannoma, and epithelioma [7]. Rhabdomyosarcoma may be treated with surgery, chemotherapy, and radiation therapy, and has recently been treated with newer chemotherapeutic drugs such as irinotecan [65]. Neuroblastoma accounts for 910% of pediatric tumors with more than 10,000 individuals affected annually. It is typically treated by a combination of surgery, chemotherapy, and radiotherapy [66]. CFC has not traditionally been associated with an increased risk of neoplasia, although malignancies, including hepatoblastoma and ALL, have been observed [6769]. Hepatoblastoma was reported in a 35-month-old CFC patient with a

MEK1 mutation and a history of hypertrophic cardiomyopathy requiring heart transplant. It is possible that the hepatoblastoma was secondary to the immunosuppression required after transplant but since hepatoblastoma can be seen in CS, it could also be related to the common perturbation of the RAS-MAPK pathway in CFC and CS [67]. Given the small numbers of clinically diagnosed CFC individuals and the lack of longitudinal studies in this patient group, the frequency of cancer may be larger than originally appreciated. It is difficult to predict what impact such inhibitor treatment may have on the congenital features of NS, CFC, or CS such as developmental delay, cardiac dysfunction, or unique facial features. Recent studies have indicated that mice with heterozygous Nf1 mutations, and, consequently, increased Ras activity, have spatial learning deficits [70]. These cognitive defects can be rescued by treatment with an FTI-inhibitor (BMS 191563) or with lovastatin, an inhibitor of cholesterol biosynthesis that is used to treat hyperlipidemia that has also been shown to inhibit Ras activity [7173]. A phase I clinical trial evaluating the use of lovastatin in adults with NF1 is currently underway [74]. The finding that pharmacologically reducing RAS-MAPK pathway activity could improve cognitive features in individuals with aberrant pathway activation is encouraging, although it is not clear what if any benefit postnatal administration of pathway inhibitors might have on individuals with NS, CS, or CFC. The impact on congenital heart defects is also difficult to predict. In a mouse model of NS, expression of an activating mutation in PTPN11 during gestation resulted in ventricular non-compaction and ventricular septal defects. When aberrant RAS-MAPK signaling was blocked by eliminating ERK during embryogenesis, cardiac anatomy was significantly improved. No effect of aberrant RAS-MAPK signaling was observed after birth [60]. These data suggest that abnormal signaling during development is necessary and sufficient to generate congenital heart defects. Inhibition of this signaling after development of the heart is

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complete may have no impact on heart structure or function. The exception to this could possibly be in individuals that have hypertrophic cardiomyopathy or valve stenosis, where RAS-MAPK inhibition could delay or prevent progression. Although the RAS-MAPK targeted drugs typically appear to be well-tolerated, even in the pediatric patient population, individuals with germline RAS-MAPK disorders have additional medical complications including congenital heart defects. Cardiotoxicity, such as acute coronary syndromes including myocardial infarction or QT prolongation, has been a side effect observed during the use of some tyrosine kinase inhibitors including sorafenib [75, 76]. This is not true of all drugs in this class; however, monitoring cardiac outcomes has not typically been built into clinical trial design of most of these inhibitors. As the molecular mechanism of these cardiac effects is not well understood, caution should be used in selecting inhibitors for treatment and consideration for evaluating adverse cardiovascular effects in trial outcomes should be taken. It is possible that these or other medical issues in individuals with NS, CFC, or CS will render these drugs intolerable at therapeutic doses. The cancer-associated RAS-MAPK gene mutations are somatic and often strongly activate the pathway. The activating mutations found in tumors and those found in NS, LS, CFC, or CS patients are not typically the same. Studies in mice have revealed that the germline presence of cancer-associated activating mutations are often not

compatible with life. Experimental evidence suggests that the mutations that lead to developmental disorders result in the activation of the RASMAPK pathway, but that the level of activation is often milder than that seen in tumors. This observation suggests that the degree of inhibition of the pathway that is required to counteract the result of mutations seen in the patients with one of these syndromes could be significantly less. Therefore, it is possible that low doses of one of these pathway inhibitors may restore a normal level of activity of the pathway. Since not all of the manifestations of the syndromes are present at birth it can be hoped that administration of low and safe doses of the pathway inhibitors may result in significant clinical benefit for the patients.

Conclusions

If successful treatments for cancers caused by somatic RAS-MAPK mutations are developed, this may be the door that leads to treatment of the effects of germline mutations. One can imagine first treating progressive features like cardiac hypertrophy, short stature, or cognitive delay and then, as methods of delivery improve, attempting prenatal treatment to prevent or slow development of congenital malformation of the heart, skeleton, kidneys, brain, or face. RAS-MAPK inhibitors hold promise that todays treatments could inform tomorrows preventions.

