martin zenker-noonan syndrome and related disorders - a matter of deregulated ras signaling...

Noonan Syndrome and Related DisordersA Matter of Deregulated Ras Signaling

Monographs in Human Genetics

Vol. 17

Series Editor

Michael Schmid Würzburg

Noonan Syndrome andRelated Disorders – A Matter of DeregulatedRas 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 ZenkerInstitute of Human GeneticsUniversity Hospital ErlangenUniversity of Erlangen–NurembergSchwabachanlage 10D–91054 Erlangen

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© Copyright 2009 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland)www.karger.comPrinted in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, BaselISSN 0077–0876ISBN 978–3–8055–8653–5e-ISBN 978–3–8055–8654–2

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

Contents

VII EditorialSchmid, M. (Würzburg)

IX PrefaceZenker, M. (Erlangen)

1 History of Noonan Syndrome and Related DisordersNoonan, J.A. (Lexington, Ky.)

9 The Clinical Phenotype of Noonan SyndromeAllanson, J.E. (Ottawa)

20 Molecular Genetics of Noonan SyndromeTartaglia, M. (Rome); Gelb, B.D. (New York, N.Y.)

40 Genotype-Phenotype Correlations in Noonan SyndromeSarkozy, A.; Digilio, M.C.; Marino, B.; Dallapiccola, B. (Rome)

55 LEOPARD Syndrome: Clinical Aspects and Molecular PathogenesisSarkozy, A.; Digilio, M.C.; Zampino, G.; Dallapiccola, B.; Tartaglia, M. (Rome); Gelb, B.D. (New York, N.Y.)

66 The Clinical Phenotype of Cardiofaciocutaneous Syndrome (CFC)Roberts, A.E. (Boston, Mass.)

73 Molecular Causes of the Cardio-Facio-Cutaneous SyndromeTidyman, W.E.; Rauen, K.A. (San Francisco, Calif.)

83 The Clinical Phenotype of Costello SyndromeKerr, B. (Manchester)

94 The Molecular Basis of Costello SyndromeSol-Church, K.; Gripp, K.W. (Wilmington, Del.)

104 Endocrine Regulation of Growth and Short Stature in Noonan SyndromeBinder, G. (Tübingen)

109 The Heart in Ras-MAPK Pathway DisordersDigilio, M.C.; Marino, B.; Sarkozy, A.; Versacci, P.; Dallapiccola, B. (Rome)

VI Contents

119 Myeloproliferative Disease and Cancer in Persons with NoonanSyndrome and Related DisordersKratz, C. (Freiburg)

128 Neurofibromatosis Type 1-Noonan Syndrome: What’s the Link?Denayer, E.; Legius, E. (Leuven)

138 Animal Models for Noonan Syndrome and Related DisordersAraki, T.; Neel, B.G. (Toronto, Ont.)

151 Towards a Treatment for RAS-MAPK Pathway DisordersJoshi, V.A.; Roberts, A.E.; Kucherlapati, R. (Boston, Mass.)

165 Author Index

166 Subject Index

This volume 17 of Monographs in Human Genetics is an in-depth discourse on the disorders of theRas-MAPK pathway (Noonan-, cardio-facio-cutaneous-, Costello-, and LEOPARD syndromes). Likethe two preceding volumes of this book series, it deals with important hereditary diseases with highclinical impact, and whose molecular causes have been unravelled in recent years. Noonan syndromebelongs to one of the most frequent monogenic disorders occurring in approximately one in 1,000 to2,500 children and therefore has significant importance in public health genomics. Molecular analyseshave led to the surprising result that all four syndromes can be traced back to specific mutations ingenes coding for molecules that interact in the Ras-MAPK pathway. This exciting discovery does notonly permit the precise diagnosis of the diseases, but also clears promising ways for potential therapiesin the future.

Martin Zenker, the Editor of the present volume, succeeded in bringing together the leading expertsworking on these diseases and received their contributions in a very short space of time. The articlestreat both the clinical and molecular data exhaustively and give the reader a very timely update and out-line of these related disorders. I thank Martin Zenker and all the authors for their time and effort to ren-der possible the publication of this book. Furthermore, I gratefully acknowledge the constantpromotion of this book series by Thomas Karger.

Michael SchmidWürzburg, August 2008

Editorial

IX

Noonan syndrome (NS), which is recognized as one of the most common monogenic disorders, wasdefined as a separate entity by Jacqueline Noonan in 1968. Thirty-three years later, the first gene forNS was identified by Marco Tartaglia and colleagues. Their discovery represented the spark for a seriesof new gene discoveries eventually showing that mutations that alter the function of molecules inter-acting in a common signalling cascade, the Ras-MAPK pathway, are responsible for NS and the clini-cally related disorders cardio-facio-cutaneous syndrome (CFCS), LEOPARD syndrome (LS), andCostello syndrome (CS). Together, these findings unexpectedly related this group of disorders to a sig-nalling pathway which was previously known for its involvement in tumorigenesis. Thereby, the asso-ciation of certain types of malignancies and tumor-like lesions with NS, LS, CFCS, and particularly CShas been elucidated. Vice versa, studies on the significance of somatic mutations in the same genes insporadic tumors have been stimulated and yielded exciting new findings. Notably, the genes mutatedin Neurofibromatosis 1 and a newly defined Neurofibromatosis 1-like phenotype encode negative reg-ulators of the same pathway. Thus, the known clinical relations between all these conditions havebecome intelligible through the achievements of molecular research.

The Editor of this volume of Monographs in Human Genetics greatly acknowledges the contribu-tions of excellent experts in the field. Their comprehensive reviews provide most updated data on thevarious clinical and molecular aspects of known disorders of the Ras-MAPK pathway. JacquelineNoonan herself is giving an historical overview in the first chapter. The book ends with a chapter oncurrent and possible future treatment options for this group of disorders. Together the contributionsto this volume nicely show the close relationship between clinical issues and molecular research andthe mutual benefit for people working in either of these fields. It is of note that the previously estab-lished clinical entities are strongly correlated with certain mutated genes or – in the case of LS – spe-cific functional consequences of certain mutations. The proposed term neuro-cardio-facial-cutaneoussyndromes for all disorders caused by germline mutations in components of the Ras-MAPK pathwaymay 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 muchmore than a progress report. The chase for genes for NS and related disorders seems to have reached a

Preface

X Preface

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% ofpatients with NS, 90% of patients with CFCS, and virtually all cases with CS. Future research will reachout for new goals by focusing on the refinement of genotype-phenotype correlations by studying largercohorts, as well as on the development of model systems to explore the precise molecular pathogenesis ofdysregulated Ras-MAPK signaling. One of the most fascinating prospects may be the possibility toinvent treatment options for NS and related disorders by pharmacological modulation of Ras-MAPK sig-naling. Concerted international efforts will be required to reach these goals.

Martin ZenkerErlangen, August 2008

Zenker M (ed): Noonan Syndrome and Related Disorders.

Monogr Hum Genet. Basel, Karger, 2009, vol 17, pp 1–8

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

AbstractDeregulation of the RAS pathway by some recently discov-

ered 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 Noo-

nan syndrome and related disorders will be reviewed, the

genes for these syndromes discussed and possible treat-

ment 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, specu-

lation as to the possible cause of the distinctive and simi-

lar facial phenotypes seen in infancy in these syndromes

will be discussed. When Noonan syndrome (NS) was de-

scribed, 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 demon-

strated that deregulation of the RAS pathway could cause

a variety of congenital malformations. This important dis-

covery 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, car-

dio-facio-cutaneous, Costello and Neurofibromatosis-NS.

In addition, speculation as to the possible cause of the dis-

tinctive 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 chil-

dren with congenital heart disease and described

nine patients who shared distinctive facial fea-

tures which included hypertelorism, downslant-

ing 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 un-

descended 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 condi-

tion 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) report-

ed a patient with a webbed neck as well as short

2 Noonan

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 Ullrich’s

but, in addition to short stature, had sexual in-

fantilism. Before Turner syndrome was shown

to be a sex chromosome abnormality, Flavell [8]

(1943) introduced the term ‘male Turner syn-

drome’. 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 syn-

drome 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 lit-

erature and reported five additional cases of his

own. These early reports were mainly by endo-

crinologists who used this term for patients with

a variety of testicular problems with or without

short stature. A vigorous attempt to find a chro-

mosomal abnormality in the ‘male Turner syn-

drome’ 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 obser-

vation that the phenotype in NS changes signifi-

cantly 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 un-

common 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 mi-

croscopic findings are similar to those seen in non-

syndromic 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 lymphangio-

grams showing lymphatic dysplasia. Lymphatic

abnormalities are reported in less than 20% of pa-

tients 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 man-

age 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 defi-

ciency 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

History of Noonan Syndrome and Related Disorders 3

disorders, including juvenile myelomonocytic

leukemia was reported in NS. In 1992 Sharland

et al. [18] reported on a large clinical study of pa-

tients with NS and discussed the feeding prob-

lems 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, howev-

er, did not link suggesting more than one gene

was involved. The search for the mutant gene be-

gan 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 pa-

tients with NS. In the past three years, three ad-

ditional genes hav e been identified, KRAS, SOS1

and RAF1. It is likely that additional genes will

be found to represent the 25–30% still without a

known mutation. Mutations in the PTPN11 gene

have a very high incidence of congenital heart dis-

ease of at least 80%. Pulmonary stenosis is most

commonly found and there is a low incidence of

hypertrophic cardiomyopathy. The most recent-

ly identified gene, RAF1, has a high incidence

of hypertrophic cardiomyopathy. Patients with

SOS1 have some cutaneous findings similar to

Cardio-facio-cutaneous syndrome. Genotype-

phenotype correlations are discussed in a sepa-

rate chapter of this book.

LEOPARD Syndrome

LEOPARD syndrome is a rare autosomal domi-

nant disorder that shares many phenotypic fea-

tures 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 café-au-lait spots as well as deaf-

ness. 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 cardio-

myopathy, LEOPARD syndrome has a very high

incidence of hypertrophic cardiomyopathy and a

lower incidence of pulmonary stenosis.

These syndromes are very difficult to distin-

guish 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 confirm-

ing the diagnosis of LEOPARD syndrome. They

all had facies similar to NS although in some the

findings were quite mild. Hypertrophic cardio-

myopathy was present in seven of the eight in-

fants and pulmonary stenosis in two of the eight.

A single patient without hypertrophic cardiomy-

opathy had a dysplastic mitral valve. Although

only one patient had lentigines, six of the eight did

have café-au-lait spots. This suggested to Digilio

that café-au-lait spots in early infancy in a pa-

tient 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 neurofibroma-

tosis 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 anoth-

er example where the overlap between neurofi-

bromatosis and NS exists.

Cardio-Facio-Cutaneous Syndrome (CFC)

CFC is a multiple anomaly syndrome with signif-

icant mental retardation. It occurs sporadically

and is characterized by failure-to-thrive, mac-

rocephaly, a distinctive face similar to NS with

4 Noonan

bitemporal constriction, hypertelorism, down-

ward slanting palpebral fissures, depressed nasal

root and low set ears. There is usually significant

cutaneous involvement consisting of dry hyperk-

eratotic scaly skin, sparse or absent eyebrows and

sparse or absent eyebrows and sparse slow grow-

ing 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 consid-

erable controversy in the literature. Many ques-

tioned 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 trian-

gular shape seen in NS. Cutaneous manifesta-

tions 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 investi-

gators 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 ap-

pears to be BRAF followed by MEK1 and MEK2

and occasional patients with a KRAS mutation.

Like NS, cardiovascular malformations are fre-

quent. About 75% [25] have some kind of a car-

diac 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 prob-

lems, 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 pa-

tients who displayed the phenotype described by

Costello but they were unaware of Costello’s re-

port. DerKaloustian et al. [28], first used the term

Costello syndrome in 1991. In 1992, Johnson et

al. [29] reported eight patients with Costello syn-

drome and reviewed 29 cases that had been pre-

viously reported under a variety of titles who

undoubtedly also had Costello syndrome.

These patients have a distinctive facial appear-

ance 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, particular-

ly bladder carcinoma and rhabdomyosarcoma.

Costello syndrome is characterized by polyhy-

dramnios, overgrowth and edema with post-

natal feeding difficulties and failure-to-thrive.

Characteristic facial features include macroceph-

aly, a high forehead, usually curly hair, hyperte-

lorism, 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 pro-

tein. In addition, they found fibroblast cultures

from Costello syndrome patients showed an in-

creased rate of proliferation. They postulated that

disturbed elastogenesis could explain the inter-

esting skin findings and that increased prolifera-

tion of fibroblasts in tissue culture might explain


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) pro-

posed 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 avail-

able from patients who had been clinically diag-

nosed with Costello syndrome at the 2003 and

2005 International Costello Syndrome Meeting

and through the Costello Syndrome Family net-

work. 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 phe-

notypic overlap between these disorders. It is now

felt that the term Costello syndrome should be lim-

ited to those individuals who do have a mutation

of the HRAS gene. Similar to NS and CFC, cardio-

vascular 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 ar-

rhythmias are relatively frequent in Costello syn-

drome, particularly in infancy.

Neurofibromatosis-Noonan Syndrome (NF-NS)

Since the mid 1980s, a number of investigators

have written about the presence of Noonan phe-

notype associated with some patients with neu-

rofibromatosis. Neurofibromatosis Type 1 (NF1)

is an autosomal dominant disorder characterized

by hamartomas in multiple organs. Mutations

or deletions in the neurofibromin-1 gene (NF1)

have been recognized as the cause of neurofibro-

matosis 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 be-

tween both syndromes.

So far, the etiology of NF-NS is unclear. There

has been one report [34] of a patient showing fea-

tures of both syndromes who was found to have

two mutations, a PTPN11 mutation which was in-

herited from the father and a de novo NF1 mu-

tation. This is the first and only report so far of

molecular occurrence of both disorders in the

same patient. Huffmeier et al. [35] recently re-

ported seven patients from five unrelated fami-

lies 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 rep-

resent 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 cardiomy-

opathy 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.

Discussion

Bentires-Alj et al. [36] suggest that the pheno-

type overlap between NS, NF1 and the other re-

lated syndromes reflects a similar underlying

6 Noonan

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 not-

ed by fetal ultrasound. It is tempting to blame the

cystic hygroma as a likely cause of the facial phe-

notype. 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 30–40

NS would be expected. Unlike Bekker et al. [38]

who found cervical cystic hygroma in midtri-

mester to be a reliable sign for in utero diagnosis

of NS Achiron noted none of his cases had evi-

dence of septated cystic hygroma and only one

of the fetuses had transient nuchal translucency.

This observation indicates lymphatic abnormali-

ties 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 translucen-

cy in utero it is necessary to propose another ex-

planation for the typical facial phenotype.

Some infants with NS and related disorders are

clearly recognized as dysmorphic at birth. The four

fetuses in Achiron’s report all developed bilateral

hydrothorax and generalized edema. All had typi-

cal 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 deliv-

ered 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 possi-

ble that the facial phenotype becomes more typi-

cal 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 com-

mon facial phenotype but it is still unknown. On

the other hand it is also clear that the RAS path-

way must play a role in early lymphatic develop-

ment. Lymphatic problems are well recognized in

NS. Chylous thorax may be present at birth or ap-

pear 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 adult-

hood. Lymphedema may present for the first time

in adulthood. Bloomfield et al. [39] reported the

first neonatal case with lymphangiography. The in-

fant was born at 33 weeks with severe edema and bi-

lateral 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 de-

velopment. Unfortunately the infant had two epi-

sodes 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 drain-

ing directly into the chest cavity. There is need for

further study of the role of RAS in lymphatic devel-

opment. While lymphatic abnormalities are clini-

cally 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, dem-

onstrating that this pathway plays an important

role in development. Once we understand exactly


References

Noonan JA: Hypertelorism with Turn- 1 er phenotype. Am J Dis Child 1968;116:373–380.Noonan JA, Ehmke DA: Associated 2 noncardiac malformations in children with congenital heart disease. J Pediatr 1963;31:150–153.Opitz JM: Editorial comment: the Noo- 3 nan syndrome. Am J Med Genet 1985;21:515–518.Kobylinski O: Über eine f lughautähn- 4 liche Ausbreitung am Halse. Arch An-thropol 1883;14:342–348.Funke O: Pterygium colli. Dtsch 5 Zeitschr Chir 1902;63:163–167.

Ullrich O: Über typische Kombinati- 6 onsbilder multipler Abartungen. Z Kinderheilkd 1930;49:271–276.Turner HH: A syndrome of infantil- 7 ism, congenital webbed neck, and cu-bitus valgus. Endocrinology 1938;25:566–574.Flavell G: Webbing of the neck with 8 Turner’s syndrome in the male. Br J Surg 1943;31:150–153.Ullrich O: Turner’s syndrome and sta- 9 tus Bonnevie-Ullrich; synthesis of ani-mal phenogenetics and clinical obser-vations on a typical complex of developmental anomalies. Am J Hum Genet 1949;1:179–202.

Heller RH: The Turner phenotype in 10 the male. J Pediatr 1965;66:48–63.Allanson JE, Hall JG, Hughes M: Noo-11 nan syndrome: the changing pheno-type. Am J Med Genet 1985;21: 507–514.Ehlers KH, Engle MA, Levin AR, Deely 12 WJ: Eccentric ventricular hypertrophy in familial and sporadic instances of 46, XX, XY Turner phenotype. Circula-tion 1972;45:639–652.Hirsch HD, Gelband H, Garcia O, Got-13 tlieb S, Tamer DM: Rapidly progressive obstructive cardiomyopathy in infants with Noonan’s syndrome. Circulation 1975;52:1161–1165.

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 hyper-

trophic cardiomyopathy, to name a few. Already

basic scientists are making important progress

in understanding the effect of specific muta-

tions 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 hy-

perproliferation in valve primordia. Nakamura

et al. [41] then generated a transgenic Q79R-Shp2

mouse model which showed again the role of en-

hanced 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 astro-

cyte 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

difficult. Registries to follow these patients will

be important. In countries with established reg-

istries, 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 path-

way 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 regis-

tries, 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 at-

tend with their affected children. At these meet-

ings, information about the syndrome is shared

with families and the physicians attending al-

ways learn much from the families. The children

are able to interact with affected peers which pro-

vides 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 follow-

up 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.

8 Noonan

Burch M, Mann JM, Sharland M, 14 Shinebourne EA, Patton MA, McKen-na WJ: Myocardial disarray in Noonan syndrome. Br Heart J 1992;68: 580–585.Vallet HL, Holtzapple PG, Eberlein 15 WR, Yakovac WC, Moshang T Jr, Bon-giovanni AM: Noonan syndrome with intestinal lymphangiectasia. J Pediatr 1972;80:269–274.Baltaxe HA, Lee JG, Ehlers KH, Engle 16 MA: Pulmonary lymphangiectasia in 2 patients with Noonan syndrome. Radi-ology 1975;155:149–153.Kitchens CS, Alexander JA: Partial 17 deficiency of coagulation factor XI as a newly recognized feature of Noonan syndrome. J Pediatr 1983;102:224–227.Sharland M, Burch M, McKenna WM, 18 Patton MA: A clinical study of Noonan syndrome. Arch Dis Child 1992;67: 178–183.Jamieson CR, van der Burgt I, Brady 19 AF, van Reen M, Elsawi MM, et al: Mapping a gene for Noonan syndrome to the long arm of chromosome 12. Nat Genet 1994;8:357–360.Tartaglia M, Kalidas K, Shaw A, Song 20 X, Musat DL, et al: PTPN11 mutations in Noonan syndrome: molecular spec-trum, genotype-phenotype correlation and phenotypic heterogeneity. Am J Hum Genet 2002;70:1555–1563.Gorlin RJ, Anderson RC, Blaw M: Mul-21 tiple lentigenes syndrome. Am J Dis Child 1969;17:652–662.Digilio MC, Sarkozy A, de Zorzi A, 22 Pacileo G, Limongelli G, et al: LEOP-ARD Syndrome: clinical diagnosis in the first year of life. Am J Med Genet A 2006;140:740–746.Reynolds JF, Neri G, Herrmann JP, 23 Blumberg B, Coldwell JG, Miles PV, Opitz JM: New multiple congenital anomalies/mental retardation syn-drome with cardio-facio-cutaneous involvement – the CFC syndrome. Am J Med Genet 1986;25:413–427.Kavamura MI, Peres CA, Alchorne 24 MM, Brunoni D: CFC index for the diagnosis of cardiofaciocutaneous syn-drome. Am J Med Genet 2002;112:12–16.

Roberts A, Allanson J, Jadico SK, Ka-25 vamura MI, Noonan J, et al: The car-diofaciocutaneous syndrome. J Med Genet 2006;43:833–842.Costello JM: A new syndrome. NZ Med 26 J 1971;74:397.Costello JM: A new syndrome: mental 27 subnormality and nasal papillomata. Aust Paediatr J 1977;13:114–118.Der Kaloustian VM, Moroz B, McIn-28 tosh N, Watters AK, Blaichman S: Cos-tello syndrome. Am J Med Genet 1991; 41:69–73.Johnson JP, Fried MH, Norton ME, 29 Rosenblatt R, Feldman G, Yang S: Pre-natal overgrowth with postnatal growth failure, dysmorphic facies, cu-taneous features, and cardiomyopathy: overlap of AMICABLE, facio-cutane-ous-skeletal (fcs) and Costello (cs) syn-dromes. Proc Greenwood Genet Cent 1992;12:98.Lurie JW: Genetics of the Costello syn-30 drome. Am J Med Genet 1994;52:358–359.Hinek A, Smith AC, Cutiongco EM, 31 Callahan JW, Gripp KW, Weksberg R: Decreased elastin deposition and high proliferation of fibroblasts from Cos-tello syndrome are related to function-al deficiency in the 67-kD elastin-binding protein. Am J Hum Genet 2000;66:859–872.Aoki Y, Niihori T, Kawame H, Kuro-32 sawa K, Ohashi H, et al: Germline mu-tations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 2005;37:1038–1040.Colley A, Donnai D, Evans DG: Neuro-33 fibromatosis/Noonan phenotype: a variable feature of type 1 neurofibro-matosis. Clin Genet 1996;49:59–64.Bertola DR, Pereira AC, Passetti F, de 34 Oliveira PS, Messiaen L, et al: Neurofi-bromatosis-Noonan syndrome: molec-ular evidence of the concurrence of both disorders in a patient. Am J Med Genet A 2005;136:242–245.

Huffmeier U, Zenker M, Hoyer J, Fah-35 sold R, Rauch A: A variable combina-tion of features of Noonan syndrome and neurofibromatosis type I are caused by mutations in the NF1 gene. Am J Med Genet A 2006;140: 2749–2756.Bentires-Alj M, Kontaridis MI, Neel 36 BG: Stops along the RAS pathway in human genetic disease. Nat Med 2006;12:283–285.Achiron R, Heggesh J, Grisaru D, Gold-37 man B, Lipitz S, Yagel S, Frydman M: Noonan syndrome: a cryptic condition in early gestation. Am J Med Genet 2000;92:159–165.Bekker MN, Go AT, van Vugt JM: Per-38 sistence of nuchal edema and distend-ed jugular lymphatic sacs in Noonan syndrome. Fetal Diagn Ther 2007;22:245–248.Bloomfield FH, Hadden W, Gunn TR: 39 Lymphatic dysplasia in a neonate with Noonan’s syndrome. Pediatr Radiol 1997;27:321–323.Krenz M, Yutzey KE, Robbins J: Noo-40 nan syndrome mutation Q79R in Shp2 increases proliferation of valve pri-mordia mesenchymal cells via extra-cellular signal-regulated kinase 1/2 signaling. Circ Res 2005;97:813–820.Nakamura T, Colbert M, Krenz M, 41 Molkentin JD, Hahn HS, Dorn GW 2nd, Robbins J: Mediating ERK 1/2 signal-ing rescues congenital heart defects in a mouse model of Noonan syndrome. J Clin Invest 2007;117:2123–2132.Gauthier AS, Furstoss O, Araki T, 42 Chan R, Neel BG, Kaplan DR, Miller FD: Control of CNS cell-fate decisions by Shp2 and its dysregulation in Noo-nan syndrome. Neuron 2007;54:245–262.

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 [email protected]



The Clinical Phenotype of Noonan Syndrome

J.E. Allanson

Children’s Hospital of Eastern Ontario, Ottawa, Canada

AbstractNoonan syndrome is an autosomal dominant condition

notable both for its common occurrence and phenotypic

variability. It is characterized by short stature, congenital car-

diac defects, unusual chest shape, broad or webbed neck,

cryptorchidism, typical facial appearance and developmen-

tal delay of variable extent. It is frequently overlooked in the

mildly affected individual and diagnosis in an adult often fol-

lows the birth of a child with more florid manifestations.


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 handi-

cap. 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 di-

agnosis is 9 years [4]. Life expectancy is likely

to be normal in the absence of serious cardiac

defects. In one natural history study, non-acci-

dental mortality was 7%, with half of all deaths

occurring in adulthood. Cause of adult death

included hypertrophic cardiomyopathy, isch-

emic heart disease, breast cancer, and cerebral

hemorrhage [5]. There are several excellent re-

views [1, 3–9].

Craniofacial Features

Facial appearance changes with age (fig. 1) [4,

10]. In the newborn, key features include tall

forehead, widespaced and down-slanting palpe-

bral fissures, ptosis or thickened eyelids, epican-

thal folds, depressed nasal root with upturned

nasal tip, deeply grooved philtrum with high,

wide peaks of the vermilion border (so-called

cupid’s bow shape), low-set and posteriorly an-

gulated ears with thick helices, small chin, and

excessive nuchal skin with a low posterior hair-

line. During infancy, the head is relatively large

in comparison to face size, with a tall and prom-

inent 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

10 Allanson

pointed chin. In adolescence and young adult-

hood, the nose has a thin, prominent bridge and

a wide base. The neck is longer with accentuated

webbing (pterygium colli) or a prominent trape-

zius. In older adults, nasolabial folds are exagger-

ated and the skin appears thin and transparent

[6, 10, 11]. The hair may be wispy or sparse dur-

ing infancy and curly or woolly in older child-

hood and adolescence.

Despite this subjective impression of age-re-

lated facial change, detailed measurements dem-

onstrate the opposite. There is a Noonan-specific

pattern of craniofacial widths, lengths, depths

and arcs that is maintained over time. This is su-

perimposed 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 dia-

mond-shaped eyebrows, and low-set posterior-

ly 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 be-

cause many published reports come from ter-

tiary and quaternary medical centres which

place emphasis on serious structural manifes-

tations, 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, 13–15]. It may be isolated or associated

with other defects. Other common structural

cardiac anomalies include atrial or ventricu-

lar septal defects and tetralogy of Fallot. Many

other cardiac defects have been reported less

commonly, including atrioventricular septal

defect, aortic stenosis or dysplasia, coarcta-

tion of the aorta [16–18], bicuspid aortic valve,

double chambered right ventricle, mitral valve

anomalies [19], Ebstein anomaly, total anoma-

lous pulmonary venous return, supravalvular

pulmonary stenosis, coronary artery dilata-

tion, coronary artery fibromuscular dysplasia

causing ischemia [20], and giant aneurysms of

the sinuses of Valsalva caused by deficiency of

medial elastin [21].

a b c

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

child and (c) young adult.

The Clinical Phenotype of Noonan Syndrome 11

Hypertrophic cardiomyopathy, both ob-

structive 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 histo-

logically, echocardiographically and clini-

cally indistinguishable from non-syndromic

hypertrophic cardiomyopathy, except that ar-

rhythmia and sudden death appear to be less

common. Nonetheless, mortality appears to

be higher in the children with Noonan syn-

drome and hypertrophic cardiomyopathy,

with progression to cardiac failure in 25% [5].

Restrictive cardiomyopathy and dilated cardi-

omyopathy 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 hypertro-

phy. 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 struc-

turally normal heart. In older individuals, ar-

rhythmia and congestive cardiac failure may

be more common than previously suspected

[27].

Growth and Feeding

Birth weight is usually normal but may be in-

creased due to subcutaneous edema. In this situ-

ation there is rapid loss of weight in the neonatal

period. Feeding difficulties occur in 77% of in-

fants [4, 5, 28], and may be mild (15%), character-

ized by poor suck, or severe (38%), requiring tube

feeding [5]. They are usually related to hypotonia

and poor coordination of oral musculature, how-

ever immature gut motility and delayed gastroin-

testinal 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

gro wth 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 152.7–153.3 cm [5,

29]. Noonan syndrome growth curves are pub-

lished [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 respon-

siveness to growth hormone [31]. One study

suggested that children with more f lorid fa-

cial, thoracic and cardiac features of Noonan

syndrome had higher peak growth hormone

levels [32]. These children did not seem to dif-

fer in pre- or post-growth hormone treatment

height when compared to children with a mild-

er 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

[34–38]. 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 fi-

nal 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 muta-

tions [33, 41, 42]. The inferior response to growth

hormone, greater likelihood of short stature, and

12 Allanson

higher (compensatory) growth hormone levels

in individuals with a PTPN11 mutation may be

explained by the fact that SHP2 normally down-

regulates growth hormone receptor signaling.

Gain-of-function mutations in PTPN11 will en-

hance this effect [33, 43].

Development and Behaviour

Early developmental milestones are often de-

layed, with average age of sitting at 10 months,

first unsupported walking at 21 months and sim-

ple two-word phrases at 31 months. Joint laxity

and hypotonia clearly contribute to the motor de-

lay. Most children will do well in a normal school

setting but 10–40% will require additional help

[4]. A large cohort of affected individuals, fol-

lowed for many years, has demonstrated a strong

association between significant feeding difficul-

ties in infancy and intellectual handicap requir-

ing 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 prob-

lems were noted in about half the children. Other

publications report learning disability with spe-

cific visual-constructional problems and verbal-

performance 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 sug-

gested an increased likelihood of stubbornness and

mood disorders [49]. Another found a majority of

a group of 26 individuals to be impulsive, hyperac-

tive 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 cog-

nition in terms of emotion recognition and alexi-

thymia. 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 com-

mon 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 nys-

tagmus in about 10%. Anterior segment changes

(prominent corneal nerves, anterior stromal dys-

trophy, cataracts, and panuveitis) are frequently

found, while coloboma [52, 53], retinitis pigmen-

tosa [54], congenital fibrosis of extraocular mus-

cles [55] and spontaneous corneal rupture [53, 56]

are rare associations. Optic nerve hypoplasia is

occasionally reported, in contrast to cardiofacio-

cutaneous syndrome, which shares many char-

acteristics 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 re-

current otitis media, however sensorineural and


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 ap-

pears long; with low-set nipples and axillary web-

bing. 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 anoma-

lies, and radio-ulnar synostosis. Joint hyper-ex-

tensibility occurs in 30% [4, 5]. The association

between Noonan syndrome and malignant hyper-

thermia is poorly understood. Malignant hyper-

thermia has been linked to a Noonan phenotype

and designated as King syndrome [60–64]. The

possibility of malignant hyperthermia is of great-

er concern in individuals with significant muscu-

lar pathology or elevated creatine kinase.

Giant cell lesions of the jaws identical to those

found in cherubism are described [65–71]. This

combination has been called Noonan-like/mul-

tiple giant-cell lesion syndrome. Cherubism may

occur as an isolated autosomal dominant disor-

der caused by mutations in SH3BP2 [72], or as

part of neurofibromatosis. In Ramon syndrome

cherubism is associated with juvenile rheuma-

toid arthritis (polyarticular pigmented villonod-

ular synovitis). Giant cell granulomas, and bone

and joint anomalies that include polyarticular

pigmented villonodular synovitis, are now rec-

ognized 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

is histologically identical to the consequences of

peri-articular bleeding caused by hemophilia (K.

Reinker, personal communication). This is in-

triguing given the bleeding diathesis that can ac-

company Noonan syndrome.

Central Nervous System

Seizures of varied types are found in 10% of in-

dividuals, with mean age of onset of 11 years [5].

Structural anomalies of the central nervous sys-

tem are unusual. Hydrocephalus is reported in

about 5% [75–78]. Communicating hydrocepha-

lus is generally described and Clericuzio and col-

leagues hypothesize that this may be related to

extra-cranial lymphatic dysplasia [75]. They refer

to studies documenting the drainage of cerebro-

spinal 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 menin-

gocele [82]. Cerebrovascular anomalies have been

described in a few individuals [83–88].

Fig. 2. The chest phenotype showing wide-spaced and

low-set nipples, pectus deformity with pectus carina-

tum superiorly and pectus excavatum inferiorly, and

rounded shoulders.

14 Allanson

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, puber-

tal development varies from normal virilization

with subsequent fertility, to delayed but normal

pubertal development, to inadequate sexual de-

velopment 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].

Gastrointestinal System

Both splenomegaly (50%) and hepatomegaly

(25%) are said to be common, although in this

author’s experience these figures seem high. The

cause is unknown, but one might suspect an as-

sociation with congestive heart failure or myelo-

dysplasia on occasion. Rarely reported anomalies

include choledochal cyst and midgut rotation

[89].

Skin

Various skin manifestations are seen in Noonan

syndrome including café-au-lait spots, pigment-

ed 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 lash-

es, 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 granu-

lar cell schwannomas are occasionally reported

but are more common in LEOPARD syndrome

[95, 96]. Rare findings include xanthomas of the

skin and oral mucous membranes, leukokerato-

sis of the lips and gingiva, molluscoid scalp skin,

and vulvar angiokeratoma.

Lymphatics

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 abnormali-

ties include generalized lymphedema, pulmonary

lymphangiectasia, chylous effusions in pleural or

peritoneal spaces, intestinal or testicular lymph-

angiectasia, and localized lymphedema of scro-

tum 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 tho-

racic duct abnormality. Lymphatic aplasia, hypo-

plasia and megalymphatics are also described.

Hematology – Oncology

Several different coagulopathies may occur, ei-

ther alone or in combination [100, 101]. They

affect about one third of individuals, however,

many more will have a history of abnormal bleed-

ing or easy bruising. The range of manifestations

is broad, from severe surgical hemorrhage to a-

symptomatic laboratory abnormalities. There is

poor correlation between bleeding history and ac-

tual defect. Laboratory findings include factor XI

deficiency, factor XII deficiency, factor VIII de-

ficiency [100, 102–106], von Willebrand disease,

and platelet dysfunction, which may be associ-

ated with trimethylaminuria or acyclooxygenase

deficiency [101, 107]. Some factor deficiencies


References

Noonan JA, Ehmke DA: Associated 1 noncardiac malformations in children with congenital heart disease. J Pediatr 1963;63:468–470.Kobylinski O: Über eine f lughautähn- 2 liche Ausbreitung am Halse. Arch An-thropol 1883;14:342–348.Mendez HMM, Opitz JM: Noonan syn- 3 drome: A review. Am J Med Genet 1985;21:493–506.Sharland M, Burch M, McKenna WM, 4 Patton MA: A clinical study of Noonan syndrome. Arch Dis Child 1992;67: 178–183.

Shaw AC, Kalidas K, Crosby AH, Jef- 5 frey S, Patton MA: The natural history of Noonan syndrome: a long-term fol-low-up study. Arch Dis Child 2007;92;128–132.Allanson JE: Noonan syndrome. J Med 6 Genet 1987;24:9–13.Char FC, Rodriguez-Fernandez HL, 7 Scott CI, Borgankoar DS, Bell BB, Rowe RD: The Noonan-syndrome: A clinical study of forty-five cases. Birth Defects 1972;8:110–118.Noonan JA: Noonan syndrome: Up- 8 date and review for the primary pedia-trician. Clin Pediatr 1994;33:548–555.

Yoshida R, Hasegawa T, Hasegawa Y, 9 Nagai T, Kinoshita E, et al: Protein-tyrosine phosphatase, non-receptor type 11 mutation analysis and clinical assessment in 45 patients with Noonan syndrome. J Clin Endocrinol Metab 2004;89:3359–3364.Allanson JE: Time and natural history: 10 the changing face. J Craniofac Genet Dev Biol 1989;9:21–28.Allanson JE, Hall JG, Hughes HE, 11 Preus M, Witt RD: Noonan syndrome: the changing phenotype. Am J Med Genet 1985;21:507–514.Allanson JE: Noonan syndrome: the 12 changing face. Proc Greenwood Genet Ctr 2001;20:78–79.

seem to improve with age. There is no evidence of

hepatic dysfunction or vitamin K-dependent co-

agulation factor deficiency. Aspirin-containing

medications should be avoided.

Congenital bone marrow hypoplasia, con-

genital hypoplastic anemia, and pancytope-

nia have been reported rarely [108, 109]. A low

frequency association with myeloprolifera-

tive disorders (MPD) exists. These include, in

particular, juvenile myelomonocytic leukemia

(JMML), but, more rarely, acute lymphoblastic

leukemia [110–113], chronic myelomonocytic

leukemia [114, 115] and proliferation of eryth-

roid precursors [116]. JMML associated with

Noonan syndrome tends to have an earlier on-

set 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 syn-

drome and MPD, but is uncommon in individu-

als with Noonan syndrome without MPD [117,

118]. Somatic mutations in PTPN11 are a com-

mon 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 [120–126].

Immunological Findings

Autoimmune thyroiditis occurs in 5% of individ-

uals with Noonan syndrome [127–130]. Other au-

toimmune disorders, such as lupus, celiac disease,

vitiligo, anterior uveitis and vasculitis are described

infrequently [4, 130]. In addition, levels of anti-thy-

roglobulin and anti-microsomal thyroid antibodies

seem to be higher than in the general population

[130]. Antiphospholipid syndrome with Moyamoya-

like 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 cys-

tic hygroma [4, 133–136]. Lack of septation of the

cystic hygroma and regression prior to mid second

trimester are associated with more favorable prog-

nosis than those with later regression [134, 135].

Other ultrasonographic markers include scalp ede-

ma, 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.

16 Allanson

Noonan JA, O’Connor W: Noonan syn-13 drome: A clinical description empha-sizing the cardiac findings. Acta Pae-diatr Jpn 1996;38:76–83.Burch M, Sharland M, Shinebourne E, 14 Smith G, Patton M, McKenna W: Car-diologic abnormalities in Noonan syn-drome: Phenotypic diagnosis and echocardiographic assessment of 118 patients. J Am Coll Cardiol 1993;22:189–192.Digilio MC, Marino B, Giannotti A, 15 Dallapiccola B: Noonan syndrome with cardiac left-sided obstructive le-sions. Hum Genet 1997;99:289.Digilio MC, Marino B, Giannotti A, 16 Dallapiccolo B: Exclusion of 22q11 de-letion in Noonan syndrome with te-tralogy of Fallot. Am J Med Genet 1996;62:413–414.Digilio MC, Marino B, Picchio F, 17 Prandstraller D, Toscana A, et al: Noo-nan syndrome and aortic coarctation. Am J Med Genet 1998;80:160–162.Ishizawa A, Oho S-I, Dodo H, Katori T, 18 Homma S-I: Cardiovascular abnor-malities in Noonan syndrome: the clinical findings and treatments. Acta Paediatr Jpn 1996;38:84–90.Marino B, Digilio MC, Toscana A, Gi-19 annotti A, Dallapiccola B: Noonan syn-drome: structural abnormalities of the mitral valve causing subaortic ob-struction. Eur J Pediatr 1995;154: 949–952.Ishikawa Y, Sekiguchi K, Akasaka Y, 20 Ito K, Akishima Y, et al: Fibromuscular dysplasia of coronary arteries resulting in myocardial infarction associated with hypertrophic cardiomyopathy in Noonan’s syndrome. Hum Pathol 2003;34:282–284.Purnell R, Williams I, Von Oppell U, 21 Wood A: Giant aneurysms of the si-nuses of Valsalva and aortic regurgita-tion in a patient with Noonan’s syn-drome. Eur J Cardiothorac Surg 2005;28:346–348. Burch M, Mann JM, Sharland M, 22 Shinebourne EA, Patton MA, McKen-na WJ: Myocardial disarray in Noonan syndrome. Br Heart J 1992;68: 586–588.Nishikawa T, Ishiyama S, Shimojo T, 23 Takeda K, Kasjima T, Momma K: Hy-pertrophic cardiomyopathy in Noonan syndrome. Acta Paediatr Jpn 1996;38:91–98.

Shimizu A, Oku Y, Matsuo K, Hashiba 24 K: Hypertrophic cardiomyopathy pro-gressing to a dilated cardiomyopathy-like feature in Noonan’s syndrome. Am Heart J 1992;123:814–816.Wilmshurst PT, Katritsis D: Restric-25 tive and hypertrophic cardiomyopa-thies in Noonan syndrome: the overlap syndromes. Heart 1996;75:94–97.Yu CM, Chow LT, Sanderson JE: Dilat-26 ed cardiomyopathy in Noonan’s syn-drome. Int J Cardiol 1996;56:83–85.Noonan JA: Noonan syndrome; in 27 Goldstein S, Reynolds CR (eds): Hand-book of Neurodevelopmental and Ge-netic Disorders in Adults. New York, Guilford Press, 2005, pp 308–319.Shah N, Rodriguez M, St Louis D, 28 Lindley K, Milla PJ: Feeding difficul-ties and foregut dysmotility in Noonan syndrome. Arch Dis Child 1999; 81:28–31.Ranke MB, Heidemann P, Knupfer C, 29 Enders H, Schmaltz AA, Bierich JR: Noonan syndrome: growth and clini-cal manifestations in 144 cases. Eur J Pediatr 1988;148:220–227.Witt DR, Keena B, Hall JG, Allanson 30 JE: Growth curves for height in Noo-nan’s syndrome. Clin Genet 1986;30:150–153.Noordam C, van der Burgt I, Sweep 31 CG, Delemarre-van de Waal HA, Sen-gers RC, Otten BJ: Growth hormone (GH) secretion in children with Noo-nan syndrome: frequently abnormal without consequences for growth or GH treatment. Clin Endocrinol 2001; 54:53–59.Noordam K, van der Burgt I, Brunner 32 HG, Otten BJ: The relationship be-tween clinical severity of Noonan’s syndrome and growth, growth hor-mone (GH) secretion and response to GH. J Pediatr Endocrinol Metab 2002;15:175–180.Binder G, Neuer K, Ranke MB, Witte-33 kindt NE: PTPN11 mutations are as-sociated with mild GH resistance in individuals with Noonan syndrome. J Clin Endocrinol Metab 2005;90: 5377–5381.Ahmed ML, Foot AB, Edge JA, Lamkin 34 VA, Savage MO, Dunger DB: Noonan’s syndrome: Abnormalities of the growth hormone IGF-1 axis and the response to treatment with human biosynthetic growth hormone. Acta Paediatr Scand 1991;80:446–450.

MacFarlane CE, Brown DC, Johnston 35 LB, Patton MA, Dunger DB, et al: Growth hormone therapy and growth in children with Noonan’s syndrome: Results of 3 years’ follow-up. J Clin Endocrinol Metab 2001;86:1953–1956.Raaijmakers R, Noordam C, 36 K’aragiannis G, Gregory JW, Hertel NT, Sipila I, Otten BJ: Response to growth hormone treatment and final height in Noonan syndrome in a large cohort of patients in the KIGS database. J Pedia-tr Endocrinol Metab 2008;21:267–273.Ogawa M, Moriya N, Ikeda H, Tanae A, 37 Tanaka T, et al: Clinical evaluation of recombinant human growth hormone in Noonan syndrome. Endocr J 2004;51:61–68.Thomas BC, Stanhope R: Long-term 38 treatment with growth hormone in Noonan’s syndrome. Acta Paediatr 1993;82:853–855.Kelnar CJH: Growth hormone therapy 39 in Noonan syndrome. Horm Res 2000;53(Suppl 1):77–81.Osio D, Dahlgren J, Wikland KA, 40 Westphal O: Improved final height with long-term growth hormone treat-ment in Noonan syndrome. Acta Pe-diatr 2005;94:1232–1237.Ferreira LV, Souza SA, Arnhold IJ, 41 Mendonca BB, Jorge AA: PTPN11 (protein tyrosine phosphatase nonre-ceptor type 11) mutations and response to growth hormone therapy in children with Noonan syndrome. J Clin Endo-crinol Metab 2005;90: 5156–5160.Limal J-M, Parfait B, Cabrol S, Bonnet 42 D, Leheup B, et al: Noonan syndrome: Relationships between genotype, growth, and growth factors. J Clin En-docrinol Metab 2005;91:300–306.Stofega MR, Herrington J, Billestrup 43 N, Carter-Su C: Mutation of the SHP-2 binding site in growth hormone (GH) receptor prolongs GH-promoted ty-rosyl phosphorylation of GH receptor, JAK2, and STAT5B. Mol Endocrinol 2000;14:1338–1350.Money J, Kalus ME: Noonan’s syn-44 drome: IQ and specific disabilities. Am J Dis Child 1979;133:846–850.Lee DA, Portnoy S, Hill P, Gillberg C, 45 Patton MA: Psychological profile of children with Noonan syndrome. Dev Med Child Neurol 2005;47:35–38.Cornish KM: Verbal-performance dis-46 crepancies in a family with Noonan syndrome. Am J Med Genet 1996;66:235–236.


Sarimski K: Developmental and be-47 havioural phenotype in Noonan syn-drome. Genet Couns 2000;11:383–390.van der Burgt I, Thoone G, Roosen-48 boom N, Assman-Hulsman C, Ga-breels F, et al: Patterns of cognitive functioning in school-aged children with Noonan syndrome associated with variability of phenotypic expres-sion. J Pediatr 1999;135:707–713. Wood A, Massarano A, Super M, Har-49 rington R: Behavioral aspects and psy-chiatric findings in Noonan’s syn-drome. Arch Dis Child 1995;72:153–155.Ghaziuddin M, Bolyard B, Alessi N: 50 Autistic disorder in Noonan syndrome. J Intellect Disabil Res 1994;38:67–72.Verhoeven W, Wingbermühle E, Egger J, 51 Van der Burgt I, Tuinier S: Noonan syndrome: psychological and psychiat-ric aspects. Am J Med Genet 2008;146A:191–196.Lee NB, Kelly L, Sharland M: Ocular 52 manifestations of Noonan syndrome. Eye 1992;6:328–334.Ascaso FJ, Del Buey MA, Huerva V, 53 Latre B, Palomar A: Noonan’s syn-drome with keratoconus and optic disc coloboma. Eur J Ophthalmol 1993;3:101–103.Schollen E, Matthijs G, Fryns JP: 54 PTPN11 mutation in a young man with Noonan syndrome and retinitis pigmentosa. Genet Couns 2003;14:259.Elgohary MA, Bradshaw P, Ahmad N: 55 Anterior uveitis and congenital fibro-sis of the extraocular muscles in a pa-tient with Noonan syndrome. J Post-grad Med 2005;51:319–321.Au YK, Collins WP, Patel JS, Asamoah 56 A: Spontaneous corneal rupture in Noonan syndrome. A case report. Ophthalmic Genet 1997;18:39–41.Cremers CWRJ, van der Burgt CJAM: 57 Hearing loss in Noonan syndrome. Int J Pediatr Otorhinol 1992;23:81–84.Qui WW, Yin SS, Stucker FJ: Audiolog-58 ic manifestations of Noonan syn-drome. Otolaryngol Head Neck Surg 1998;118:319–323.Naficy S, Shepard NT, Telian SA: Mul-59 tiple temporal bone anomalies associ-ated with Noonan syndrome. Otolar-yngol Head Neck Surg 1997;116: 265–267.Chitayat D, Hodgkinson KA, Ginsburg 60 O, Dimmick J, Watters GV: King syn-drome: a genetically heterogeneous phe-notype due to congenital myopathies. Am J Med Genet 1992;43:954–956.