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Victoria A. Joshi 65 Landsdowne St. Cambridge, MA 02139 (USA) Tel. +1 617 768 8324, Fax +1 617 768 8513, E-Mail vjoshi@partners.org

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Author Index

Allanson, J.E. 9 Araki, T. 138 Binder, G. 104

Kerr, B. 83 Kratz, C. 119 Kucherlapati, R. 151 Legius, E. 128

Sarkozy, A. 40, 55, 109 Schmid, M. VII Sol-Church, K. 94 Tartaglia, M. 20, 55 Tidyman, W.E. 73 Versacci, P. 109

Dallapiccola, B. 40, 55, 109 Denayer, E. 128 Digilio, M.C. 40, 55, 109 Gelb, B.D. 20, 55 Gripp, K.W. 94 Joshi, V.A. 151

Marino, B. 40, 109 Neel, B.G. 138 Noonan, J.A. 1 Rauen, K.A. 73 Roberts, A.E. 66, 151

Zampino, G. 55 Zenker, M. IX

165

Subject Index

Animal models 138 AZD6244 156 BRAF 33, 76, 114 Caf-au-lait spots 128 Cancer predisposition 94 Cardiac valve defects 138 Cardiofaciocutaneous syndrome (CFC, CFCS) 66, 73, 114, 119 clinical diagnosis 66, 73 clinical features 67 cardiovascular features 68, 114 development 67 ectodermal features 69 facial features 67 gastrointestinal features 70 growth 67 malignancy 70 neurological features 68 ophthalmologic features 69 renal features 70 skeletal feature 70 history 3 molecular causes 73 natural history 71 treatment 80 CFC, see Cardiofaciocutaneous syndrome CFCS, see Cardiofaciocutaneous syndrome Congenital heart defect (CHD) 10, 109 Costello syndrome (CS) 83, 94, 114, 122 development 87 diagnosis 89

endocrine abnormalities 87 history 4, 83 HRAS mutations 94, 101 hypertrophic cardiomyopathy (HCM) 86, 114 natural history 85 phenotype 88 skeletal abnormalities 88 tumor risk 85 Cryptorchidism 14 CS, see Costello syndrome Drosophila models 142 Gain of function 94 Genotype-phenotype correlation 40, 61, 106 Germline mutation 94, 98, 123, 138 Growth hormone (GH) 104 therapy 105 HRAS 94, 119 G12 variants 96 Germline mutation 98 Rare variants 97 Somatic mosaicism 99 Hypertrophic cardiomyopathy (HCM) 86, 111, 114 IGF-I 105 Inhibitor 151 Juvenile myelomonocytic leukemia (JMML) 15, 120, 138 KRAS 13, 27, 45, 78

166

Learning difficulties 128 LEOPARD syndrome (LS) 3, 20, 55, 109, 114 clinical aspects 55 disease genes 59 history 3, 55 molecular pathogenesis 55 Leukemia 138 Lonafarnib 156 LS, see LEOPARD syndrome MEK1/2 36, 52, 77, 114 Mental retardation 138 Mouse models 144 Myeloproliferative disorder (MPD) 15, 119, 138 Neuroblastoma 119 Neuro-Cardio-Facial-Cutaneous (NCFC) Syndromes 134, 138 Neurofibromatosis-Noonan syndrome (NFNS) 5, 128 history 5 Neurofibromatosis type 1 (NF1) 5, 128 NF1, see neurofibromatosis type 1 NFNS, see Neurofibromatosis-Noonan syndrome Noonan syndrome (NS) 1, 9, 20, 40, 59, 109, 119, 128, 138 animal models 138 cardiovascular anomalies 10 central nervous system 13 craniofacial features 9 development 12 gastrointestinal system 14 genes 21 genitourinary system 14 genotype-phenotype correlation 40, 106 growth 11, 104 hearing 12 hematology 14 history 1 immunological findings 15 lymphatics 14

molecular genetics 20 musculoskeletal findings 13 ocular anomalies 12 short stature 104 skin 14 NS (see Noonan syndrome) Oncogene 94 Parental origin 98 PD325901 156 Pectus 13 Protein-tyrosine phosphatase (PTP) 138, 140 PTPN11 11, 23, 41, 59, 104, 109, 113, 119, Puberty 104 Pulmonary stenosis 10, 110, 115 RAF1 33, 49, 61, 109, 114 Ras-MAPK pathway 75, 94, 110, 128, 151 RAS function 100 signaling 21, 62 structure 100 Rhabdomyosarcoma 85, 119 SH2 domain 140 short stature 104 SHP2 105, 139 Signal transduction 76 Somatic mosaicism 99 Sorafenib 156 SOS1 14, 31, 46 Tipifarnib 156 Treatment 80, 115, 151 Watson syndrome 128 Zebrafish model 143

Subject Index

167