Hunter A, Pinsky L: An evaluation of 61 the possible association of malignant hyperpyrexia with Noonan syndrome using serum creatine phosphokinase levels. J Pediatr 1975;96:412–415.King JO, Denborough MA: Anesthetic-62 induced malignant hyperpyrexia in children. J Pediatr 1973;83:37–40.Rissam HS, Mittal SR, Wahi PL, Bid-63 wai PS: Post-operative hyperpyrexia in a case of Noonan’s syndrome. Indian Heart J 1982;34:180–182.Steenson AJ, Torkelson RD: King’s syn-64 drome with malignant hyperthermia. Am J Dis Child 1987;141:271–273.Addante RR, Breen GH: Cherubism in 65 a patient with Noonan’s syndrome. J Oral Maxillofac Surg 1996;54:210–213.Betts NJ, Stewart JC, Fonseca RJ, Scott 66 RF: Multiple central giant cell lesions in a Noonan-like phenotype. Oral Surg Oral Med Oral Pathol 1993;76:601–607.Chuong R, Kaban LB, Kozakewich H, 67 Perez-Atayde A: Central giant cell le-sions of the jaws: a clinicopathologic study. J Oral Maxillofac Surg 1986;44:708–713.Dunlap C, Neville B, Vickers RA, 68 O’Neil D, Barker B: The Noonan syn-drome/cherubism association. Oral Surg Oral Med Oral Pathol 1989;67: 698–705.Hoyer PF, Neukam FW: Cherubismus 69 – eine osteofibröse Kiefererkrankung im Kindesalter. Klin Paediatr 1982;194:128–131.Sugar AW, Ezsias A, Bloom AL, Mor-70 cos WE: Orthognathic surgery in a patient with Noonan’s syndrome. J Oral Maxillofac Surg 1994;52:421–425.Weldon L, Cozzi G: Multiple giant cell 71 lesions of the jaws. J Oral Maxillofac Surg 1982;40:520–522.Mangion J, Rahman N, Edkins S, Bar-72 foot R, Nguyen T, et al: The gene for cherubism maps to chromosome 4p16.3. Am J Hum Genet 1999;65:151–157.Jafarov T, Ferimazova N, Reichenberg-73 er E: Noonan-like syndrome mutations in PTPN11 in patients diagnosed with cherubism. Clin Genet 2005;68: 190–191. Wolvius EB, de Lange J, Smeets EEJ, 74 van der Wal KGH, van den Akker HP: Noonan-like/multiple giant cell lesion syndrome: Report of a case and review of the literature. J Oral Maxillofac Surg 2006;64:1289–1292.

Clericuzio CL, Roberts A, Kucherlapati 75 RS, Tworog-Dube E, Allanson JE: Communicating hydrocephalus in Noonan syndrome: A consequence of lymphatic dysplasia? Proc Greenwood Gen Ctr 2008;27:81.Fryns JP: Progressive hydrocephalus in 76 Noonan syndrome. Clin Dysmorphol 1997;6:379.Henn W, Reichert H, Nienhaus Z, 77 Zankl M, Lindinger A, et al: Progres-sive hydrocephalus in two members of a family with autosomal dominant Noonan phenotype. Clin Dysmorphol 1997;6:153–156.Heye N, Dunne JW: Noonan’s syn-78 drome with hydrocephalus, hindbrain herniation, and upper cervical intrac-ord cyst. J Neurol Neurosurg Psychia-try 1995;59:338–339.Walter BA, Valera VA, Takahashi S, 79 Ushiki T: The olfactory route for cere-brospinal f luid drainage into the pe-ripheral nervous system. Neuropathol Appl Neurobiol 2006;32:388–396.Ball MJ, Peiris A: Chiari (type I) mal-80 formation and syringomyelia in a pa-tient with Noonan’s syndrome. J Neu-rol Neurosurg Psychiatry 1982; 45:753–754.Holder-Espinasse M, Winter RM: Type 81 1 Arnold-Chiari malformation and Noonan syndrome. A new diagnostic feature. Clin Dysmorphol 2003;12:275.Hughes HE, Hughes RM, Summers A, 82 Hochhauser L: Noonan syndrome and lateral meningoceles: another link with neurofibromatosis. Proc Green-wood Genet Ctr 1987;6:159.Hara T, Sasaki T, Miyauchi H, Takaku-83 ra K: Noonan phenotype associated with intracerebral hemorrhage and cerebral vascular anomalies: Case re-port. Surg Neurol 1993;39:31–36.Hinnant CA: Thromboembolic in-84 farcts occurring after mild traumatic brain injury in a paediatric patient with Noonan’s syndrome. Brain Injury 1994;8:719–727.Hinnant CA: Noonan syndrome asso-85 ciated with thromboembolic brain infarcts and posterior circulation ab-normalities. Am J Med Genet 1995;56:241–244.Robertson S, Tsang B, Aftimos S: Cere-86 bral infarction in Noonan syndrome. Am J Med Genet 1997;71:111–114.

18 Allanson

Schon F, Bowler J, Baraitser M: Cere-87 bral arteriovenous malformation in Noonan’s syndrome. Postgrad Med J 1992;68:37–40.Tanaka Y, Masuno M, Iwamoto H, 88 Aida N, Ijiri R, et al: Noonan syndrome and cavernous hemangioma of the brain. Am J Med Genet 1999;82:212–214.George CD, Patton MA, El Sawi M, 89 Sharland M, Adam EJ: Abdominal ul-trasound in Noonan syndrome: A study of 44 patients. Pediatr Radiol 1993;23:316–318.Elsawi MM, Pryor JP, Klufio G, Barnes 90 C, Patton MA: Genital tract function in men with Noonan syndrome. J Med Genet 1994;31:468–470.Daoud MS, Dahl PR, Su WP: Noonan 91 syndrome. Semin Dermatol 1995;14:140–144.Pierini DO, Pierini AM: Keratosis pi-92 laris atrophicans faciei (ulerythema ophryogenes): A cutaneous marker in the Noonan’s syndrome. Br J Dermatol 1979;100:409–416.Tartaglia M, Pennacchio LA, Zhao C, 93 Yadav KK, Fodale V, et al: Gain-of-function SOS1 mutations cause a dis-tinctive form of Noonan syndrome. Nat Genet 2007;39:75–79.Zenker M, Horn M, Wieczorek D, Al-94 lanson J, Pauli S, et al: SOS1 is the sec-ond most common Noonan gene but plays no major role in cardio-facio-cu-taneous (CFC) syndrome. J Med Genet 2007;44:651–656.Lohmann DR, Gillessen-Kaesbach G: 95 Multiple cutaneous granular cell tu-mours in a patient with Noonan syn-drome. Clin Dysmorphol 2001;19:301–302.Sahn EE, Dunlavey ES, Parsons JL: 96 Multiple cutaneous granular cell tu-mors in a child with possible neurofi-bromatosis. J Am Acad Dermatol 1997;36:327–330.Witt DR, Hoyme HE, Zonana J, Man-97 chester DK, Fryns JP, et al: Lymphede-ma in Noonan syndrome: Clues to pathogenesis and premature diagnosis and review of the literature. Am J Med Genet 1987;27:841–856.Bloomfield FH, Hadden W, Gunn TR: 98 Lymphatic dysplasia in a neonate with Noonan’s syndrome. Pediatr Radiol 1997;27:321–323.

Evans DG, Lonsdale RN, Patton MA: 99 Cutaneous lymphangioma and amega-karyocytic thrombocytopenia in Noo-nan syndrome. Clin Genet 1991;39:228–232.Sharland M, Patton MA, Talbot S, Chi-100 tolie A, Bevan DH: Coagulation-factor deficiencies and abnormal bleeding in Noonan’s syndrome. Lancet 1992;339:19–21.Witt DR, McGillivray BC, Allanson JE, 101 Hughes HE, Hathaway WE, et al: Bleeding diathesis in Noonan syn-drome: a common association. Am J Med Genet 1988;31:305–317.de Haan M, van der Kamp JJP, Briet E, 102 Dubbeldam J: Noonan syndrome: par-tial factor XI deficiency. Am J Med Genet 1988;29:277–282.Emmerich J, Aiach M, Capron L, 103 Fiessinger JN: Noonan’s syndrome and coagulation-factor deficiencies. Lancet 1992;339:431.Kitchens CS, Alexander JA: Partial 104 deficiency of coagulation factor XI as a newly recognized feature of Noonan syndrome. J Pediatr 1983;102:224–227.Massarano A, Wood A, Tait RC, Ste-105 vens R, Super M: Noonan syndrome: Coagulation and clinical aspects. Acta Paediatr 1996;85:1181–1185.Singer ST, Hurst D, Addiego JE Jr: 106 Bleeding disorders in Noonan syn-drome: three case reports and review of the literature. J Pediatr Hematol Oncol 1997;19:130–134.Humbert JR, Hammond KB, Hathaway 107 WE: Trimethylaminuria: the fish-odour syndrome. Lancet 1970;2:770–771.Feldman KW, Ochs HD, Price TH, 108 Wedgwood RJ: Congenital stem cell dysfunction associated with Turner-like phenotype. J Pediatr 1976;88:979–998.Sackey K, Sakati N, Aur RJA, Shebib S, 109 Sabbah RS, Rifai S: Multiple dysmor-phic features and pancytopenia: a new syndrome? Clin Genet 1985;27:606–610.Attard-Montalto SP, Kingston JE, Eden 110 T: Noonan’s syndrome and acute lym-phoblastic leukaemia. Med Pediatr On-col 1994;23:391–392.Johannes JM, Garcia ER, De Vaan GA, 111 Weening RS: Noonan’s syndrome in association with acute leukemia. Pe-diatr Hematol Oncol 1995;12:571–575.

Piombo M, Rosana C, Pasino M, 112 Marasini M, Cerruti P, Comelli A: Acute lymphoblastic leukemia in Noo-nan syndrome: report of two cases. Med Pediatr Oncol 1993;21:454–455.Roti G, La Starza R, Ballanti S, 113 Crescenzi B, Romoli S, et al: Acute lymphoblastic leukaemia in Noonan syndrome. Br J Haematol 2006;133:448–450.Bader-Meunier B, Tchernia G, Mielot 114 F, Fontaine JL, Thomas C, et al: Occur-rence of myeloproliferative disorder in patients with Noonan syndrome. J Pe-diatr 1997;130:885–889.Fukuda M, Horibe K, Miyajima Y, Mat-115 sumoto K, Nagashima M: Spontaneous remission of juvenile chronic myelo-monocytic leukemia in an infant with Noonan syndrome. J Pediatr Hematol Oncol 1997;19:177–178.Kratz CP, Nathrath M, Freisinger P, 116 Dressel P, Assmuss H-P, et al: Lethal proliferation of erythroid precursors in a neonate with a germline PTPN11 mutation. Eur J Pediatr 2006;165:182–185.Kratz CP, Niemeyer CM, Castleberry 117 RP, Cetin M, Bergstrasser E, et al: The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and Noonan syndrome. Blood 2005;15:2183–2185.Jongmans M, Sistemans EA, Rikken A, 118 Nillesen WM, Tamminga R, et al: Ge-notypic and phenotypic characteriza-tion of Noonan syndrome: New data and review of the literature. Am J Med Genet A 2005;134:165–170. Tartaglia M, Niemeyer CM, Fragale A, 119 Song X, Buechner J, et al: Somatic mu-tations in PTPN11 in juvenile myelo-monocytic leukemia, myelodysplastic syndromes and acute myeloid leuke-mia. Nat Genet 2003;34:148–150. Becker CE, Rosen SW, Engelman K: 120 Pheochromocytoma and hyporespon-siveness to thyrotropin in a 46,XY male with features of Turner pheno-type. Ann Intern Med 1969;70: 325–333.Cotton JL, Williams RG: Noonan syn-121 drome and neuroblastoma. Arch Pe-diatr Adolesc Med 1995;149:1280–1281.Jung A, Bechthold S, Pfluger T, Renner 122 C, Ehrt O: Orbital rhabdomyosarcoma in Noonan syndrome. J Pediatr Hema-tol Oncol 2003;25:330–332.


Kaplan MS, Opitz JM, Gosset FR: Noo-123 nan’s syndrome: A case with elevated serum alkaline phosphatase levels and malignant schwannoma of the left forearm. Am J Dis Child 1968;116:359–366.Khan S, McDowell H, Upadhyaya M, 124 Fryer A: Vaginal rhabdomyosarcoma in a patient with Noonan syndrome. J Med Genet 1995;32:743–745.Lopez-Miranda B, Westra SJ, Yazdani 125 S, Boechar MI: Noonan syndrome as-sociated with neuroblastoma: a case report. Pediatr Radiol 1997;27:324–326.Ijiri R, Tanaka Y, Keisuke K, Masuno 126 M, Imaizumi K: A case of Noonan’s syndrome with possible associated neuroblastoma. Pediatr Radiol 2000;30:432–433.Chaves-Carballo E, Hayles AB: Ullrich-127 Turner syndrome in the male: review of the literature and report of a case with lymphocytic (Hashimoto’s) thy-roiditis. Mayo Clin Proc 1966;41:843–854.

Vesterhus P, Aarskog D: Noonan’s syn-128 drome and autoimmune thyroiditis. J Pediatr 1973;83:237–240.Amoroso A, Garzia P, Vadacca M, Gal-129 luzzo S, Del Porto F, et al: The unusual association of three autoimmune dis-eases in a patient with Noonan syn-drome. J Adol Health 2003;32:94–97.Lopez-Rangel E, Malleson PN, Li-130 renman DS, Roa B, Wiszniewska J, Lewis ME: Systemic lupus erythemato-sus and other autoimmune disorders in children with Noonan syndrome. Am J Med Genet A 2006;139:239–242.Ganesan V, Kirkham FJ: Noonan syn-131 drome and Moyamoya. Pediatr Neurol 1997;16:256–258.Yamashita Y, Kusaga A, Koga Y, Nag-132 amitsu S-I, Matsuishi T: Noonan syn-drome, Moyamoya-like vascular changes, and antiphospholipid anti-bodies. Pediatr Neurol 2004;31: 364–366.

Achiron R, Heggesh J, Grisaru D, Gold-133 man B, Lipitz S, et al: Noonan syn-drome: A cryptic condition in early gestation. Am J Med Genet 2000;92:159–165.Benacerraf BR, Greene MF, Holmes 134 LB: The prenatal sonographic features of Noonan’s syndrome. J Ultrasound Med 1989;8:59–64.Donnenfeld A, Nazir MA, Sindoni F, 135 Librizzi RJ: Prenatal sonographic doc-umentation of cystic hygroma regres-sion in Noonan syndrome. Am J Med Genet 1991;39:461–465.Zarabi M, Mieckowski GC, Mazer J: 136 Cystic hygroma associated with Noo-nan’s syndrome. J Clin Ultrasound 1983;11:398–400.Bawle EV, Black V: Nonimmune hy-137 drops fetalis in Noonan’s syndrome. Am J Dis Child 1986;140:758–760.

Judith E. Allanson

Department of Genetics, Children’s Hospital of Eastern Ontario

401 Smyth Road

Ottawa, ON K1H 8L1 (Canada)




Molecular Genetics of Noonan Syndrome

M. Tartagliaa � B.D. Gelbb

aDipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanità, Rome, Italy; bCenter for Molecular Cardiology, Departments of Pediatrics and Genetics & Genomic Sciences,

Mount Sinai School of Medicine, New York, N.Y., USA

AbstractNoonan syndrome (NS) is a genetically heterogeneous

disorder that can result from mutations in the PTPN11, SOS1,

KRAS, RAF1 and MEK1 genes, which encode transducers par-

ticipating in the RAS-MAP kinase (MAPK) signaling path-

way. 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 phos-

phatase 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 mis-

sense PTPN11 mutations have been identified as somatic

lesions in hematological malignancies and germline de-

fects in LEOPARD syndrome (LS), which is clinically relat-

ed 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 dis-

sociation rate. As documented for PTPN11, the distribu-

tions 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-thre-

onine 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 14–3–

3 recognition sequence around Ser259 accounting for 75%

of the mutations. Since 14–3–3 binding at residue Ser259

stabilizes RAF1’s catalytically inactive conformation and

impairs its translocation to the plasma membrane, muta-

tions affecting this motif promote increased RAF1 activ-

ity. 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 rel-

evant fraction of pediatric cases with isolated hypertro-

phic 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.


Molecular Genetics of Noonan Syndrome 21

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 con-

dition until recently. Autosomal dominant inher-

itance 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 re-

port appearing in 1992. Since NS shares some fea-

tures 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 genetical-

ly 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 four-

generation Belgian family transmitting the trait,

achieving independent linkage to NS1, and refin-

ing the critical interval further to approximate-

ly 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 candi-

date because it mapped to the NS1 critical region

and because its protein product, SHP-2, occu-

pied a critical role in several intracellular signal

transduction pathways controlling diverse devel-

opmental 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 [10–13]. Based on those efforts, it has

now been established that PTPN11 mutations ac-

count for approximately 50% of individuals with

NS, are almost always missense changes that af-

fect 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 individ-

uals with Noonan-like syndrome and multiple gi-

ant 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 dif-

ferent class of missense mutations in PTPN11 oc-

curs as somatic events in myeloid and lymphoid

malignancies [17–19], and the identity of PTPN11

mutations conferring susceptibility to these hema-

tological 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 rela-

tively large panel of germline or somatic mutations

identified multiple mechanisms promoting SHP-2

gain of function [17, 21–27].

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 oth-

er 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 suffi-

ciently 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

22 Tartaglia � Gelb

three additional NS disease genes, KRAS, SOS1

and RAF1, in the last two years [28–33]. KRAS

codes for one of the three members of the RAS

family, while SOS1 and RAF1 are, respectively,

a RAS-specific guanine nucleotide exchange fac-

tor (GEF) and an effector of RAS with serine/

threonine kinase activity functioning as the up-

stream 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 [29–34].

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 cardiofa-

ciocutaneous 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 abnormal-

ities, 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 in-

dividuals, indicating that other disease genes re-

sponsible for a relatively large portion of Noonan

syndrome remain to be identified. These genes

are likely to encode proteins with role in the RAS-

MAPK signaling pathway.

While other genes are expected to be identified

in the next following years, mutational screening

efforts focused on genes that encode transduc-

ers participating in the RAS-MAPK signaling

pathway and are mutated in disorders clinical-

ly 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

SOS1

SOS1: NS

PTPN11: NS, LS NF1: NF1, NFNS

Neurofibromin

RAF1: NS, LS

BRAF: CFC

MEK1: CFCS, NS

MEK2: CFCS

HRAS: CS

KRAS: NS, CFCS

GDP-RAS

RAF

MEK

GRB2SHC

SHP2

GDP-RAS

ERK

Gene expression

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 ge-

neric dimerized cell-surface recep-

tors binding to their ligand.


that activates ERK proteins, has been identified

[37]. According to this study, MEK1 gene muta-

tions 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 knowl-

edge 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,

N-SH2

1 2

ATG TGA

3 4 5 6 7 8 9 10 11 12 13 14 15 16

3a 104112 216 221 524

C-SH2 PTP

Germline transmitted Somatically acquired

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 func-

tional 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 do-

mains. (b) Location of mutated residues in the three dimensional structure of SHP-2 in its cata-

lytically 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).


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 [38–40]. SHP-2’s structure

is composed of two tandemly arranged amino-

terminal SH2 domains (N-SH2 and C-SH2), a

single catalytic domain (PTP) and a carboxy-

terminal tail containing two tyrosyl phospho-

rylation sites and a proline-rich stretch (fig. 2).

Both the N-SH2 and C-SH2 domains selective-

ly bind to short amino acid motifs containing

a phosphotyrosyl residue and promote SHP-

2’s 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 us-

ing a separate site [41]. As these subdomains

show negative cooperativity, the N-SH2 domain

functions as an intramolecular switch control-

ling 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 phos-

photyrosyl 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 SHP-

2 can either positively or negatively modulate

signal flow depending upon its binding partner

and interactions with downstream signaling net-

works, it is now established that SHP-2 positively

controls the activation of the RAS-MAPK cas-

cade induced by a number of growth factors and

cytokines [38–40]. In most cases, SHP-2’s func-

tion in intracellular signaling appears to be dis-

tal to activated receptors and upstream to RAS.

While the mechanisms of SHP-2’s action and its

physiological substrates are still poorly defined,

accumulated evidence supports the view that

both membrane translocation and PTPase activ-

ity are required for SHP-2 function.

Available records based on more than 500

germline defects indicate that NS-causing

PTPN11 mutations are almost always mis-

sense 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 (ta-

ble 1 and fig. 2). Most of the mutations affect

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 (%)

Somatic origin (n = 256)

n (%)

I A/I switching 243 (42.4) 217 (84.8)

II A/I switching and catalysis 66 (11.5) 3 (1.2)

III A/I switching and specificity 27 (4.7) 27 (10.5)

IV A/I switching and/or catalysis 195 (34.0) 4 (1.6)

V SH2 pY-binding 28 (4.9) 5 (1.9)

VI SH2 orientation or mobility 12 (2.1) –

others – 2 (0.4) –

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


residues involved in the N-SH2/PTP interdo-

main 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 physiologi-

cal activation by impairing the switch between

the active and inactive conformation, favoring a

shift in the equilibrium toward the latter, with-

out altering SHP-2’s catalytic capability. Recent

biochemical and molecular modeling data con-

sistently support this view [24, 27, 42]. A number

of mutations, however, affect residues contrib-

uting to the stability of the catalytically inactive

conformation but also participating in catalysis

or controlling substrate specificity. For a num-

ber of these defects it can be speculated that the

individual substitution does not markedly per-

turb 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 evi-

dence 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) per-

centage of cases results from de novo mutations.

To investigate the parental origin of de novo mu-

tations in NS, Tartaglia and co-workers studied 46

families, each consisting of an affected individual

heterozygous for a PTPN11 mutation and unaf-

fected parents [43]. Among the fourteen informa-

tive families identified in the study, the mutation

was of paternal origin in all cases. Moreover, ad-

vanced paternal age was noted among fathers of

sporadic NS cases with or without PTPN11 mu-

tations, consistent with many, but not all, other

autosomal dominant disorders with paternal ori-

gin of spontaneous mutations. Notably, a sex-ra-

tio 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 attrib-

utable 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 fertil-

ity 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 typi-

cal 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, mutation-

al analysis of three unrelated families inherit-

ing 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 intro-

duced 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 phenotyp-

ic 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 includ-

ing 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 pres-

ence 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


‘LEOPARD syndrome: Clinical aspects and mo-

lecular pathogenesis’ in this volume).

The elucidation of the pathogenesis of NS, par-

ticularly with respect to the developmental pertur-

bations, depends upon studies of animal models.

Araki and co-workers generated and character-

ized a knock-in mouse bearing the Asp61Gly mu-

tation in the Ptpn11 gene [49]. Consistent with

biochemical data on human SHP-2 mutants ex-

pressed transiently in cell culture, embryo fibro-

blasts 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 em-

bryos were grossly edematous and hemorrhagic,

had diffuse liver necrosis and severe cardiac de-

fects. Among the Ptpn11D61G/+ embryos, approx-

imately 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 heterozy-

gotes, including proportional growth failure, car-

diofacial dysmorphism, and a mild leukocytosis

with increased neutrophils and lymphocytes in

adult mice. Splenomegaly was present due to ex-

tramedullary hematopoiesis. There was a my-

eloid 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 engen-

dered a mild myeloproliferative disease similar to

that observed in some NS patients.

New information concerning gain-of-func-

tion Shp-2 and development has emerged through

work with transgenic flies [50]. The Drosophila

homolog of PTPN11, corkscrew (csw), acts down-

stream of several receptor tyrosine kinases

controlling developmental processes [51]. While

ubiquitous expression of leukemia-associated csw

transgenic alleles engendered embryonic or lar-

val 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, epistat-

ic studies with genes relevant for Egfr-Ras-Mapk

signaling showed that the N308D allele interact-

ed genetically with nearly all genes in the path-

way, documenting dependence on the activation

of the receptor by its ligand for ectopic wing vein

formation [50].

Children with NS are predisposed to a spec-

trum of hematologic abnormalities, including

juvenile myelomonocytic leukemia (JMML), a

clonal myeloproliferative disorder of childhood

characterized by excessive proliferation of im-

mature myelomonocytic cells that infiltrate he-

matopoietic and non-hematopoietic tissues [52,

53]. The hallmark of JMML cells is the hypersen-

sitive pattern of myeloid progenitor colony growth

in response to GM-CSF, which is due to a selective

inability to down-regulate RAS. Indeed, approxi-

mately 50% of children with JMML exhibit either

oncogenic RAS mutations or neurofibromin loss

of function, the latter is a GTPase activating pro-

tein (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 germ-

line mutations in the majority of cases, as well as

the occurrence of genotype-phenotype correla-

tions [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 as-

sociation 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


acquired as a somatic event, might play a role in

leukemogenesis. Indeed, somatic missense muta-

tions in PTPN11 have been demonstrated to oc-

cur in approximately one-third of isolated JMML

as well as variable proportions of other myeloid

and lymphoid malignancies of childhood [17–19,

22, 27, 54, 55]. The prevalence of PTPN11 muta-

tions among adult patients with myeloid or lym-

phoid disorders appears to be considerably lower

than observed among pediatric cases [27, 56–59]

(our unpublished data), even though SHP-2 over-

expression 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 le-

sions identified in this heterogeneous group of

hematologic malignancies are missense chang-

es that alter residues located at the interface be-

tween the N-SH2 and PTP domains. Remarkably,

the available molecular data indicate that speci-

ficity in the amino acid substitution is relevant to

the functional deregulation of SHP-2 and disease

pathogenesis (table 1 and fig. 2). Indeed, compar-

ison of the molecular spectra observed with the

NS and leukemias indicate a clear-cut genotype-

phenotype correlation, strongly supporting the

idea that the germline transmitted PTPN11 mu-

tations 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 NS-

associated mutant proteins [24, 27, 42]. Moreover,

the leukemia-associated PTPN11 mutations up-

regulate RAS signaling and induce cell hyper-

sensitivity 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 develop-

mental-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 mild-

er gain-of-function effects, which are sufficient

to perturb development processes but inadequate

to deregulate hematopoietic precursor cell prolif-

eration. The PTPN11 mutations observed in iso-

lated JMML and other hematologic malignancies

produce mutant SHP-2 proteins with higher gains

in function. Since these molecular lesions are ob-

served almost exclusively as somatic defects, it

is likely that they affect embryonic development

and/or fetal survival. The PTPN11 mutations ob-

served 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 addi-

tional NS genes existed. Based on the link between

the functions of SHP-2 and RAS, two groups in-

dependently 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 indi-

viduals among 212 PTPN11 mutation-negative

subjects with NS. Of note, a generally more se-

vere 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 muta-

tions associated with these developmental disor-

ders as well as their phenotypic spectrum have

been investigated further, refining the picture of

a clustered distribution of germline disease-as-

sociated KRAS defects, and confirming the high

clinical variability [35, 37]. On the whole, avail-

able data indicate that NS-causing KRAS muta-

tions are missense and account for less than 3% of


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 pro-

duces two transcripts through alternative splic-

ing, 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 molec-

ular 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 transduc-

ers (fig. 1). GDP/GTP cycling is controlled by

GAPs, which accelerate the intrinsic GTPase ac-

tivity, and GEFs, which promote release of GDP.

1 2 3 4 5 6

AUG UAA

UAAAUGG domainSwitch I

G1PM1 PM2 PM3

G2 G3

Switch II

Isoform B

a b

Isoform A

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 re-

quired 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 re-

gion is shown in grey, together with the C-terminal motifs that direct post-translational process-

ing 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 confor-

mation. (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.


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&ln=KRAS (November 30, 2007) (somatic mutations).

Amino acid

Amino acid change


n (%)

Somatic origin (n = 10,754)

n (%)

Lys5 Asn 1 (3.3) 2 (<0.1)

Glu 1 (3.3) –

Gly12 Ala – 566 (5.2)

Cys – 1319 (12.3)

Asp – 3861 (35.9)

Phe – 16 (<0.2)

Leu – 3 (<0.1)

Asn – 6 (<0.1)

Arg – 479 (4.5)

Ser 1 (3.3) 654 (6.1)

Val – 2507 (23.3)

Gly13 Ala – 20 (0.2)

Cys – 98 (<1.0)

Asp – 928 (8.6)

Arg – 23 (0.2)

Ser – 41 (0.4)

Val – 13 (0.1)

Val14 Ile 6 (20.0) 2 (<0.1)

Gln22 Lys – 4 (<0.1)

Arg 1 (3.3) 1 (<0.01)

Glu 1 (3.3) –

Pro34 Arg 1 (3.3) –

Leu 1 (3.3) –

Gln 1 (3.3) –

Ile36 Met 1 (3.3) –

Thr58 Ile 3 (10.0) –

Gly60 Ala – 1 (<0.01)

Arg 2 (6.7) –


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 post-

translational 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, un-

dergo major conformational changes upon GTP/

GDP exchange and mediate binding to effectors,

GAPs and GEFs [64, 66]. As observed for the so-

matically acquired oncogenic NRAS, KRAS and

HRAS mutations, some of the NS-causing KRAS

defects were found to up-regulate protein func-

tion 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-associat-

ed Gly12Asp mutant (that has negligible GTPase

activity). Interestingly, both the Val14Ile and

Thr58Ile KRAS proteins were partially respon-

sive to neurofibromin and p120 GAP, although

to different extents. Consistent with these data,

cells expressing each of the two mutants were hy-

per-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 pro-

tein exhibited a response to GAPs that was com-

parable to that observed for the wild type protein.

These mutants also differed in their capability to

bind guanine nucleotides with the Asp153Val pro-

tein exhibiting a normal GTP/GDP dissociation

Gln61 Glu – 9 (<0.1)

His – 75 (0.7)

Lys – 13 (0.1)

Leu – 22 (0.2)

Pro – 11 (0.1)

Arg – 22 (0.2)

Val152 Gly 1 (3.3) –

Asp153 Val 6 (20.0) –

Phe156 Ile 3 (10.0) –

other – 58 (0.5)

Table 2. (continued)

Amino acid

Amino acid change


n (%)

Somatic origin (n = 10,754)

n (%)


rate while Phe156Leu KRAS showed a dramat-

ically increased rate of guanine nucleotide dis-

sociation. Although these studies documented a

complex pattern of intrinsic biochemical proper-

ties, 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-respon-

sive 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 docu-

mented in one-third of NS or CFCS subjects with a

KRAS germline mutation. This exon codes for resi-

dues 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 func-

tions [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 pro-

cessing of the two KRAS isoforms leads to alterna-

tive 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 per-

turb 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 GDP-

bound 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 ho-

mology 3 (SH3) domain of GRB2, and following

growth factor stimulation, the GRB2-SOS com-

plex binds directly to specific tyrosyl-phospho-

rylated 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 sig-

naling, two groups considered SOS1 and SOS2 as

excellent candidate genes, and independently dis-

covered 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 resi-

dues), which are implicated in the activation of

RAC, a small GTPase of the RHO/CDC42 fam-

ily [76]. The C-terminal half of the protein con-

tains the RAS exchanger motif (REM) domain

(≈200 residues) and the Cdc25 domain (≈300 res-

idues), 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


proteins that deliver SOS1 to the membrane upon

receptor activation (fig. 4). Additional anchorage

sites on the membrane are provided by the phos-

phatidylinositol 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 20–30% of sub-

jects without a defect in PTPN11 or KRAS [32,

33, 36]. Thus far, all of the mutations identified

are missense and affect multiple domains, clus-

tering 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 interact-

ing regions of the DH (Thr266 and Met269) and

REM (Trp729 and Ile733) domains (14% of mu-

tations). A single amino acid change (Glu846Lys)

within the Cdc25 domain accounts for approxi-

mately 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 ac-

tivity through the binding of nucleotide-bound

RAS [80]. Whereas the former is located entire-

ly within the Cdc25 domain, the allosteric site is

bracketed by the Cdc25 domain and REM do-

mains. 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,

T266KM269RM269T

D309YY337C

W432RE433KG434RC441Y

E108K

a

b

1 198

Histonefolds DH PH Rem Cdc25 PxxP

404 550 750 1,050 1,200 1,333

P478LP478R

S548RL550PR552GR552KR552S

Y702H

F623IW729LI733F

E846K

Fig. 4. SOS1 domain structure and

location of affected residues in NS.

(a) The predicted amino acid substi-

tutions 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 do-

main; 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 indicat-

ed with their lateral chains (histone

folds, violet; HD, blue; PH, green; he-

lical linker, red; Rem, orange; Cdc25,

cyan). Based on Sondermann et al.

[78], which utilized structural data

and computational modeling.


the inhibitory effect of the DH domain is relieved

by a still undefined event(s) allowing RAS bind-

ing 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 autoinhib-

ited 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 interac-

tion is expected to affect the relative orientation

of the DH-PH unit and the REM domain. A sim-

ilar perturbing effect is predicted for the other

amino acid substitutions involving residues lo-

cated in the helical linker connecting the PH and

REM domains (fig. 4). While the mutation clus-

ter affecting residues 432 to 441 may disrupt the

autoinhibited conformation by destabilizing the

PH domain’s 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 in-

teracts directly with Met269, thereby positioning

the DH domain in its autoinhibitory conforma-

tion. Biochemical data confirmed these predic-

tions and demonstrated that NS-causing SOS1

mutations promote gain-of-function. Roberts

and co-workers documented that transient ex-

pression 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 ac-

tivation 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 relat-

ed to SOS autoinhibition, either through interac-

tion 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 RAS-

GTP levels. It should be noted that the DH-PH

module of SOS has also been implicated in the ac-

tivation of the Rho GTPase RAC [76]. The extent

to which SOS1 gain-of-function mutations affect

different RAS-dependent or RAC-dependent sig-

nals 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 mem-

ber of a small family of serine-threonine kinas-

es that includes two additional members, ARAF

and BRAF [83–85]. 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 ac-

tivity 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 [86–89].

The RAF1 gene (OMIM 164760) encodes a 74-

kDa protein characterized by three functional do-

mains, known as conserved regions 1 to 3 (CR1–3)


(fig. 5). The N-terminal CR1 contains the domain

involved in GTP-RAS binding and a cysteine-

rich region that mediates RAF1 interaction with

the cytosolic surface of the cell membrane. CR2

contains a negative regulatory domain control-

ling 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 co-

lon, 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 muta-

tions in RAF1 in subjects with NS who were nega-

tive for a mutation in PTPN11, KRAS or SOS1.

Pandit and co-workers documented a heterozy-

gous condition for a RAF1 lesion in 18 out of

231 individuals (8%) [30], while a higher preva-

lence (10/30) was reported by Razzaque and co-

workers [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 af-

fects the consensus 14–3–3 recognition sequence

(Arg256Ser257Thr258pSer259Thr260Pro261) or an

adjacent residue within the CR2 region. Of note,

Arg256, Ser257, Ser259 and Pro261, which are the in-

variant 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 muta-

tions affecting residues within the activation seg-

ment 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 tu-

mors 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) locat-

ed 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 can-

cer, one somatic missense mutation (Ser259Ala)

in RAF1 has been observed in an ovarian carci-

noma [91].

Since a high prevalence of hypertrophic car-

diomyopathy 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 mis-

sense 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 in-

active conformation, the N-terminal portion of

the protein is thought to interact with and inac-

tivate the kinase domain at the C-terminus. This

autoinhibited conformation is stabilized by 14–

3–3 protein dimers that bind to phosphorylat-

ed Ser259 and Ser621 [83–85]. 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 phos-

phorylate regulatory residues that, in their un-

phosphorylated state, contribute to stabilizing

the catalytically inactive conformation of the

protein. To examine the functional consequenc-

es of RAF1 mutations, Pandit and co-work-

ers expressed mutants from each of the three


clusters transiently in Cos-1 cells. Pro261Ser

and Leu613Val RAF1 proteins, representative

of the HCM-associated clusters, displayed in-

creased kinase activity compared to wild type

protein basally and after EGF stimulation [30].

Consistent with that study, Razzaque and co-

workers observed enhanced kinase activity for

the Ser257Leu, Pro261Ser, Pro261Ala, Val263Ala

and Leu613Val RAF1 mutants [31]. In contrast,

Asp486Asn and Thr491Ile, representing the mu-

tation cluster in the activation segment and not

associated with HCM, were observed to be ki-

nase impaired [30]. Of note, while the expres-

sion of the Ser257Leu, Pro261Ser, Pro261Ala,

Val263Ala and Leu613Val RAF1 mutants result-

ed in a constitutively increased activation of the

MAPK cascade, expression of the Asp486Asn

mutant, which was kinase dead, caused a re-

duced 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 reduc-

tion 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 explana-

tion. Pandit and co-workers also investigated

the status of 14–3–3 binding for the Pro261Ser

and Leu613Val RAF1 mutants, and demonstrat-

ed that the increased activation promoted by the

BRAF

V600A/D/E/G/K/L/M/RT599l

L597L/Q/R/S/VD594/G/E/K/V

K601E/NG469A/R/S/V

G466A/E/R/V

G464/E/R/V

CR1 CR2 CR3

CR1 CR2 CR3

G596V

F595LD638E

N581D

Activation segment

S259AR226I

P207S Q335HS259 S621S427G

I448V E478K

G534R

E501K/G

K499F

L485FS467AF468S

G469E

R256SS257L

S259FT260R/I

P261S/L/AV263A

D486N/GT491I/R

S612T

L613V

Q257R

A246P

RBD CRD

RAF1

Fig. 5. RAF1 domain structure and

location of affected residues in hu-

man 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 resi-

dues (blue) that can be phosphory-

lated as part of RAF1’s regulation.

The mutations observed in Noonan

and LEOPARD syndromes and those

associated with cancer are shown

above and below the cartoon, re-

spectively. 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 (preva-

lence 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 syn-

drome-causing RAF1 T491I change,

is also reported.


amino acid substitution was associated with a

loss of 14–3–3-mediated inactivation. This find-

ing 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 14–3–3 and had increased kinase activ-

ity [93]. On the contrary, the Leu613Val mutant

bound to 14–3–3 normally at Ser621 and had

normal phosphorylation of Ser259 so the mech-

anism through which mutations in this cluster

activated RAF1’s MEK kinase activity remains

to be explained.

MEK1

A missense mutation in MEK1, predicting the

Asp67Asn amino acid substitution, has recent-

ly been reported in two unrelated subjects with

sporadic NS [37]. One additional previously un-

reported variant (Glu44Gly), which was identi-

fied in a third sporadic case and her apparently

clinically unaffected mother but not in 200 pop-

ulation-matching controls, is likely to represent a

private polymorphism. According to this study,

MEK1 gene mutations would account for ap-

proximately 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 speci-

ficity 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 nega-

tive 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-redun-

dant roles. In particular, genetic evidence from

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 pro-

mote transformation [98], mutations in these

genes have not been reported in human can-

cers [99].

A few missense mutations in MEK1 (Phe53Ser

and Tyr130Cys) and MEK2 (Phe57Cys) had previ-

ously been reported in a small fraction of subjects

with CFCS [100]. Functional characterization of

the three mutants documented that their tran-

sient expression in 293T cells were more active

than the wild type protein in stimulating ERK

phosphorylation basally.

Concluding Remarks

In the last few years, we have witnessed the elu-

cidation of genetic causes of NS, which can now

be viewed as a disorder of dysregulated RAS-

MAPK signaling. Further studies are needed to

identify the still missing genes, probably func-

tionally related, responsible for the remaining

35% of affected individuals as well as to under-

stand in detail NS disease pathogenesis. These

steps are preludes to developing improved di-

agnostics 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’ lab-oratories is supported in part by grants from Telethon-Italy (GGP07115), ‘Programma di Collaborazione Italia-USA/malattie rare’ and from Associazione ONLUS ‘Morgan Di Gianvittorio per la cura e la ric-erca nei tumori e leucemie in età 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.).


References

van Der Burgt I, Brunner H: Genetic 1 heterogeneity in Noonan syndrome: evidence for an autosomal recessive form. Am J Med Genet 2000;94:46–51.Sharland M, Taylor R, Patton MA, Jef- 2 fery S: Absence of linkage of Noonan syndrome to the neurofibromatosis type 1 locus. J Med Genet 1992;29:188–190.Flintoff WF, Bahuau M, Lyonnet S, 3 Gilgenkrantz S, Lacombe D, et al: No evidence for linkage to the type 1 or type 2 neurofibromatosis loci in Noo-nan syndrome families. Am J Med Genet 1993;46:700–705.Jamieson CR, van der Burgt I, Brady 4 AF, van Reen M, Elsawi MM, et al: Mapping a gene for Noonan syndrome to the long arm of chromosome 12. Nat Genet 1994;8:357–360.Brady AF, Jamieson CR, van der Burgt 5 I, Crosby A, van Reen M, et al: Further delineation of the critical region for Noonan syndrome on the long arm of chromosome 12. Eur J Hum Genet 1997;5:336–337.Legius E, Schollen E, Matthijs G, Fryns 6 JP: Fine mapping of Noonan/cardio-facio cutaneous syndrome in a large family. Eur J Hum Genet 1998;6:32–37.Ion A, Crosby AH, Kremer H, Ken- 7 mochi N, Van Reen M, et al: Detailed mapping, mutation analysis, and in-tragenic polymorphism identification in candidate Noonan syndrome genes MYL2, DCN, EPS8, and RPL6. J Med Genet 2000;37:884–886.Tartaglia M, Mehler EL, Goldberg R, 8 Zampino G, Brunner HG, et al: Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syn-drome. Nat Genet 2001;29:465–468.Chen B, Bronson RT, Klaman LD, 9 Hampton TG, Wang JF, et al: Mice mu-tant for Egfr and Shp2 have defective cardiac semilunar valvulogenesis. Nat Genet 2000;24:296–299.Tartaglia M, Kalidas K, Shaw A, Song 10 X, Musat DL, et al: PTPN11 mutations in Noonan syndrome: molecular spec-trum, genotype-phenotype correla-tion, and phenotypic heterogeneity. Am J Hum Genet 2002;70:1555–1563.Sarkozy A, Conti E, Seripa D, Digilio MC, 11 Grifone N, et al: Correlation between PTPN11 gene mutations and congenital heart defects in Noonan and LEOPARD syndromes. J Med Genet 2003;40:704–708.

Zenker M, Buheitel G, Rauch R, Koen-12 ig R, Bosse K, et al: Genotype-pheno-type correlations in Noonan syndro-me. J Pediatr 2004;144:368–374.Jongmans M, Sistermans EA, Rikken 13 A, Nillesen WM, Tamminga R, et al: Genotypic and phenotypic character-ization of Noonan syndrome: New data and review of the literature. Am J Med Genet A 2005;134:165–170.Lee JC, Tartaglia M, Gelb BD, Fridrich 14 K, Sachs S, et al: Phenotypic and geno-typic characterization of Noonan-like/multiple giant cell lesion syndrome. J Med Genet 2005;42:e11.Digilio MC, Conti E, Sarkozy A, Min-15 garelli 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.Legius E, Schrander-Stumpel C, Schol-16 len E, Pulles-Heintzberger C, Gewillig M, Fryns JP: PTPN11 mutations in LE-OPARD syndrome. J Med Genet 2002;39:571–574.Tartaglia M, Niemeyer CM, Fragale A, 17 Song X, Buechner J, et al: Somatic mu-tations in PTPN11 in juvenile myelo-monocytic leukemia, myelodysplastic syndromes and acute myeloid leuke-mia. Nat Genet 2003;34:148–150.Tartaglia M, Martinelli S, Cazzaniga 18 G, Cordeddu V, Iavarone I, et al: Ge-netic evidence for lineage- and differ-entiation stage-related contribution of somatic PTPN11 mutations to leuke-mogenesis in childhood acute leuke-mia. Blood 2004;104:307–313.Tartaglia M, Martinelli S, Iavarone I, 19 Cazzaniga G, Spinelli M, et al: Somatic PTPN11 mutations in childhood acute myeloid leukaemia. Br J Haematol 2005;129:333–339.Kratz CP, Niemeyer CM, Castleberry 20 RP, Cetin M, Bergsträsser E, et al: The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and noonan syndrome/myeloprolifera-tive disease. Blood 2005;106: 2183–2185.Fragale A, Tartaglia M, Wu J, Gelb BD: 21 Noonan syndrome-associated SHP2/PTPN11 mutants cause EGF-dependent prolonged GAB1 binding and sus-tained ERK2/MAPK1 activation. Hum Mutat 2004;23:267–277.

Loh ML, Vattikuti S, Schubbert S, 22 Reynolds MG, Carlson E, et al: Muta-tions in PTPN11 implicate the SHP-2 phosphatase in leukemogenesis. Blood 2004;103:2325–2331.Chan RJ, Leedy MB, Munugalavadla V, 23 Voorhorst CS, Li Y, Yu M, Kapur R: Human somatic PTPN11 mutations induce hematopoietic-cell hypersensi-tivity to granulocyte-macrophage col-ony-stimulating factor. Blood 2005;105:3737–3742.Keilhack H, David FS, McGregor M, 24 Cantley LC, Neel BG: Diverse biochem-ical properties of Shp2 mutants. Impli-cations for disease phenotypes. J Biol Chem 2005;280:30984–30993.Schubbert S, Lieuw K, Rowe SL, Lee 25 CM, Li X, et al: Functional analysis of leukemia-associated PTPN11 muta-tions in primary hematopoietic cells. Blood 2005;106:311–317.Mohi MG, Williams IR, Dearolf CR, 26 Chan G, Kutok JL, et al: Prognostic, therapeutic, and mechanistic implica-tions of a mouse model of leukemia evoked by Shp2 (PTPN11) mutations. Cancer Cell 2005;7:179–191.Tartaglia M, Martinelli S, Stella L, 27 Bocchinfuso G, Flex E, et al: Diversity and functional consequences of ger-mline and somatic PTPN11 mutations in human disease. Am J Hum Genet 2006;78:279–290.Carta C, Pantaleoni F, Bocchinfuso G, 28 Stella L, Vasta I, et al: Germline mis-sense mutations affecting KRAS iso-form b are associated with a severe Noonan syndrome phenotype. Am J Hum Genet 2006;79:129–135.Schubbert S, Zenker M, Rowe SL, Böll 29 S, Klein C, et al: Germline KRAS muta-tions cause Noonan syndrome. Nat Genet 2006;38:331–336.Pandit B, Sarkozy A, Pennacchio LA, Car-30 ta C, Oishi K, et al: Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyo-pathy. Nat Genet 2007;39:1007–1012.Razzaque MA, Nishizawa T, Komoike 31 Y, Yagi H, Furutani M, et al: Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet 2007;39:1013–1017.


Roberts AE, Araki T, Swanson KD, Mont-32 gomery KT, Schiripo TA, et al: Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet 2007;39:70–74.Tartaglia M, Pennacchio LA, Zhao C, 33 Yadav KK, Fodale V, et al: Gain-of-func-tion SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet 2007;39:75–79.Schubbert S, Bollag G, Lyubynska N, 34 Nguyen H, Kratz CP, et al: Biochemical and functional characterization of germ line KRAS mutations. Mol Cell Biol 2007;27:7765–7770.Zenker M, Lehmann K, Schulz AL, Barth 35 H, Hansmann D, et al: Expansion of the genotypic and phenotypic spectrum in patients with KRAS germline mutations. J Med Genet 2007;44:131–135.Zenker M, Horn D, Wieczorek D, Allan-36 son J, Pauli S, et al: SOS1 is the second most common Noonan gene but plays no major role in cardio-facio-cutaneous syn-drome. J Med Genet 2007;44:651–656.Nava C, Hanna N, Michot C, Pereira S, 37 Pouvreau N, et al: Cardio-facio-cutane-ous and Noonan syndromes due to muta-tions in the RAS/MAPK signalling path-way: genotype-phenotype relationships and overlap with Costello syndrome. J Med Genet 2007;44:763–771.Neel BG, Gu H, Pao L: The ‘Shp’ing 38 news: SH2 domain-containing ty-rosine phosphatases in cell signaling. Trends Biochem Sci 2003;28:284–293.Tartaglia M, Niemeyer CM, Shannon KM, 39 Loh ML: SHP-2 and myeloid malignan-cies. Curr Opin Hematol 2004;11:44–50.Chan RJ, Feng GS: 40 PTPN11 is the first identified proto-oncogene that en-codes a tyrosine phosphatase. Blood 2007;109:862–867.Hof P, Pluskey S, Dhe-Paganon S, Eck 41 MJ, Shoelson SE: Crystal structure of the tyrosine phosphatase shp-2. Cell 1998;92:441–450.Bocchinfuso G, Stella L, Martinelli S, 42 Flex E, Carta C, et al: Structural and functional effects of disease-causing amino acid substitutions affecting res-idues Ala72 and Glu76 of the protein tyrosine phosphatase SHP-2. Proteins 2007;66:963–974.Tartaglia M, Cordeddu V, Chang H, 43 Shaw A, Kalidas K, et al: Paternal ger-mline origin and sex-ratio distortion in transmission of PTPN11 mutations in Noonan syndrome. Am J Hum Gen-et 2004;75:492–497.

Elsawi MM, Pryor JP, Klufio G, Barnes 44 C, Patton MA: Genital tract function in men with Noonan syndrome. J Med Genet 1994;31:468–470.Bertola DR, Kim CA, Pereira AC, Mota 45 GF, Krieger JE, et al: Are Noonan syn-drome and Noonan-like/multiple giant cell lesion syndrome distinct entities? Am J Med Genet 2001;98:230–234.Cohen MM Jr, Gorlin RJ: Noonan-like/46 multiple giant cell lesion syndrome. Am J Med Genet 1991;40:159–166.Gorlin RJ, Anderson RC, Moller JH: 47 The LEOPARD (multiple lentigines) syndrome revisited. Birth Defects Orig Artic Ser 1971;7:110–115.Voron DA, Hatfield HH, Kalkhoff MD: 48 Multiple lentigines syndrome: case report and review of the literature. Am J Med 1976;60:447–456.Araki T, Mohi MG, Ismat FA, Bronson 49 RT, Williams IR, et al: Mouse model of Noonan syndrome reveals cell type- and gene dosage-dependent effects of Ptpn11 mutation. Nat Med 2004;10:849–857.Oishi K, Gaengel K, Krishnamoorthy 50 S, Kamiya K, Kim IK, et al: Transgenic Drosophila models of Noonan syn-drome causing PTPN11 gain-of-func-tion mutations. Hum Mol Genet 2006;15:543–553.Perkins LA, Johnson MR, Melnick MB, 51 Perrimon N: The nonreceptor protein tyrosine phosphatase corkscrew func-tions in multiple receptor tyrosine ki-nase pathways in Drosophila. Dev Biol 1996;180:63–81.Emanuel PD, Shannon KM, Castleber-52 ry RP: Juvenile myelomonocytic leuke-mia: Molecular understanding and prospects for therapy. Mol Med Today 1996;2:468–475.Arico M, Biondi A, Pui CH: Juvenile 53 myelomonocytic leukemia. Blood 1997;90:479–488.Loh ML, Reynolds MG, Vattikuti S, 54 Gerbing RB, Alonzo TA, Carlson et al: PTPN11 mutations in pediatric pa-tients with acute myeloid leukemia: Results from the children’s cancer group. Leukemia 2004;18:1831–1834.Paulsson K, Horvat A, Strömbeck B, 55 Nilsson F, Heldrup J, et al: Mutations of FLT3, NRAS, KRAS, and PTPN11 are frequent and possibly mutually exclu-sive in high hyperdiploid childhood acute lymphoblastic leukemia. Genes Chromosomes Cancer 2008;47:26–33.

Loh ML, Martinelli S, Cordeddu V, 56 Reynolds MG, Vattikuti S, et al: Ac-quired PTPN11 mutations occur rarely in adult patients with myelodysplastic syndromes and chronic myelomonocyt-ic leukemia. Leuk Res 2005;29:459–462.Hugues L, Cavé H, Philippe N, Pereira 57 S, Fenaux P, Preudhomme C: Muta-tions of PTPN11 are rare in adult my-eloid malignancies. Haematologica 2005;90:853–854.Nomdedéu J, Carricondo MT, Lasa A, 58 Perea G, Aventin A, Sierra J: Low frequen-cy of exon 3 PTPN11 mutations in adult de novo acute myeloid leukemia. Analysis of a consecutive series of 173 patients. Haematologica 2005;90:412–413.Bentires-Alj M, Paez JG, David FS, 59 Keilhack H, Halmos B, et al: Activating mutations of the noonan syndrome-associated SHP2/PTPN11 gene in hu-man solid tumors and adult acute my-elogenous leukemia. Cancer Res 2004;64:8816–8820.Xu R, Yu Y, Zheng S, Zhao X, Dong Q, 60 et al: Overexpression of Shp2 tyrosine phosphatase is implicated in leukemo-genesis in adult human leukemia. Blood 2005;106:3142–3149.Martinelli S, Carta C, Flex E, Binni F, 61 Cordisco EL, et al: Activating PTPN11 mutations play a minor role in pediat-ric and adult solid tumors. Cancer Genet Cytogenet 2006;166:124–129.Niihori T, Aoki Y, Narumi Y, Neri G, 62 Cavé H, et al: Germline KRAS and BRAF mutations in cardio-facio-cuta-neous syndrome. Nat Genet 2006;38: 294–296.Friday BB, Adjei AA: K-ras as a target 63 for cancer therapy. Biochim Biophys Acta 2005;1756:127–144.Mitin N, Rossman KL, Der CJ: Signal-64 ing interplay in ras superfamily func-tion. Curr Biol 2005;15:R563–R574.Wennerberg K, Rossman KL, Der CJ: 65 The Ras superfamily at a glance. J Cell Sci 2005;118:843–846.Barbacid M: Ras genes. Annu Rev Bio-66 chem 1987;56:779–827.Silvius JR: Mechanisms of Ras protein 67 targeting in mammalian cells. J Mem-br Biol 2002;190:83–92.Plowman SJ, Williamson DJ, 68 O’Sullivan MJ, Doig J, Ritchie AM, et al: While k-ras is essential for mouse development, expression of the k-ras 4a splice variant is dispensable. Mol Cell Biol 2003;23:9245–9250.


Johnson L, Greenbaum D, Cichowski 69 K, Mercer K, Murphy E, et al: K-ras is an essential gene in the mouse with partial functional overlap with N-ras. Genes Dev 1997;11:2468–2481.Koera K, Nakamura K, Nakao K, Mi-70 yoshi J, Toyoshima K, et al: K-ras is es-sential for the development of the mouse embryo. Oncogene 1997;15:1151–1159.Quilliam LA, Khosravi-Far R, Huff SY, 71 Der CJ: Guanine nucleotide exchange factors: activators of the Ras superfamily of proteins. Bioessays 1995;17:395–404.Quilliam LA, Rebhun JF, Castro AF: A 72 growing family of guanine nucleotide exchange factors is responsible for acti-vation of Ras-family GTPases. Prog Nu-cleic Acid Res Mol Biol 2002;71:391–444.Nimnual A, Bar-Sagi D: The two hats 73 of SOS. Sci STKE 2002;145:PE36.Chardin P, Camonis JH, Gale NW, van 74 Aelst L, Schlessinger J, Wigler MH, Bar-Sagi D: Human Sos1:a guanine nucle-otide exchange factor for Ras that binds to GRB2. Science 1993;260:1338–1343.Hart TC, Zhang Y, Gorry MC, Hart PS, 75 Cooper M, et al: A mutation in the SOS1 gene causes hereditary gingival fibromatosis type 1. Am J Hum Genet 2002;70:943–954.Nimnual AS, Yatsula BA, Bar-Sagi D: 76 Coupling of Ras and Rac guanosine triphosphatases through the Ras ex-changer Sos. Science 1998;279: 560–563.Chen RH, Corbalan-Garcia S, Bar-Sagi 77 D: The role of the PH domain in the signal-dependent membrane targeting of Sos. EMBO J 1997;16:1351–1359.Sondermann H, Nagar B, Bar-Sagi D, 78 Kuriyan J: Computational docking and solution x-ray scattering predict a membrane-interacting role for the his-tone domain of the Ras activator son of sevenless. Proc Natl Acad Sci USA 2005;102:16632–16637.Wang DZ, Hammond VE, Abud HE, 79 Bertoncello I, McAvoy JW, Bowtell DD: Mutation in Sos1 dominantly enhanc-es a weak allele of the EGFR, demon-strating a requirement for Sos1 in EGFR signaling and development. Genes Dev 1997;11:309–320.

Margarit SM, Sondermann H, Hall BE, 80 Nagar B, Hoelz A, et al: Structural evi-dence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS. Cell 2003;112:685–695.Sondermann H, Soisson SM, Boykev-81 isch S, Yang SS, Bar-Sagi D, Kuriyan J: Structural analysis of autoinhibition in the Ras activator Son of sevenless. Cell 2004;119:393–405.Rapp UR, Goldsborough MD, Mark 82 GE, Bonner TI, Groffen J, Reynolds FH Jr, Stephenson JR: Structure and bio-logical activity of v-raf, a unique onco-gene transduced by a retrovirus. Proc Natl Acad Sci USA 1983;80:4218–4222.Wellbrock C, Karasarides M, Marais R: 83 The RAF proteins take centre stage. Nat Rev Mol Cell Biol 2004;5:875–885.Schreck R, Rapp UR: Raf kinases: on-84 cogenesis and drug discovery. Int J Cancer 2006;119:2261–2271.Leicht DT, Balan V, Kaplun A, Singh-85 Gupta V, Kaplun L, Dobson M, Tzivion G: Raf kinases: function, regulation and role in human cancer. Biochim Biophys Acta 2007;1773:1196–1212.Pritchard CA, Bolin L, Slattery R, Mur-86 ray R, McMahon M: Post-natal lethal-ity and neurological and gastrointesti-nal defects in mice with targeted disruption of the A-Raf protein kinase gene. Curr Biol 1996;6:614–617.Mikula M, Schreiber M, Husak Z, Kucero-87 va L, Rüth J, et al: Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene. EMBO J 2001;20:1952–1962.Yamaguchi O, Watanabe T, Nishida K, 88 Kashiwase K, Higuchi Y, et al: Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apop-tosis. J Clin Invest 2004;114:937–943.Wojnowski L, Zimmer AM, Beck TW, 89 Hahn H, Bernal R, Rapp UR, Zimmer A: Endothelial apoptosis in Braf-deficient mice. Nat Genet 1997;16:293–297.Emuss V, Garnett M, Mason C, Marais 90 R: Mutations of C-RAF are rare in hu-man cancer because C-RAF has a low basal kinase activity compared with B-RAF. Cancer Res 2005;65: 9719–9726.

Greenman C, Stephens P, Smith R, 91 Dalgliesh GL, Hunter C, et al: Patterns of somatic mutation in human cancer genomes. Nature 2007;446:153–158.Wan PT, Garnett MJ, Roe SM, Lee S, 92 Niculescu-Duvaz D, et al: Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004;116:855–867.Light Y, Paterson H, Marais R: 14–3–3 93 antagonizes Ras-mediated Raf-1 re-cruitment to the plasma membrane to maintain signaling fidelity. Mol Cell Biol 2002;22:4984–4996.Zheng CF, Guan KL: Properties of 94 MEKs: the kinases that phosphorylate and activate the extracellular signal-regulated kinases. J Biol Chem 1993;268:23933–23939.Giroux S, Tremblay M, Bernard D, Car-95 din-Girard JF, Aubry S, et al: Embry-onic death of Mek1-deficient mice re-veals a role for this kinase in angiogenesis in the labyrinthine re-gion of the placenta. Curr Biol 1999;9:369–372.Bélanger LF, Roy S, Tremblay M, Brott 96 B, Steff AM, et al: Mek2 is dispensable for mouse growth and development. Mol Cell Biol 2003;23:4778–4787.Cowley S, Paterson H, Kemp P, Mar-97 shall CJ: Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transfor-mation of NIH 3T3 cells. Cell 1994;77:841–852.Mansour SJ, Matten WT, Hermann AS, 98 Candia JM, Rong S, et al: Transforma-tion of mammalian cells by constitu-tively active MAP kinase kinase. Sci-ence 1994;265:966–970.Bansal A, Ramirez RD, Minna JD: Mu-99 tation analysis of the coding sequences of MEK-1 and MEK-2 genes in human lung cancer cell lines. Oncogene 1997;14:1231–1234.Rodriguez-Viciana P, Tetsu O, Tidy-100 man WE, Estep AL, Conger BA, et al: Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome. Science 2006;311:1287–1290.

Marco Tartaglia

Department of Cell Biology and Neurosciences

Istituto Superiore di Sanità

00161 Rome (Italy)

Tel. �3906 4990 2569, Fax �3906 4938 7143, E-Mail [email protected]



Genotype-Phenotype Correlations in Noonan Syndrome

A. Sarkozya,b � M.C. Digilioc � B. Marinod �

B. Dallapiccolaa,b

aIRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo and CSS-Mendel Institute, Rome, bDepartment of Experimental Medicine, La Sapienza University, Rome, cMedical Genetics,

Bambino Gesù Hospital, Rome, and dPediatric Cardiology, La Sapienza University, Rome, Italy

AbstractNoonan syndrome (NS) is an autosomal dominant disorder

mainly characterized by short stature, distinct facial anom-

alies 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 pul-

monary valve stenosis, typical facial features, cryptorchid-

ism and bleeding diathesis. Mutations in the SOS1 gene are

associated with clinical features partly overlapping those

found in PTPN11 mutation-positive individuals, but distin-

guished 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 asso-

ciated with hypertrophic cardiomyopathy, mental retarda-

tion, short stature, and skin features of LEOPARD syndrome.

Patients with KRAS mutations are sporadic, affected by vari-

able mental retardation and may manifest features overlap-

ping those of Costello and CFCS, while MEK1 mutations have

been found so far only in two unrelated NS individuals.


Noonan syndrome (NS) is an autosomal dominant

disorder characterized by short stature, distinct

facial anomalies, congenital heart defects (CHD),

developmental delay and several additional clini-

cal features [reviewed in 1]. NS shows a wide phe-

notypic 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 condi-

tion, occurring in approximately 1/1,000–2,500

individuals [2–3]. However, mild expressions are

likely to be overlooked. In particular, facial fea-

tures 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 relative-

ly 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-cu-

taneous (CFCS) and Neurofibromatosis type 1-

Noonan (NFNS) syndromes [5]. Patients with

these disorders present overlapping features, such

as facial abnormalities, CHD and short stature,

together with other common abnormalities such

Genotype-Phenotype Correlations in Noonan Syndrome 41

as skin and genital anomalies and variable degree

of mental retardation. Recently, the term ‘neuro-

cardio-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 nega-

tive cases [6–13]. In addition to genetic heteroge-

neity, NS also displays a wide allelic heterogeneity

in all known genes.

The wide clinical spectrum of NS and its over-

lap with other related disorders have been debat-

ed 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 attempt-

ing to prove any relationship between the allel-

ic 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 dis-

ease-genes and try to establish possible genotype-

phenotype 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 addi-

tional prospective studies. Genotype-phenotype

correlations are further complicated by the ob-

vious 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 research-

ers in this difficult task.

NS and the PTPN11 Gene

Germline mutations in the PTPN11 gene, encod-

ing the SHP2 protein, are responsible for about

45–50% of NS patients [14]. The incidence of

PTPN11 gene mutations is higher in families than

in sporadic patients (60 vs. 40%). Several reports

of PTPN11 screening have been published world-

wide and detailed clinical description of mutated

patients provided [15–23]. However, the frequen-

cy of mutations and clinical characteristics may

have been biased not only by the different selec-

tion criteria, but also by the different clinical set-

tings in which the patients have been enrolled (i.e.

endocrinological vs. cardiological units). In addi-

tion, 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 [15–24]. 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 [24–26]. Conversely, patients with NLS dis-

play 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 PTPN11-

positive NS individuals show a stature lower than

the 3rd centile. Of note, published data are main-

ly 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%), downslant-

ing palpebral fissures (68%) and ptosis (53%)

[16–18] (fig. 1a). Other studies have indicated the

presence of characteristic facial features in up to

90% of the cases [17, 21–22]. 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

42 Sarkozy � Digilio � Marino � Dallapiccola

more than 80% of the PTPN11-positive NS pa-

tients. Pulmonary valve stenosis (PVS) is the most

common cardiac defect (68%), followed by atrial

septal defect (ASD; 24%) and hypertrophic car-

diomyopathy (HCM; 9%). Atrio-ventricular sep-

tal 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 dif-

ficulties 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 sev-

eral exons, encoding different protein domains.

Even in the presence of a wide allelic heterogene-

ity, 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-nega-

tive patients has shown a significant association

between PTPN11 mutations and a few clinical

features (table 1). A consistent correlation was es-

tablished 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 con-

trast, 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 associa-

tion 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 defor-

mities, short neck, mental retardation/learning

difficulties (table 1). Conversely, typical facial

features, cryptorchidism and bleeding diathesis

are more common in the mutated patients (see ta-

ble 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 mu-

tated patients appear significantly shorter [30].

b ca

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


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 no no no 43/130 (33)

Fetal macrosomia no no no no

Short stature (<3rd) 169/223 (76) 64/95 (67) – 169/223 (76) 84/115 (73) –

Macrocephaly 9/23 (39) no 9/23 (39) 19/151 (12) 0.0036

Downslanting palpebral

fissures

39/57 (68) 4/8 (50) – 39/57 (68) no

Ptosis 31/58 (53) 3/8 (38) – 31/58 (53) no

Low-set ears with thickened

helices

61/72 (85) 14/31 (45) <0.0001 61/72 (85) no

Thick lips/macrostomia no no no no

Pterygium colli 40/130 (31) 10/31(32) – 40/130 (31) no

Short neck 16/37 (43) 5/8 (62) – 16/37 (43) no

Short/webbed neck 23/44 (52) no 23/44 (52) no

Thorax abnormalities 139/215 (65) 59/84 (70) – 139/215 (65) 144/151 (95) <0.0001

Cardiac defects 236/285 (83) 42/66 (64) 0.0012 236/285 (83) 132/151 (87) –

Pulmonary valve stenosis 247/362 (68) 47/96 (49) 0.0007 247/362 (68) 93/151 (62) –

Septal defect 81/340 (24) 21/94 (22) – 81/340 (24) 29/151 (19) –

Hypertrophic cardiomyopathy 31/362 (9) 27/96 (28) <0.0001 31/362 (9) 30/151 (20) 0.0005

Atrioventricular septal defect 4/127 (3) no 4/127 (3) no

Facial keratosis pilaris (6) no (6) 21/151 (14) 0.0605

Curly hair (34) no (34) 44/151 (29) –

Cryptorchidism 82/104 (79) 33/58 (57) 0.0040 82/104 (79) 64/83 (77) –

Mental retardation 76/185 (41) 38/82 (46) – 76/185 (41) 32/105 (30) –

Bleeding diathesis 62/142 (44) 1/23 (4) 0.0001 62/142 (44) 37/151 (25) 0.0006

Distinct facial features 100/113 (88) 15/23 (65) 0.0096 100/113 (88) no

a See references 15–23.b See references 15, 17 and 18.c See reference 31.


These individuals display unremarkable GH se-

cretion after pharmacological stimuli, low se-

rum IGF-1 and ALS concentrations, but normal

IgFBP-3 level, which argue for a post receptor sig-

nalling GH resistance, specific for IgF-1 and ALS

and not for IgFBP-3 stimulation. Long term fol-

low-up of mutated patients indicate that the dis-

tribution 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 fre-

quencies of short stature, cardiac involvement,

PVS, cryptorchidism and mental retardation

similar to those in the general NS population

[31]. However, the prevalence of pectus anoma-

lies and HCM is less common in the mutated pa-

tients compared to the general NS population (p <

0.0001 and p = 0.0005, respectively), while bleed-

ing 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 sub-

jects 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 mani-

festations 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 sen-

sorineural 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 learn-

ing disabilities in the heterozygotes for the com-

mon 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 fa-

cies, dowslanting palpebral fissures, hyperte-

lorism, webbed neck, PVS, bleeding diathesis,

prominent corneal nerves, ectodermal involve-

ment with sparse and coarse hair, sparse eye-

brows and eyelashes, and developmental delay

[32]. This mutation was also found in his mother

and sister, who presented with a mild NS pheno-

type. Zenker et al. reported the G409A mutation

in several members of a family segregating a vari-

able and mild NS phenotype, comprising chest

deformities, pterygium colli, growth retardation

and mild craniofacial features [33]. The NS phe-

notype was not evident in the older sister and in

her mother who were heterozygous for this muta-

tion. It has been suggested that this partial phe-

notype resulted from incomplete penetrance of

this mutation, which has been never documented

so far in NS families. Altogether, these data indi-

cate that PTPN11 amino acid exchanges in a spe-

cific 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 cas-

es), is rare in NS subjects without juvenile myelo-

monocytic leukaemia (JMML) (2%) [24]. These


observations indicate that patients with the T73I

substitution are at risk of developing myeloprolifer-

ative disorders. However, haematological tumours

have been reported in NS patients carrying differ-

ent mutations [14]. Worthy of mention, the spec-

tra 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 malignan-

cies 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 observa-

tion points to a more severe perturbing effect on

the development and/or foetal survival of com-

pound heterozygotes and homozygotes for NS-

causing 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 de-

scribed in LS and NLS [14, 15, 25–27]. 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, includ-

ing cysts and other skeletal anomalies [14, 15,

27].

Functional data on PTPN11 mutants docu-

ment a close correlation between the identity of

the lesion and the disease and demonstrate that

NS-causative mutations are less effective in pro-

moting 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 genotype-

phenotype correlation results. However, it is still

unclear why the gain-of-function and apparent

loss-of-function of SHP-2 mutants result in sim-

ilar 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 pheno-

typic spectrum may be extremely wide, ranging

from mild to full-blown or severely affected NS

patients, as well as to NLS and LS. Allele specific-

ity of PTPN11 mutations in NS and LS is useful

in addressing the correct diagnosis in young and

borderline individuals and provides some sup-

port 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 differ-

ent mutations have been described in 18 sporadic

NS individuals, including a few with a pheno-

type 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 genotype-

phenotype correlation [7, 11, 36].


KRAS Gene Mutation

In general, the phenotype of NS patients carry-

ing a KRAS mutation is variable although often

severe, being characterized by short stature and

developmental delay in almost all individuals. No


familial case has been described, suggesting a re-

duced fitness of the patients with KRAS muta-

tion. 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 identi-

fied 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 ex-

hibited typical craniofacial features, short stat-

ure, macrocephaly, short/webbed neck, thorax

deformity and characteristic ocular anomalies.

These individuals had a mild to moderate men-

tal retardation and three of them also presented

with additional cerebral abnormalities, including

mild hydrocephalus, intracranial vascular anom-

alies and Dandy-Walker malformation [36]. Nava

et al. described four additional NS patients with

KRAS mutations [11]. All of them showed typi-

cal NS facies, no major skin involvement, marked

developmental delay and short stature, while PVS

was present in two, and mitral valve defect as-

sociated 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 spec-

trum [11].


The KRAS mutations reported in NS so far clus-

ter 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 muta-

tions are among the most common molecular le-

sions in human cancers, NS patients with KRAS

mutations usually are not affected by tumours.

Similar to the PTPN11 gene, there is great di-

vergence between the spectrum of somatic and

germline KRAS mutations. However, functional

data are showing that NS-associated KRAS mu-

tants, which are less active than their oncogenic

counterpart, may have different intrinsic GTPase

activities that might underlie the extreme pheno-

type in a few of these patients [7].

NS and the SOS1 Gene

Germline mutations in the SOS1 gene, encoding

a GEF protein, cause about 10–15% of all NS cas-

es (table 2) [9, 10, 28]. This figure changes in the

different studies, possibly because of non homo-

geneous inclusion criteria. In fact, the frequen-

cy 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 clini-

cally 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].


SOS1 Gene Mutation

Tartaglia et al. provided a detailed clinical de-

scription of 16 SOS1 mutation-positive indi-

viduals [9]. They showed a high prevalence of


CHDs (81%), primarily PVS (62%) and septal de-

fects (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 pi-

laris (50%) and curly hair (88%) were quite com-

mon, as well as macrocephaly (56%). Conversely,

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 8/15 (53) 43/130 (33) 8/15 (53) no

Fetal macrosomia 9/15 (60) no 9/15 (60) no

Short stature (<3rd) 19/53 (36) 84/115 (73) 0.0110 19/53 (36) 169/223 (76) <0.001

Macrocephaly 9/16 (56) 19/151 (12) 0.0002 9/16 (56) 9/23 (39) <0.0001

Downslanting palpebral fissures 15/16 (94) no 15/16 (94) 39/57 (68) 0.0537

Ptosis 16/16 (100) no 16/16 (100) 31/58 (53) 0.0003

Low-set ears with thickened helices 16/16 (100) no 16/16 (100) 61/72 (85) –

Thick lips/macrostomia 14/16 (88) no 14/16 (88) no

Pterygium colli no no no 40/130 (31)

Short neck no no no 16/37 (43)

Short/webbed neck 15/16 (94) no 15/16 (94) 23/44 (52) –

Thorax anomalies 33/40 (83) 144/151 (95) 0.08125 33/40 (83) 139/215 (65) 0.0280

Cardiac defects 23/28 (82) 132/151 (87) – 23/28 (82) 236/285 (83) –

Pulmonary valve stenosis 41/56 (73) 93/151 (62) – 41/56 (73) 247/362 (68) –

Septal defect 10/56 (18) 29/151 (19) – 10/56 (18) 81/340 (24) –

Hypertrophic cardiomyopathy 6/56 (11) 30/151 (20) – 6/56 (11) 31/362 (9) –

Atrioventricular septal defect no no no 4/127 (3)

Facial keratosis pilaris 8/16 (50) 21/151 (14) 0.0015 8/16 (50) (6) <0.0001

Curly hair 14/16 (88) 44/151 (29) <0.0001 14/16 (88) (34) <0.0001

Cryptorchidism 16/30 (53) 64/83 (77) 0.0194 16/30 (53) 82/104 (79) 0.0093

Mental retardation 10/51(19) 32/105 (30) – 10/51(19) 76/185 (41) 0.0050

Bleeding diathesis 13/56 (23) 37/151 (25) – 13/56 (23) 62/142 (44) 0.0090

Distinct facial features no no no 100/113 (88)

a See references 9, 10 and 28.b See reference 31.c See references 15–23.


mental retardation was present in only one indi-

vidual (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 macro-

cephaly and ectodermal manifestations were con-

cerned, but was clearly different, because both the

mental development and linear growth were pre-

served [9]. However, Roberts et al. have not con-

firmed this conclusion [10]. In fact, their 15 SOS1

mutated individuals displayed both facial charac-

teristics and other features overlapping those in

the general NS population. CHDs were present

in 83% of the patients, including PVS in 73%, fol-

lowed by HCM (20%) and ASD (13%). Short stat-

ure was present in 31%, cryptorchidism in 50%

and bleeding disorder/easy bruising in 17–40% 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 scru-

tiny of published pictures of the SOS1 mutated pa-

tients, both sparse eyebrows and curly hair could

be appreciated in a number of them [10]. The high

frequency of ectodermal anomalies in SOS1 posi-

tive 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 fre-

quency 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 anoma-

lies. Differences in patient selection and clinical

evaluation may account for some of these dis-

crepancies. In fact, Zenker et al. recruited several

patients from a paediatric endocrinology depart-

ment 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 man-

datory in order to delineate the eventual pheno-

type specificities and the differences in respect to

the general NS population. In table 2, the cumu-

lative frequencies of the 56 SOS1-positive patients

are reported. The overall prevalence of short stat-

ure 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 pto-

sis, ectodermal symptoms, normal intelligence

and low frequency of short stature.


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 involv-

ing the R552 residue, with three major mutation

clusters. Detailed clinical description was not

reported for all patients, and accordingly, gen-

otype-phenotype correlation is not available at

present between the different protein domains,

exons or single mutations.


The clinical spectrum associated with SOS1

gene mutations is broad, and although clear-

ly within the NS phenotype, resembles in part

CFCS for some dysmorphisms, including mac-

rocephaly and ectodermal manifestations, but is

clearly different in respect to development and

linear growth, which are preserved. The com-

parison between SOS1 mutation-positive cohorts

and either the PTPN11-positive or the general NS

population, displays a number of consistent dif-

ferences (table 2). The frequencies of short stat-

ure in SOS1-positive and PTPN11-positive patient

cohorts are significantly different (36 vs. 76%, p

< 0.001). Similarly, the prevalence of ptosis, tho-

racic 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, men-

tal retardation and bleeding diathesis. A statisti-

cally significant difference for short stature was

observed also comparing SOS1-positive individ-

uals 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 down-

stream signalling. Accordingly, NS-associated

mutants are hypermorphs. Comparison of the ef-

fect of five representative mutants on EGF stim-

ulated 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 knock-

in 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 mod-

el 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 re-

ported by two independent groups in less than

5% of sporadic and familial NS patients [12, 13].

Prevalence of RAF1 mutations, although some-

how discordant in the two cohorts (8 vs. 33%, to-

tal 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].


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 palpe-

bral fissures, ptosis and low set ears with thick-

ened helices (about 90% each) (fig. 1c). The neck

was short (76%) and the thorax abnormal in up

to 71% of the cases. Cryptorchidism was pres-

ent 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 café-au-lait spots were found in 8/21 pa-

tients (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 mis-

sense RAF1 mutation (T260I) in a male subject

in a cohort of 241 individuals with isolated HCM,

who developed ventricular hypertrophy at age of


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 report-

ed 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 in-

terrupted. Short stature was present in 91%, typi-

cal 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 par-

ticular HCM (78%), short stature (90%) and men-

tal retardation (55%) (table 3). NS patients also

present typical facial features (100%), in partic-

ular ptosis and downslanting palpebral fissures

(90%), short and webbed neck (83%), thoracic

anomalies (71%), macrocephaly (76%) and cryp-

torchidism (39%). Skin anomalies include curly

hair (22%) and hyperpigmented lesions in 1/3 of

the patients in the Pandit et al. series [12].


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 resi-

due S257, T260-P261 and L613. Clinical descrip-

tion of single mutated patients was provided and

accordingly, some genotype-phenotype correla-

tion 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-pos-

itive individuals, RAF1 mutated patients display

more frequently macrocephaly, ptosis and short

or webbed neck, but, similarly to SOS1 mutated

patients, less cryptorchidism and bleeding diath-

esis (table 3). Multiple nevi, lentigines and café-

au-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 predis-

position 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 resi-

dues, 3 of whom had PVS. Conversely, 25/28 NS

individuals with mutations clustering to resi-

dues S259 and S612 had HCM, and only two of

them had PVS [12, 13]. This allele specificity was

in part supported by functional studies indicat-

ing 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 ac-

tivity in respect to the wild-type protein both ba-

sally and after EGF stimulation, while the latter

was kinase impaired [12]. Razzaque et al. created

a morpholino antisense oligonucleotide knock-

down zebrafish model of Raf1 gene, and showed

that the Raf1 morphant had enlarged heart tube,

especially in the atrial region, which also was un-

bent compared to the wild type, further confirm-

ing 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


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 RAF1+ (%)a All NS (%)b p-Value RAF1+ (%)a PTPN11+ (%)c p-Value RAF1+ (%)a SOS1+ (%)d p-Value

Polyhydramnios 6/19 (32) 43/130 (33) – 6/19 (32) no 6/19 (32) 8/15 (53) –

Fetal macrosomia 6/20 (30) no 6/20 (30) no 6/20 (30) 9/15 (60) –

Short stature (<3rd) 28/31 (90) 84/115 (73) 0.0547 28/31 (90) 169/223 (76) – 28/31 (90) 19/53 (36) <0.0001

Macrocephaly 16/21 (76) 19/151 (12) <0.0001 16/21 (76) 9/23 (39) 0.0173 16/21 (76) 9/16 (56) –

Downslanting palpebral

fissures

19/21 (90) no 19/21 (90) 39/57 (68) – 19/21 (90) 15/16 (94) –

Ptosis 19/21 (90) no 19/21 (90) 31/58 (53) 0.0031 19/21 (90) 16/16 (100) –

Low-set ears with thickened

helices

18/21 (86) no 18/21 (86) 61/72 (85) – 18/21 (86) 16/16 (100) –

Thick lips/macrostomia 9/21 (43) no 9/21 (43) no 9/21 (43) 14/16 (88) 0.0073

Pterygium colli no no no 40/130 (31) no no

Short neck no no no 16/37 (43) no no

Short/webbed neck 25/30 (83) no 25/30 (83) 23/44 (52) 0.0069 25/30 (83) 15/16 (94) –

Thorax anomalies 20/28 (71) 144/151 (95) 0.0004 20/28 (71) 139/215 (65) – 20/28 (71) 33/40 (83) –

Cardiac defects 30/32 (93) 132/151 (87) – 30/32 (93) 236/285 (83) – 30/32 (93) 23/28 (82) –

Pulmonary valve stenosis 4/32 (13) 93/151 (62) <0.0001 4/32 (13) 247/362 (68) <0.0001 4/32 (13) 41/56 (73) <0.0001

Septal defect 10/32 (31) 29/151 (19) – 10/32 (31) 81/340 (24) – 10/32 (31) 10/56 (18) –

Hypertrophic cardiomyopathy 25/32 (78) 30/151 (20) <0.0001 25/32 (78) 31/362 (9) <0.0001 25/32 (78) 6/56 (11) <0.0001

Atrioventricular septal defect no no no 4/127 (3) no no

Facial keratosis pilaris 2/21 (10) 21/151 (14) – 2/21 (10) 6% – 2/21 (10) 8/16 (50) 0.0095

Curly hair 7/32 (22) 44/151 (29 – 7/32 (22) 34% – 7/32 (22) 14/16 (88) <0.0001

Cryptorchidism 7/18 (39) 64/83 (77) 0.0032 7/18 (39) 82/104 (79) 0.0010 7/18 (39) 16/30 (53) –

Mental retardation 17/31 (55) 32/105 (30) 0.0187 17/31 (55) 76/185 (41) – 17/31 (55) 10/51(19) 0.0015

Bleeding diathesis 1/21 (5) 37/151 (25) 0.0482 1/21 (5) 62/142 (44) 0.0005 1/21 (5) 13/56 (23) –

Distinct facial features 11/11 (100) no 11/11 (100) 100/113 (88) – 11/11 (100) no

a See references 12 and 13.b See reference 31.c See references 15–23.d See references 9, 10 and 28.


change, identified in two NS patients aged 3 and

5 years [12, 13]. Accordingly, there is no appar-

ent allele specificity for LS associated with RAF1

mutations. However, the NS patients with S257L

reported by Pandit et al. were all young (aged 3–8

years), showing high prevalence of skin features

(5/7, 71%) [12]. Accordingly, a long term follow-

up 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 occur-

ring 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 ac-

tivation 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 14–3–3-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].

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 in-

telligence, a hyperactivity-attention deficit dis-

order, 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

6 years. Both patients carried the novel D67N

change. Interestingly, even though the same mu-

tation has been reported concurrently in a sin-

gle CFCS patient as well, clinical feature of these

NS patients are overlapping only in part those of

CFCS [11].

Conclusions

A wide variation of phenotypes and natural histo-

ries is apparent among the NS patients heterozy-

gous for PTPN11, KRAS, SOS1, RAF1 and MEK1

gene mutations. The cumulative data point to

some genotype-phenotype correlations. In par-

ticular, PTPN11 gene mutations, which are re-

sponsible for almost half of these patients, result

in a wide spectrum of anomalies, including a high

prevalence of PVS, typical facial features, cryp-

torchidism and bleeding diathesis. Mutations in

SOS1, the second most common anomaly, are as-

sociated with a low frequency of short stature and

mental retardation, and a high frequency of pto-

sis, 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 muta-

tions 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 clini-

cal 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 pheno-

type, in agreement with the specific functional

role of the mutant.


References

Allanson J: Noonan syndrome; in 1 Cassidy SB, Allanson JE (eds): Man-agement of Genetic Syndromes (Wiley-Liss, NY 2005).Sharland M, Burch M, McKenna WM, 2 Patton MA: A clinical study of Noonan syndrome. Arch Dis Child 1992;67: 178–183.Allanson JE: Noonan syndrome. J Med 3 Genet 1987;24:9–13.Allanson JE, Hall JG, Hughes HE, 4 Preus M, Witt RD: Noonan syndrome: the changing phenotype. Am J Med Genet 1985;21:507–514.Bentires-Alj M, Kontaridis MI, Neel 5 BG: Stops along the RAS pathway in human genetic disease. Nat Med 2006;12:283–285.Tartaglia M, Mehler EL, Goldberg R, 6 Zampino G, Brunner HG, et al: Muta-tions in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 2001;29:465–468.Schubbert S, Zenker M, Rowe SL, Boll 7 S, Klein C, et al: Germline KRAS muta-tions cause Noonan syndrome. Nat Genet 2006;38:331–336.

Carta C, Pantaleoni F, Bocchinfuso G, 8 Stella L, Vasta I, et al: Germline mis-sense mutations affecting KRAS Iso-form B are associated with a severe Noonan syndrome phenotype. Am J Hum Genet 2006;79:129–135.Tartaglia, Pennacchio LA, Zhao C, Ya- 9 dav KK, Fodale V, et al: Gain-of-func-tion SOS1 mutations cause a distinc-tive form of Noonan syndrome. Nat Genet 2007;39:75–79. Roberts AE, Araki T, Swanson KD, 10 Montgomery KT, Schiripo TA, et al: Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet 2007;39:70–74. Nava C, Hanna N, Michot C, Pereira S, 11 Pouvreau N, et al: Cardio-facio-cuta-neous and Noonan syndromes due to mutations in the RAS/MAPK signal-ling pathway: genotype phenotype re-lationships and overlap with Costello syndrome. J Med Genet 2007;44:763–771.Pandit B, Sarkozy A, Pennacchio LA, 12 Carta C, Oishi K, et al: Gain-of-func-tion RAF1 mutations cause Noonan and LEOPARD syndromes with hyper-trophic cardiomyopathy. Nat Genet 2007;39:1007–1012.

Razzaque MA, Nishizawa T, Komoike 13 Y, Yagi H, Furutani M, et al: Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet 2007;39:1013–1017.Tartaglia M, Gelb BD: Noonan syn-14 drome and related disorders: genetics and pathogenesis. Annu Rev Genomics Hum Genet 2005;6:45–68.Tartaglia M, Kalidas K, Shaw A, Song 15 X, Musat DL, et al: PTPN11 mutations in Noonan syndrome: molecular spec-trum, genotype-phenotype correla-tion, and phenotypic heterogeneity. Am J Hum Genet 2002;70:1555–1563.Musante L, Kehl HG, Majewski F, Mei-16 necke P, Schweiger S, et al: Spectrum of mutations in PTPN11 and geno-type-phenotype correlation in 96 pa-tients with Noonan syndrome and five patients with cardio-facio-cutaneous syndrome. Eur J Hum Genet 2003;11:201–206.Zenker M, Buheitel G, Rauch R, Koen-17 ig R, Bosse K, et al: Genotype-pheno-type correlations in Noonan syndro-me. J Pediatr 2004;144:368–374.

The results of genotype-phenotype correla-

tions 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 pa-

tients, independently from their clinical charac-

teristics. 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 per-

formed in patients with PVS, CFCS ectodermal

features, normal growth and development, while

the RAF1 gene screening should be done in indi-

viduals 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 pa-

tients should follow the mutation prevalence of

the RAS gene cascade, including first SOS1 and

then RAF1, KRAS and, finally, MEK1, at least un-

til 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 Italia-Usa, Malattie Rare’ to A.S.) and Italian Ministry of University and Research (‘Progetto Ateneo 2007’, to B.D.; ‘Idea Progettuale 2006’ to B.D.).


Maheshwari M, Belmont J, Fernbach S, 18 Ho T, Molinari L, et al: PTPN11 muta-tions in Noonan syndrome type I: de-tection of recurrent mutations in exons 3 and 13. Hum Mutat 2002;20:298–304.Sarkozy A, Conti E, Seripa D, Digilio 19 MC, Grifone N, et al: Correlation be-tween PTPN11 gene mutations and congenital heart defects in Noonan and LEOPARD syndromes. J Med Gen-et 2003;40:704–708.Jongmans M, Sistermans EA, Rikken 20 A, Nillisen WM, Tamminga R, et al: Genotypic and phenotypic character-ization of Noonan syndrome: new data and review of the literature. Am J Med Genet A 2005;134:165–170.Shaw AC, Kalidas K, Crosby AH, Jef-21 fery S, Patton MA: The natural history of Noonan syndrome: a long-term fol-low-up study. Arch Dis Child 2007;92:128–132.Bertola DR, Pereira AC, Albano LM, 22 De Oliveira PS, Kim CA, Krieger JE: PTPN11 gene analysis in 74 Brazilian patients with Noonan syndrome or Noonan-like phenotype. Genet Test 2006;10:186–191.Sznajer Y, Keren B, Baumann C, Perei-23 ra S, Alberti C, et al: The spectrum of cardiac anomalies in Noonan syn-drome as a result of mutations in the PTPN11 gene. Pediatrics 2007;119: e1325–e1331.Tartaglia M, Martinelli S, Stella L, 24 Bocchinfuso G, Flex E, et al: Diversity and functional consequences of ger-mline and somatic PTPN11 mutations in human disease. Am J Hum Genet 2006;78:279–290.

Digilio MC, Conti E, Sarkozy A, Min-25 garelli 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.Sarkozy A, Conti E, Digilio MC, Ma-26 rino B, Morini E, et al: Clinical and molecular analysis of 30 patients with multiple lentigines LEOPARD syn-drome. J Med Genet 2004;41:e68.Sarkozy A, Obregon MG, Conti E, Es-27 posito G, Mingarelli R, Pizzuti A, Dal-lapiccola B: A novel PTPN11 gene mu-tation bridges Noonan syndrome, multiple lentigines/LEOPARD syn-drome and Noonan-like/multiple giant cell lesion syndrome. Eur J Hum Genet 2004;12:1069–1072.Zenker M, Horn D, Wieczorek D, Al-28 lanson J, Pauli S, et al: SOS1 is the sec-ond most common Noonan gene but plays no major role in cardio-facio-cu-taneous syndrome. J Med Genet 2007;44:651–656.Ferreira LV, Souza SA, Arnhold IJ, 29 Mendonca BB, Jorge AA: PTPN11 (protein tyrosine phosphatase, nonre-ceptor type 11) mutations and re-sponse to growth hormone therapy in children with Noonan syndrome. J Clin Endocrinol Metab 2005;90:5156–5160.Limal J-M, Parfait B, Carbol S, Bonnet 30 D, Leheup B, et al: Noonan syndrome: relationships between genotype, growth, and growth factors. J Clin En-docrinol Metab 2006;91:300–306.

Sharland M, Burch M, McKenna WM, 31 Paton MA: A clinical study of Noonan syndrome. Arch Dis Child 1992;67: 178–183.Bertola DR, Pereira AC, de Oliveira PS, 32 Kim CA, Krieger JE: Clinical variabil-ity in a Noonan syndrome family with a new PTPN11 gene mutation. Am J Med Genet A 2004;130:378–383.Zenker M, Voss E, Reis A: Mild vari-33 able Noonan syndrome in a family with a novel PTPN11 mutation. Eur J Med Genet 2007;50:43–47.Becker K, Hughes H, Howard K, Arm-34 strong M, Roberts D, et al: Early fetal death associated with compound heterozygosity for Noonan syndrome-causative PTPN11 mutations. Am J Med Genet A 2007;143:1249–1252.Digilio MC, Sarkozy A, de Zorzi A, 35 Pacileo G, Limongelli G, et al: LEOP-ARD syndrome: clinical diagnosis in the first year of life. Am J Med Genet A 2006;140:740–746.Zenker M, Lehmann K, Schulz AL, 36 Barth H, Hansmann D, et al: Expan-sion of the genotypic and phenotypic spectrum in patients with KRAS ger-mline mutations. J Med Genet 2007;44:131–135.Araki T, Mohi MG, Ismat FA, Bronson 37 RT, Williams IR, et al: Mouse model of Noonan syndrome reveals cell type- and gene dosage-dependent effects of Ptpn11 mutation. Nat Med 2004;10:849–857.

Anna Sarkozy

CSS-Mendel Institute

Viale Regina Elena 261

IT–00198 Rome (Italy)

Tel. +39 0644160536, Fax +39 0644160548, E-Mail [email protected]



LEOPARD Syndrome: Clinical Aspects and Molecular Pathogenesis

A. Sarkozya � M.C. Digiliob � G. Zampinoc �

B. Dallapiccolaa � M. Tartagliad � B.D. Gelbe

aIRCCS-CSS, San Giovanni Rotondo, and Dipartimento di Medicina Sperimentale e Patologia,

Università ‘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 Gesù’, Rome, cIstituto di Clinica Pediatrica, Università Cattolica del Sacro Cuore,

Rome, and dDipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanità, Rome, Italy; eCenter for Molecular Cardiology, Departments of Pediatrics and Genetics & Genomic Sciences,

Mount Sinai School of Medicine, New York, N.Y., USA

AbstractLEOPARD syndrome (LS) is an autosomal dominant disor-

der for which the acronymic name denotes major clinical

characteristics including lentigines, facial dysmorphism,

heart defects, cryptorchidism, short stature and sen-

sorineural deafness. Café-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 Noo-

nan syndrome (NS) and distinguishing the two in infants

and young children before lentigines emerge can be chal-

lenging. LS, like NS, arises from dysregulated RAS/mitogen-

activated protein kinase (MAPK) signaling. Mutations in the

PTPN11 gene, which encodes the protein tyrosine phos-

phatase 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 associ-

ated 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 RAS-

MAPK signaling. While biochemical differences between

their alleles exist, a fuller explanation of their respective

disease pathogenesis awaits elucidation.


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 multi-

ple lentigines, increasing in number from birth

to puberty, pectus carinatum, hypertelorism and

mandibular prognathism [1]. In 1962, cardiac ab-

normalities and short stature were first associat-

ed with this condition [2]. Gorlin et al. reviewed

all reported patients supporting the concept of a

more generalized condition and coined the ac-

ronym LEOPARD, a mnemonic for the major

features of this disorder: multiple Lentigines,

Electrocardiographic (ECG) conduction ab-

normalities, Ocular hypertelorism, Pulmonic

56 Sarkozy � Digilio � Zampino � Dallapiccola � Tartaglia � Gelb

stenosis, Abnormal genitalia, Retardation of

growth, and sensorineural Deafness [3, 4]. LS is

also known as multiple lentigines syndrome, car-

diocutaneous syndrome, lentiginosis profusa or

progressive cardiomyopathic lentiginosis [5–8].

The population prevalence of LS is unknown

but estimates would depend on the age of assess-

ment because the diagnosis can be challenging

in infants and young children. LS is an auto-

somal dominant, fully penetrant trait with many

reported familial cases. It is likely that the per-

centage of sporadic cases is similar to the 35–50%

rate observed with other autosomal dominant

disorders.

Clinical Description

As suggested by the disease’s acronym, affected

patients present with a wide clinical spectrum [3,

4, 9, 10], including multiple lentigines, facial dys-

morphism, 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 fea-

tures, 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%), pto-

sis (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 triangu-

lar face, prominent eyes and ptosis, become evi-

dent 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 atrioven-

tricular 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 repolar-

ization abnormalities (42%) [12]. Conduction de-

fects, 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 pa-

tients with cardiac involvement (about 60% of all

LS cases) [11, 12, 14 and our unpublished data]

and represents the major potentially life-threat-

ening aspect of this condition. The left ventric-

ular 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 pres-

ence of a significant left ventricular-aortic gradi-

ent include beta-blockade and calcium channel

blockers, while surgical removal of the left ven-

tricular outflow obstruction is indicated in the

absence of any significant improvement follow-

ing pharmacological treatment. HCM can result

in fatal events and, accordingly, a careful risk as-

sessment and prophylaxis against sudden death

in patients at risk is recommended [12, 13, 16].

Mitral valve anomalies are often diagnosed in as-

sociation with HCM. Morphological mitral valve

abnormalities, such as mitral valve prolapse or

LEOPARD Syndrome: Clinical Aspects and Molecular Pathogenesis 57

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

10–20% of LS cases [12, 14], a figure much low-

er 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 atrioventricu-

lar septal defects, coronary artery abnormalities,

apical aneurysm, non-compaction of the left ven-

tricle, multiple ventricular septal defects, isolated

left ventricular enlargement and endocardial fi-

broelastosis [9, 12].

Multiple lentigines are the most distinctive

feature of LS, but are not pathognomonic of this

condition as they occur in association with oth-

er conditions such as the Carney Complex and

Peutz-Jeghers syndrome [1, 9]. Lentigines are dis-

persed 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, len-

tigines are characterized by pigment accumula-

tion 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 4–5 years, increas-

ing into the thousands by puberty, and seem to

arise independent of sun exposure. Quite excep-

tionally, older children may not exhibit multiple

lentigines, but no adult without lentigines has

been reported. In our cohort of patients, two in-

dividuals with the clinical and molecular diagno-

sis of LS still had no lentigines at age of 11 and 14

years, respectively. Cafè-au-lait spots (CLS) oc-

cur in about half of LS patients. They normally

b

c da

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


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 pa-

tients may manifest some hypopigmented skin

lesions [6]. Total UVA-UVB protection is recom-

mended 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 hy-

perelastic (67%) [11].

In LS, birth weight is generally normal or

above the average (37%) [11]. Retardation of

growth appears subsequently, having been re-

ported in a variable proportion of these patients

(13–60%) [9–11, 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 puber-

ty. In the presence of a significant growth delay,

GH therapy may be indicated until adult height

is reached. Cardiac status, however, should be as-

sessed 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 (30–75% of affected individuals) [11].

These defects are common in newborns (75%) [7].

Other less frequent skeletal abnormalities include

mandibular prognathism, winging of the scapu-

lae, scoliosis and joint hyperflexibility [19].

Sensorineural deafness is detected in 15–25%

of the cases [9, 10, 14]. Deafness is usually diag-

nosed at birth or during childhood, but may de-

velop 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 develop-

ment [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 bi-

lateral cryptorchidism, and, less frequently, hy-

pospadias 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 kid-

ney, are infrequent (12%) [10, 11].

Hematological/oncological complications,

such as myelodysplasia, acute myelogenous leuke-

mia and neuroblastoma, have been reported oc-

casionally in LS [14, 24, 25]. Recently, a malignant

melanoma was diagnosed in a woman with LS,

who had a germline PTPN11 mutation and a so-

matic 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 periph-

eral aneurysms, congenital intrahepatic porto-

systemic venous shunt, and dental anomalies,

such as delayed dental development, possible

agenesis of permanent teeth or supernumerary

teeth [28–32].

Clinical Diagnosis

Voron et al. outlined diagnostic criteria for LS [10].

According to these recommendations, the clini-

cal diagnosis of LS may be suspected in the pres-

ence 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 emerg-

ing data about the progressive nature of this con-

dition indicate, however, that these criteria may

not permit the diagnosis of LS in young patients

manifesting partial phenotypes, particularly


when their case is sporadic. With the advent of

molecular testing, the frequency of misdiagno-

sis 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 syn-

drome (NS; OMIM 163950) including Noonan-

like/multiple giant cell lesions syndrome (NL/

MGCLS; OMIM 163955), neurofibromatosis type

1 (NF1; OMIM 162200) including neurofibroma-

tosis-Noonan syndrome (NFNS; OMIM 601321),

Costello syndrome (CS; OMIM 218040), and car-

dio-facio-cutaneous syndrome (CFCS; OMIM

115150). All of these conditions have been recent-

ly grouped in the ‘neuro-cardio-facio-cutaneous’

syndrome (NCFCS) family [33]. These disorders

share facial anomalies, CHD and growth retar-

dation, as well as skin, skeletal and genitouri-

nary anomalies, and variable degrees of mental

retardation. A few distinct features, however, are

useful handles for differentiating them. LS over-

laps with NS most closely [20, 21]. Unlike LS, NS

patients manifest characteristic facial features at

birth and during childhood and affected individ-

uals 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 cu-

taneous manifestations, such as CLS and multiple

lentigines, HCM and deafness. Differentiating LS

from NS can be extremely difficult in young indi-

viduals 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 pa-

tients supports this.

NFNS is a rare condition characterized by fea-

tures of NS and neurofibromatosis type 1, includ-

ing CLS, neurofibromas, central nervous system

and skeletal anomalies [34, 35]. Molecular dis-

tinction 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 re-

currence 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 cyto-

plasmic Src homology 2 (SH2) domain-contain-

ing protein tyrosine phosphatases functioning as

intracellular signal transducers. SHP-2’s struc-

ture comprises two tandemly arranged SH2 do-

mains 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 selec-

tively bind to short amino acid motifs containing

phosphotyrosyl residues and promote SHP-2’s as-

sociation with cell surface receptors, cell adhesion


molecules and scaffolding adapters. The N-SH2

domain also interacts with the PTP domain us-

ing a separate site, which functions as an intra-

molecular switch controlling SHP-2’s 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 phos-

photyrosyl ligand promotes a conformational

change of the domain that weakens the auto-in-

hibiting intramolecular interaction, making the

catalytic site available to substrate, thereby acti-

vating the phosphatase [39].

SHP-2 positively modulates signal flow in most

circumstances but can also function as a nega-

tive regulator depending upon its binding part-

ner 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 [40–46], as well

as IL-1 and TNF-dependent NF-κB activation

[47] and PDGF- and FGF2-induced Ca2+ signal-

ing [48], while it negatively regulates STAT func-

tion [49–51] and IFNα signaling [52]. Similarly,

SHP-2 seems to play either a positive or a nega-

tive role in PI3K signaling downstream from ac-

tivated growth factor receptors [53, 54]. In most

cases, SHP-2’s function in intracellular signaling

appears to be immediately distal to activated re-

ceptors 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 estab-

lished that PTPN11 mutations cause NS [56].

Subsequently, they and others documented that

PTPN11 mutations underlie approximately 50%

of NS cases [57–61]. 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 muta-

tions affect residues located in the N-SH2 or PTP

domains and involved in the N-SH2/PTP inter-

domain 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 recur-

rent occurrences.

Digilio and co-workers screened eight inde-

pendent 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 phe-

notype 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 di-

agnosis 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 cas-

es. Y279C and T468M represent the most com-

mon defects, although a few additional mutations

N-SH2 C-SH2

Y279S

Y279S A461TG464A

R498W,L

Q506PQ510E,G

T468M

PTP

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 = pro-

tein tyrosine phosphatase.


(Y279S, A461T, G464A, R498W/L, Q506P, and

Q510E/G) have been documented subsequently

(fig. 2) [14, 18, 38, 62–66]. These results suggest

specificity for these mutations for the develop-

ment of lentigines, CLS, HCM, conduction ab-

normalities, 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 phospho-

rylate the dual specificity kinase MEK, which in

turn activates MAPK. Unlike ARAF and BRAF,

RAF1 is expressed ubiquitously. The RAF pro-

teins 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 resi-

dues, and CR3 contains the kinase domain.

RAF1 is highly regulated with numerous Ser

and Thr residues that can be phosphorylated, re-

sulting in activation or inactivation [68]. Among

these, Ser259, which resides in CR2 (fig. 3), is par-

ticularly important. In its inactive conformation,

the N-terminal portion of RAF1 is thought to in-

teract with and inactivate the kinase domain at

the C-terminus. This conformation is stabilized

by 14–3–3 protein dimers that bind to phosphory-

lated 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 RAS-

GTP at the membrane and subsequent propa-

gation of the signal through the RAS-MAPK

cascade via RAF1’s 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 pre-

mised on their discovery of RAF1 mutations in

approximately 3% of patients with NS. They discov-

ered missense defects in two patients, predicting

S257L and L613V substitutions in RAF’s CR2 and

C-terminal domains, respectively.

Genotype-Phenotype Correlations

LS patients with PTPN11 mutations show a full-

blown 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 com-

pared to those with missense mutations affecting

residue Tyr279 (26 vs. 47% and 9 vs. 24%, respec-

tively) [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 develop-

ment. Other reports indicate that mutations af-

fecting residue Gln510 are often associated with

an important cardiac phenotype, characterized

by rapidly progressive severe biventricular ob-

structive HCM, often with prenatal onset [71, 72

and our personal observation]. LS patients with-

out 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 infer-

ences can be made.

S621S259

S257L L613V

CR3CR2CR1

RBD CRD Activationsegment

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 RAF1’s regulation

below. CR = Conserved region; RBD = Ras binding do-

main; CRD = cyteine-rich domain.


Consequences of LEOPARD Syndrome-

Causing Mutations on RAS Signaling

PTPN11

Three LS-associated SHP-2 mutants, Y279C,

T468M and Q510P, have been expressed in bac-

teria and/or eukaryotic cells, and phosphatase ac-

tivities assayed from isolated SHP-2 proteins or

immunocomplexes [73–75]. All studies demon-

strated extremely low basal phosphatase activi-

ties for the Y279C and T468M mutants and that

these proteins could be stimulated with phos-

phopeptides 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 mu-

tants decreased activation of MAPK in a domi-

nant 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 ob-

served for SHP-2 mutants relevant for NS.

NS-associated SHP-2 mutants engender gain-

of-function effects with increased phosphatase

activity and increased activation of RAS-MAPK

signaling as measured by MAPK phosphoryla-

tion status [74, 76, 77]. This has been confirmed

in a mouse model in which an NS-causing mu-

tation, D61G, was introduced into the Ptpn11

gene [78]. The D61G heterozygous mice, which

exhibit many of the hallmark features of the dis-

order, 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 engi-

neered to be hemizygous for Shp-2 have no phe-

notype [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 ob-

served in LS [74]. Thus, loss of SHP-2 activity

per se seems unlikely to explain the pathogen-

esis of this disorder. Rather, mechanisms such as

the dominant negative effects on MAPK activa-

tion and increased binding to docking partners

[73] are being explored in ongoing animal mod-

eling studies.

RAF1

The two RAF1 mutants observed in LS, S257L

and L613V, have been expressed in eukaryot-

ic cells [70, 80]. The S257L RAF1 had normal

phosphorylation at Ser259, but failed to bind

14–3–3 and had increased kinase activity [80].

This mutant protein was more readily recruit-

ed from the cytosol to the plasma membrane by

activated RAS and resulted in increased down-

stream signaling to MAPK. The mechanism for

this gain of function relates to loss of 14–3–3

binding at Ser259, which normally serves to in-

activate RAF1.

The L613V RAF1 mutant also possessed in-

creased kinase activity and induced increased

MAPK activation [70]. Binding of 14–3–3 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 be-

haved 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 dispar-

ity of effects noted among the SHP-2 mutants

associated with LS and NS. Aside from provid-

ing 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 de-

termines whether an individual manifests LS ver-

sus NS.


References

Zeisler EP, Becker SW: Generalized 1 lentigo: Its relation to systemic nonel-evated nevi. Archiv Derm Syphilol 1936;33:109–125.Moynahan EJ: Multiple symmetrical 2 moles, with psychic and somatic infan-tilism and genital hypoplasia: First male case of a new syndrome. Proc R Soc Med 1962;55:959–960.Gorlin RJ, Anderson RC, Blaw M: Mul- 3 tiple lentigenes syndrome. Am J Dis Child 1969;117:652–662.Gorlin RJ, Anderson RC, Moller JH: 4 The Leopard (multiple lentigines) syn-drome revisited. Birth Defects Orig Artic Ser 1971;07:110–115.Polani PE, Moynahan EJ: Progressive 5 cardiomyopathic lentiginosis. Q J Med 1972;41:205–225.Selmanowitz VJ, Orentreich N, Felsen- 6 stein JM: Lentiginosis profusa syn-drome (multiple lentigines syndrome). Arch Dermatol 1971;104:393–401.Seuanez H, Mane-Garzon F, Kolski R: 7 Cardio-cutaneous syndrome (the ‘LEOPARD’ Syndrome). Review of the literature and a new family. Clin Genet 1976;9:266–276.Walther RJ, Polansky BJ, Grotis IA: 8 Electrocardiographic abnormalities in a family with generalized lentigo. N Engl J Med 1966;275:1220–1225.Coppin BD, Temple IK: Multiple len- 9 tigines syndrome (LEOPARD syn-drome or progressive cardiomyopathic lentiginosis). J Med Genet 1997;34: 582–586.Voron DA, Hatfield HH, Kalkhoff RK: 10 Multiple lentigines syndrome. Case report and review of the literature. Am J Med 1976;60:447–456.Digilio MC, Sarkozy A, de Zorzi A, 11 Pacileo G, Limongelli G, et al: Leopard syndrome: Clinical diagnosis in the first year of life. Am J Med Genet A 2006;140:740–746.

Limongelli G, Pacileo G, Marino B, 12 Digilio MC, Sarkozy A, et al: Preva-lence and clinical significance of car-diovascular abnormalities in patients with the LEOPARD syndrome. Am J Cardiol 2007;100:736–741.Somerville J, Bonham-Carter RE: The 13 heart in lentiginosis. Br Heart J 1972;34:58–66.Sarkozy A, Conti E, Digilio MC, Ma-14 rino B, Morini E, et al: Clinical and molecular analysis of 30 patients with multiple lentigines LEOPARD syn-drome. J Med Genet 2004;41:e68.Elliott P, McKenna WJ: Hypertrophic 15 cardiomyopathy. Lancet 2004;363: 1881–1891.Woywodt A, Welzel J, Haase H, Duer-16 holz A, Wiegand U, Potratz J, Sheikhzadeh A: Cardiomyopathic len-tiginosis/LEOPARD syndrome pre-senting as sudden cardiac arrest. Chest 1998;113:1415–1417.Kontoes PP, Vlachos SP, Marayiannis 17 KV: Intense pulsed light for the treat-ment of lentigines in LEOPARD syn-drome. Br J Plast Surg 2003;56: 607–610.Keren B, Hadchouel A, Saba S, Sznajer 18 Y, Bonneau D, et al: PTPN11 muta-tions in patients with LEOPARD syn-drome: A french multicentric experi-ence. J Med Genet 2004;41:e117.Gorlin RJ, Cohen MM, Jr., Hennekam 19 RCM (eds.): Syndromes of the head and neck. LEOPARD syndrome (mul-tiple lentigines syndrome, progressive cardiomyopathic lentiginosis); pp. 555–558 (Oxford University Press, New York 2001).Allanson JE: Noonan syndrome; in 20 Cassidy SB, Allanson JE (eds.): Man-agement of Genetic Syndromes, pp. 385–398 (John Wiley & Sons, Inc., Hoboken, NJ 2005).van der Burgt I: Noonan syndrome. 21 Orphanet J Rar Dis 2007;2:1–6.

Agha A, Hashimoto K: Multiple len-22 tigines (Leopard) syndrome with Chi-ara I malformation. J Dermatol 1995;22:520–523.Bonioli E, Di Stefano A, Costabel S, 23 Bellini C: Partial agenesis of corpus callosum in LEOPARD syndrome. Int J Dermatol 1999;38:855–862.Merks JH, Caron HN, Hennekam RC: 24 High incidence of malformation syn-dromes in a series of 1,073 children with cancer. Am J Med Genet A 2005;134:132–143.Ucar C, Calyskan U, Martini S, Hein-25 ritz W: Acute myelomonocytic leuke-mia in a boy with LEOPARD syndrome (PTPN11 gene mutation positive). J Pediatr Hematol Oncol 2006;28: 123–125.Seishima M, Mizutani Y, Shibuya Y, 26 Arakawa C, Yoshida R, Ogata T: Malig-nant melanoma in a woman with LEOPARD syndrome: Identification of a germline PTPN11 mutation and a somatic BRAF mutation. Br J Derma-tol 2007;157:1297–1299.Choi WW, Yoo JY, Park KC, Kim KH: 27 LEOPARD syndrome with a new asso-ciation of congenital corneal tumor, choristoma. Pediatr Dermatol 2003;20:158–160.Digilio MC, Capolino R, Marino B, 28 Sarkozy A, Dallapiccola B: Congenital intrahepatic portosystemic venous shunt: An unusual feature in LEOP-ARD syndrome and in neurofibroma-tosis type 1. Am J Med Genet A 2005;134:457–458.Ho IC, O’Donnell D, Rodrigo C: The 29 occurrence of supernumerary teeth with isolated, nonfamilial leopard (multiple lentigines) syndrome: Report of case. Spec Care Dentist 1989;9:200–202.Munshi A, Munshi AK: Leopard syn-30 drome – report of a variant case. J In-dian Soc Pedod Prev Dent 1999;17:5–8.

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.).


Yagubyan M, Panneton JM, Lindor 31 NM, Conti E, Sarkozy A, Pizzuti A: LEOPARD syndrome: A new polyan-eurysm association and an update on the molecular genetics of the disease. J Vasc Surg 2004;39:897–900.Yam AA, Faye M, Kane A, Diop F, 32 Coulybaly-Ba D, et al: Oro-dental and craniofacial anomalies in LEOPARD syndrome. Oral Dis 2001;7:200–202.Bentires-Alj M, Kontaridis MI, Neel 33 BG: Stops along the RAS pathway in human genetic disease. Nat Med 2006;12:283–285.Friedman JM, Birch PH: Type 1 neuro-34 fibromatosis: A descriptive analysis of the disorder in 1,728 patients. Am J Med Genet 1997;70:138–143.Opitz JM, Weaver DD: The neurofibro-35 matosis-Noonan syndrome. Am J Med Genet 1985;21:477–490.Sarkozy A, Schirinzi A, Lepri F, Bot-36 tillo I, De Luca A, et al: Clinical lump-ing and molecular splitting of LEOP-ARD and NF1/NF1-Noonan syndromes. Am J Med Genet A 2007;143:1009–1011.Digilio MC, Conti E, Sarkozy A, Min-37 garelli 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.Legius E, Schrander-Stumpel C, Schol-38 len E, Pulles-Heintzberger C, Gewillig M, Fryns JP: PTPN11 mutations in LEOPARD syndrome. J Med Genet 2002;39:571–574.Hof P, Pluskey S, Dhe-Paganon S, Eck 39 MJ, Shoelson SE: Crystal structure of the tyrosine phosphatase SHP-2. Cell 1998;92:441–450.Cunnick JM, Meng S, Ren Y, Desponts 40 C, Wang HG, Djeu JY, Wu J: Regula-tion of the mitogen-activated protein kinase signaling pathway by SHP2. J Biol Chem 2002;277:9498–9504.Maroun CR, Naujokas MA, Holgado-41 Madruga M, Wong AJ, Park M: The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis down-stream from the met receptor tyrosine kinase. Mol Cell Biol 2000;20: 8513–8525.

Saxton TM, Henkemeyer M, Gasca S, 42 Shen R, Rossi DJ, et al: Abnormal me-soderm patterning in mouse embryos mutant for the SH2 tyrosine phos-phatase Shp-2. EMBO J 1997;16: 2352–2364.Shi ZQ, Yu DH, Park M, Marshall M, 43 Feng GS: Molecular mechanism for the Shp-2 tyrosine phosphatase function in promoting growth factor stimula-tion of Erk activity. Mol Cell Biol 2000;20:1526–1536.MacGillivray M, Herrera-Abreu MT, 44 Chow CW, Shek C, Wang Q, et al: The protein tyrosine phosphatase SHP-2 regulates interleukin-1-induced ERK activation in fibroblasts. J Biol Chem 2003;278:27190–27198.Yu WM, Hawley TS, Hawley RG, Qu 45 CK: Catalytic-dependent and -inde-pendent roles of SHP-2 tyrosine phos-phatase in interleukin-3 signaling. Oncogene 2003;22:5995–6004.Easton JB, Royer AR, Middlemas DS: 46 The protein tyrosine phosphatase, Shp2, is required for the complete acti-vation of the RAS/MAPK pathway by brain-derived neurotrophic factor. J Neurochem 2006;97:834–845.You M, Flick LM, Yu D, Feng GS: Mod-47 ulation of the nuclear factor kappa B pathway by Shp-2 tyrosine phos-phatase in mediating the induction of interleukin (IL)-6 by IL-1 or tumor necrosis factor. J Exp Med 2001;193:101–110.Uhlen P, Burch PM, Zito CI, Estrada 48 M, Ehrlich BE, Bennett AM: Gain-of-function/Noonan syndrome SHP-2/Ptpn11 mutants enhance calcium os-cillations and impair NFAT signaling. Proc Natl Acad Sci USA 2006;103: 2160–2165.Zhang EE, Chapeau E, Hagihara K, 49 Feng GS: Neuronal Shp2 tyrosine phos-phatase controls energy balance and metabolism. Proc Natl Acad Sci USA 2004;101:16064–16069.Chen Y, Wen R, Yang S, Schuman J, 50 Zhang EE, et al: Identification of Shp-2 as a Stat5A phosphatase. J Biol Chem 2003;278:16520–16527.You M, Yu DH, Feng GS: Shp-2 ty-51 rosine phosphatase functions as a neg-ative regulator of the interferon-stimu-lated Jak/STAT pathway. Mol Cell Biol 1999;19:2416–2424.

Du Z, Shen Y, Yang W, Mecklenbrauk-52 er I, Neel BG, Ivashkiv LB: Inhibition of IFN-alpha signaling by a PKC- and protein tyrosine phosphatase SHP-2-dependent pathway. Proc Natl Acad Sci USA 2005;102:10267–10272.Wu CJ, O’Rourke DM, Feng GS, John-53 son GR, Wang Q, Greene MI: The ty-rosine phosphatase SHP-2 is required for mediating phosphatidylinositol 3-kinase/Akt activation by growth fac-tors. Oncogene 2001;20:6018–6025.Zhang SQ, Tsiaras WG, Araki T, Wen 54 G, Minichiello L, Klein R, Neel BG: Receptor-specific regulation of phos-phatidylinositol 3′-kinase activation by the protein tyrosine phosphatase Shp2. Mol Cell Biol 2002;22:4062–4072.Zhang SQ, Yang W, Kontaridis MI, 55 Bivona TG, Wen G, et al: Shp2 regu-lates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment. Mol Cell 2004;13: 341–355.Tartaglia M, Mehler EL, Goldberg R, 56 Zampino G, Brunner HG, et al: Muta-tions in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 2001;29:465–468.Jongmans M, Sistermans EA, Rikken 57 A, Nillesen WM, Tamminga R, et al: Genotypic and phenotypic character-ization of Noonan syndrome: New data and review of the literature. Am J Med Genet A 2005;134:165–170.Musante L, Kehl HG, Majewski F, Mei-58 necke P, Schweiger S, et al: Spectrum of mutations in PTPN11 and geno-type-phenotype correlation in 96 pa-tients with Noonan syndrome and five patients with cardio-facio-cutaneous syndrome. Eur J Hum Genet 2003;11:201–206.Tartaglia M, Kalidas K, Shaw A, Song 59 X, Musat DL, et al: PTPN11 mutations in Noonan syndrome: Molecular spec-trum, genotype-phenotype correla-tion, and phenotypic heterogeneity. Am J Hum Genet 2002;70:1555–1563.Yoshida R, Hasegawa T, Hasegawa Y, 60 Nagai T, Kinoshita E, et al: Protein-tyrosine phosphatase, nonreceptor type 11 mutation analysis and clinical assessment in 45 patients with Noonan syndrome. J Clin Endocrinol Metab 2004;89:3359–3364.


Zenker M, Buheitel G, Rauch R, Koen-61 ig R, Bosse K, et al: Genotype-pheno-type correlations in Noonan syndro-me. J Pediatr 2004;144:368–374.Yoshida R, Nagai T, Hasegawa T, 62 Kinoshita E, Tanaka T, Ogata T: Two novel and one recurrent PTPN11 mu-tations in LEOPARD syndrome. Am J Med Genet 2004;130A:432–434.Sarkozy A, Conti E, Seripa D, Digilio 63 MC, Grifone N, et al: Correlation be-tween PTPN11 gene mutations and congenital heart defects in Noonan and LEOPARD syndromes. J Med Genet 2003;40:704–708.Sarkozy A, Obregon MG, Conti E, Es-64 posito G, Mingarelli R, Pizzuti A, Dal-lapiccola B: A novel PTPN11 gene mu-tation bridges Noonan syndrome, multiple lentigines/LEOPARD syn-drome and Noonan-like/multiple giant cell lesion syndrome. Eur J Hum Genet 2004;12:1069–1072.Conti E, Dottorini T, Sarkozy A, Tiller 65 GE, Esposito G, Pizzuti A, Dallapiccola B: A novel PTPN11 mutation in LEOP-ARD syndrome. Hum Mutat 2003;21:654.Du-Thanh A, Cave H, Bessis D, Puso C, 66 Guilhou JJ, Dereure O: A novel PTPN11 gene mutation in a patient with LEOPARD syndrome. Arch Der-matol 2007;143:1210–1211.Baccarini M: Second nature: Biological 67 functions of the Raf-1 ‘kinase’. FEBS Lett 2005;579:3271–3277.Wellbrock C, Karasarides M, Marais R: 68 The RAF proteins take centre stage. Nat Rev Mol Cell Biol 2004;5:875–885.

Muslin AJ, Tanner JW, Allen PM, Shaw 69 AS: Interaction of 14–3–3 with signal-ing proteins is mediated by the recog-nition of phosphoserine. Cell 1996;84:889–897.Pandit B, Sarkozy A, Pennacchio LA, 70 Carta C, Oishi K, et al: Gain-of-func-tion RAF1 mutations cause Noonan and LEOPARD syndromes with hyper-trophic cardiomyopathy. Nat Genet 2007;39:1007–1012.Digilio MC, Sarkozy A, Pacileo G, Li-71 mongelli G, Marino B, Dallapiccola B: PTPN11 gene mutations: Linking the Gln510Glu mutation to the ‘LEOPARD syndrome phenotype’. Eur J Pediatr 2006;165:803–805.Takahashi K, Kogaki S, Kurotobi S, 72 Nasuno S, Ohta M, et al: A novel muta-tion in the PTPN11 gene in a patient with Noonan syndrome and rapidly progressive hypertrophic cardiomyo-pathy. Eur J Pediatr 2005;164:497–500.Kontaridis MI, Swanson KD, David FS, 73 Barford D, Neel BG: PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activat-ing, effects. J Biol Chem 2006;281: 6785–6792.Tartaglia M, Martinelli S, Stella L, 74 Bocchinfuso G, Flex E, et al: Diversity and functional consequences of ger-mline and somatic PTPN11 mutations in human disease. Am J Hum Genet 2006;78:279–290.

Hanna N, Montagner A, Lee WH, 75 Miteva M, Vidal M, et al: Reduced phosphatase activity of SHP-2 in LEOPARD syndrome: Consequences for PI3K binding on Gab1. FEBS Lett 2006;580:2477–2482.Fragale A, Tartaglia M, Wu J, Gelb BD: 76 Noonan syndrome-associated SHP2/PTPN11 mutants cause EGF-depen-dent prolonged GAB1 binding and sus-tained ERK2/MAPK1 activation. Hum Mutat 2004;23:267–277.Keilhack H, David FS, McGregor M, 77 Cantley LC, Neel BG: Diverse biochem-ical properties of Shp2 mutants. Impli-cations for disease phenotypes. J Biol Chem 2005;280:30984–30993.Araki T, Mohi MG, Ismat FA, Bronson 78 RT, Williams IR, et al: Mouse model of Noonan syndrome reveals cell type- and gene dosage-dependent effects of Ptpn11 mutation. Nat Med 2004;10:849–857.Arrandale JM, Gore-Willse A, Rocks S, 79 Ren JM, Zhu J, et al: Insulin signaling in mice expressing reduced levels of Syp. J Biol Chem 1996;271: 21353–21358.Light Y, Paterson H, Marais R: 14–3–3 80 antagonizes Ras-mediated Raf-1 re-cruitment to the plasma membrane to maintain signaling fidelity. Mol Cell Biol 2002;22:4984–4996.

Bruce D. Gelb

Center for Molecular Cardiology, Departments of Pediatrics and Genetics & Genomic Sciences

Mount Sinai School of Medicine

New York, NY 10029 (USA)




The Clinical Phenotype of Cardiofaciocutaneous Syndrome (CFC)

A.E. Roberts

Department of Cardiology and Division of Genetics, Children’s Hospital Boston, and

Harvard Medical School Partners HealthCare Center for Genetics and Genomics, Boston, Mass., USA

AbstractThe first publication describing eight patients with cardio-

faciocutaneous syndrome (CFC) focused on the findings of

growth and psychomotor retardation, characteristic facial

appearance, congenital heart defect, and ectodermal dys-

plasia [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 distin-

guishing features. The neurological and ectodermal find-

ings appear to be the most specific and sensitive for CFC.


Clinical Diagnosis

In evaluating 56 published cases of CFC syn-

drome, 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 fea-

tures, growth retardation, cardiac defect, sparse/

curly hair, neurological impairment/develop-

mental delay, gastrointestinal dysfunction, ocular

abnormalities/dysfunction, history of polyhy-

dramnios, 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 syn-

drome and from Costello syndrome. The Index

is based upon 82 clinical traits and their frequen-

cies in the population with CFC. For a given pa-

tient 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 be-

low two standard deviations from the mean [3].

With the recent discovery of the molecular ge-

netic 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] calculat-

ed an average CFC Index for three KRAS patients

(16.7), 16 BRAF patients (16.0) and six MEK1/2 pa-

tients (16.8), all consistent with the diagnosis.

CFC Clinical Phenotype 67

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 char-

acteristic of CFC.

Growth

The birth weight is most often within the aver-

age range and about a third fall above the 75th

centile [5]. However, postnatal growth retarda-

tion 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 prob-

lem, nutrition is not the only factor. Fifteen of fif-

ty-eight children (26%) with CFC were reported

to have a feeding problem [7], lower than the es-

timated 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.

Development

Generalized hypotonia is common. When chil-

dren are followed over time, there is significant

psychomotor delay in infancy and early child-

hood which improves with age [10]. The cogni-

tive 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 (usual-

ly mild to moderate) has been described in most

cases of CFC with few exceptions. Wieczorek’s

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

with molecular genetic confirmation (a patho-

genic BRAF, KRAS, MEK1 or MEK2 mutation)

found that 95% had speech delay and 100% had

MR [11]. Another 25 cases with a CFC gene mu-

tation were reported and also had a 100% preva-

lence of MR [4].

Ward et al. [10] reported a family with features

of CFC but also with features thought to be dis-

tinctive 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 (val-

vular and infundibular pulmonic stenosis, brittle

and wooly hair with patchy alopecia, dry and hy-

pohydrotic skin, and characteristic facial traits)

who did not have mental retardation. In retro-

spect it is possible that these are misclassified cas-

es of CFC and are actually SOS1 positive Noonan

syndrome cases. Noonan syndrome caused by

mutations in the SOS1 gene often have accom-

panying hair and skin findings seen in CFC and

mental retardation is not found in a majority of

patients [13].

Facial Features

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, hy-

pertelorism, downslanting palpebral fissures, epi-

canthal 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 discrimi-

nate 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 fea-

tures of 25 cases of CFC syndrome with a BRAF,

68 Roberts

KRAS, MEK1 or MEK2 mutation and found each

feature (hypertelorism, low and posteriorly rotat-

ed 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 analy-

sis of 35 children with CFC showed increased fa-

cial 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 nor-

mal hearts (79% in an analysis of 53 cases [5] and

84% in an analysis of 25 CFC gene mutation posi-

tive cases [4] had a congenital heart malforma-

tion). 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 cas-

es 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, thick-

ened mitral valve and mitral valve prolapse have

also been reported [5].

It appears that adults without previous evi-

dence of cardiomyopathy may be at risk for late

onset. Two young adults with severe hypertro-

phic 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 20’s. A 52-year-old woman with CFC was

not diagnosed with hypertrophic cardiomyopa-

thy until her late 40’s [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 hy-

poplasia, and three had an abnormal EEG [1].

Subsequent reports have added cortical atrophy

Fig. 1. Almost 2-year-old girl with BRAF positive CFC and

hydrocephalus. Note the high forehead with bitempo-

ral constriction, posteriorly rotated ears with thick helix,

high ‘boxy’ appearance of the cranial vault, hypopla-

sia of supraorbital ridge, hypertelorism, downslanting

palpebral fissues, and depressed nasal bridge with an-

teverted nares.


[8], seizure disorder [8], hypoplasia of the cor-

pus 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 arach-

noid cyst [19]. In the same study, corticospinal

tract findings (hyperreflexia, extensor plantar

response) were observed in 7 out of 22 partici-

pants who were evaluated by detailed neurologi-

cal exam [19]. Among CFC gene mutation positive

cases that have been reported, 16–36% [11, 4] have

been found to have seizures and 56% hypotonia

[4]. More common than clinical seizures are ab-

normal EEG findings including decreased anteri-

or voltage, spike-wave or polyspike pattern, sharp

and slow waves, generalized dysrhythmia grade

I and II, irritative waves, or generalized disorga-

nization [1, 8, 17]. The finding of congenital hy-

pertrophy 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 mitochon-

dria. There has been one case report of moyamoya

syndrome in a child with CFC [22].

Ectodermal Features

Follicular hyperkeratosis of the arms, legs, and

face and sparse, slow-growing curly hair are

considered to be hallmark ectodermal findings

of CFC [14]. In the original eight cases pub-

lished, the hair and skin manifestations noted

included sparse, friable, curly hair, patchy alo-

pecia, ichthyosis, hyperkeratosis, and absent

eyebrows [1]. Ulerythema ophryogenes (absent

eyebrows with hyperkeratosis) is also a com-

mon finding. In a retrospective analysis of the

first 25 cases, café-au-lait spots, acanthosis ni-

gricans, syringocystadenoma papilliferum, he-

mangioma, 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 report-

ed to have ulcerating hemangioma [27]. Of 25

CFC gene mutation positive cases, 36% had ab-

sent 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 pal-

moplantar 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 indi-

viduals, it is thought that with age the hair be-

comes thicker and more typical in texture and

the dryness of the skin and the follicular kera-

tosis improve with age [14].

Ophthalmologic Features

Half of the original case series had an eye abnor-

mality: esotropia and/or strabismus [1]. Young

and colleagues have examined and reported the

most patients to date [14, 28, 29]. One third of

70 Roberts

patients have strabismus (exotropia more com-

mon 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 in-

tracranial pressure [14]. There were single cases of

cataract, vertical strabismus, dissociated vertical

deviation and inferior oblique muscle overaction,

nasolacrimal duct obstruction, and keratoconus

[14].

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 ref lux, and

dysmotility. Nasogastric tube feeding may be

required and some go on to require gastrostomy

tube placement. Umbilical hernia was report-

ed 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 gastrointes-

tinal abnormality including feeding problems

(15 cases), splenomegaly (8 cases), hepatomeg-

aly (4 cases), umbilical hernia (7 cases), or in-

guinal hernia (3 cases). There was a single case

of anal stenosis. Extensive food allergies have

also been reported in one child [6]. A 17-year-

old woman was reported to have fatty liver with

both macro- and microvesicular steatosis of

unclear etiology [30]. There was also a case re-

port of antral narrowing on upper GI with de-

layed emptying and biopsies showing foveolar

hyperplasia (though no evidence of infiltration

on culture or staining) and functional megaco-

lon from severe constipation [31]. There have

been two cases of malrotation reported [7, 31].

One of the originally described cases, now in

her early 30’s, became critically ill when she

had pneumonia complicated by intestinal ob-

struction [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 re-

port of persistent hypercalciuria, nephrolithiasis,

medullary nephrocalcinosis, obstructing ureteral

calculi and bladder calculi, and renal cysts [31].

Skeletal Features

There are very few reports of orthopaedic issues

for people with CFC syndrome. This could re-

flect a lack of study rather than a true absence of

orthopaedic problems. Ades et al. [32] reported

a single case of bilateral progressive femoral val-

gus deformity. There was also a case of a 17-year-

old 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 predis-

position 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-month-

old MEK1 mutation positive patient with CFC

and a history of hypertrophic cardiomyopathy

requiring heart transplant presented with meta-

static hepatoblastoma [34]. It is possible that the

hepatoblastoma was secondary to the immuno-

suppression required after transplant but since


References

Reynolds JF, Neri G, Herrmann JP, 1 Blumberg B, Coldwell JG, et al: New multiple congenital anomalies/mental retardation syndrome with cardio-fa-cio-cutaneous involvement – the CFC syndrome. Am J Med Genet 1986;25:413–427.Grebe TA, Clericuzio C: Neurologic 2 and gastrointestinal dysfunction in cardio-facio-cutaneous syndrome: identification of a severe phenotype. Am J Med Genet 2000;95:135–143.Kavamura MI, Peres CA, Alchorne 3 MMA, Brunoni D: CFC index for the diagnosis of cardiofaciocutaneous syn-drome. Am J Med Genet 2002;112:12–16.

Narumi Y, Aoki Y, Niihori T, Neri G, 4 Cave H, et al: Molecular and clinical characterization of cardio-facio-cuta-neous (CFC) syndrome. Am J Med Genet A 2007;143:799–807.Wieczorek D, Majewski F, Gillessen- 5 Kaesbach G: Cardio-facio-cutaneous (CFC) syndrome – a distinct entity? Report of three patients demonstrating the diagnostic difficulties in delinea-tion of the CFC syndrome. Clin Genet 1997;52:37–46.Neri G, Sabatino G, Bertini E, Genu- 6 ardi M: Brief clinical report: the CFC syndrome – report of the first two cas-es outside of the United States. Am J Med Genet 1987;27:767–771.

McDaniel CH, Fujimoto A: Intestinal 7 malrotation in a child with cardio-fa-cio-cutaneous syndrome. Am J Med Genet 1997;70:284–286.Raymond G, Holmes LB: Cardio-facio- 8 cutaneous syndrome: neurological features in two children. Dev Med Child Neurol 1993;35:727–741.Kavamura MI, Pomponi MG, Zollino 9 M, Lecce R, Murdola M, et al: PTPN11 mutations are not responsible for the cardiofaciocutaneous syndrome. Eur J Med Genet 2003;11:64–68.Ward KA, Moss C, McKeown C: The 10 cardio-facio-cutaneous syndrome: a manifestation of the Noonan syn-drome? Br J Dermatol 1994;131: 270–274.

hepatoblastoma can be seen in Costello syn-

drome, it could also be related to the common

perturbation of the Ras/MAPK pathway in CFC

and Costello syndromes.

Natural History

With so few cases reported and very little long

term follow-up, the natural history of CFC re-

mains 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 neu-

ropathy [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 40’s. Of note, she presented

at age 47 with chest pain and was diagnosed with

hypertrophic cardiomyopathy.

Conclusions

CFC is a rare genetic disorder with cardiac, ec-

todermal, 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 stud-

ied, it will be difficult to determine if these single

case report findings are coincidental or truly re-

lated to CFC. Recent molecular genetic discover-

ies 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.

72 Roberts

Nava C, Hanna N, Michot C, Pereira S, 11 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;44:763–771.Manoukian S, Lalatta F, Selicorni A, 12 Tadini G, Cavalli R, Neri G: Cardio-facio-cutaneous (CFC) syndrome: re-port of an adult without mental retar-dation. Am J Med Genet 1996;63:382–385.Tartaglia M, Pennacchio LA, Zhao C, 13 Yadav KK, Fodale V, et al: Gain-of-function SOS1 mutations cause a dis-tinctive form of Noonan syndrome. Nat Genet 2007;39:75–79.Roberts A, Allanson J, Jadico SK, Ka-14 vamura MI, Noonan J, et al: The car-diofaciocutaneous syndrome. J Med Genet 2006;43:833–842.Allanson J, Opitz JM, Carey JC, 15 Viskochil D, Noonan J, et al: Cardio-facio-cutaneous syndrome: a distinct entity. Proc Greenwood Genet Ctr 2002;21:67.McGaughran J: Cardio-facio-cutane-16 ous syndrome: first presentation in a 52-year old woman. Am J Med Genet A 2003;116:210–212.Sabatino G, Verrotti A, Domizio S, An-17 gelozzi B, Chiarelli F, Neri G: The car-dio-facio-cutaneous syndrome: a long-term follow-up of two patients, with special reference to the neurologic fea-tures. Child Nerv Syst 1997;13: 238–241.Ion A, Tartaglia M, Song X, Kalidas K, 18 van der Burgt I, et al: Absence of PTPN11 mutations in 28 cases of car-diofaciocutaneous (CFC) syndrome. Hum Genet 2002;111:421–427.

Yoon G, Rosenberg J, Blaser S, Rauen 19 KA: Neurological complications of cardio-facio-cutaneous syndrome. Dev Med Child Neurol 2007;49:894–899.Manci EA, Martinez JE, Horenstein 20 MG, Gardner TM, Ahmed A, et al: Car-diofaciocutaneous syndrome (CFC) with congenital peripheral neuropathy and nonorganic malnutrition: an au-topsy study. Am J Med Genet A 2005;137:1–8.Aeby A, Snajer Y, Cave H, Rebuffat E, 21 Van Coster R, et al: Cardiofaciocutane-ous (CFC) syndrome associated with mucular coenzyme Q10 deficiency. J Inherit Metab Dis 2007;30:827.Mathews CA, George P, Hood AF: Car-22 diofaciocutaneous syndrome. Arch Dermatol 1993;129:46–47.Ishiguro Y, Kubota T, Takenaka J, 23 Maruyama K, Okumura A, et al: Car-dio-facio-cutaneous syndrome and moyamoya syndrome. Brain Dev 2002;24:245–249.Turnpenny PD, Dean JCS, Auchterlo-24 nie IA, Johnston AW: Cardiofaciocuta-neous syndrome with new ectodermal manifestations. J Med Genet 1992;29:428–429.Leal-Ugarte E, Macias-Gomez NM, 25 Gutierrez-Angulo M, Barros-Nunez P: Cardio-facio-cutaneous syndrome with hemihidrosis: ectodermal dyspla-sias spectrum? Int J Dermatol 2006;45:1481–1482.Weiss G, Confino Y, Shemer A, Traut 26 H: Cutaneous manifestations in the cardiofaciocutaneous syndrome, a variant of the classical Noonan syn-drome. Report of a case and review of the literature. JEADV 2004;18:324–327.

Tang B, Reardon W, Black GC, Kerr 27 BA: Congenital ulcerating hemangio-ma in a baby with KRAS mutation and cardio-facio-cutaneous syndrome. Clin Dysmorphol 2007;16:203–206.Young TL, Ziylan S, Schaffer DB: The 28 ophthalmologic manifestations of the cardio-facio-cutaneous syndrome. J Pediatr Ophthalmol Strabismus 1993;30:48–52.Young TL: Cardio-facio-cutaneous 29 syndrome conference ophthalmologic findings summary. Rockville Mary-land, June 2003;http://www.cfcsyn-drome.org/conference-summary.htm.Nanda S, Rajpal M, Reddy BSN: Car-30 dio-facio-cutaneous syndrome: report of a case with a review of the literature. Int Soc Dermatol 2004;43:447–450.Herman TE, McAlister WH: Gastroin-31 testinal and renal abnormalities in cardio-facio-cutaneous syndrome. Pediatr Radiol 2005;35:202–205.Ades LC, Sillence DO, Rogers M: Car-32 diofaciocutaneous syndrome. Clin Dysmorphol 1992;1:145–150.Van Den Berg H, Hennekam RCM: 33 Acute lymphoblastic leukemia in a patient with cardiofaciocutaneous syn-drome. J Med Genet 1999;36:799–800.Al-Rahawan MM, Chute DJ, Sol-34 Church K, Gripp KW, Stabley DL, et al: Hepatoblastoma and heart transplan-tation in a patient with cardio-facio-cutaneous syndrome. Am J Med Genet A 2007;143:1481–1488.DeRoos ST, Ryan MM, Ouvrier RA: 35 Peripheral neuropathy in cardiofacio-cutaneous syndrome. Pediatr Neurol 2007;36:250–252.

Amy E. Roberts, MD

Cardiovascular Genetics, Department of Cardiology, Farley 2, Children’s Hospital Boston

300 Longwood Ave

Boston, MA 02115 (USA)




Molecular Causes of the Cardio-Facio-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

AbstractCardio-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 B-

Raf, MEK1 or MEK2. Mutations in K-Ras, a small GTPase, have

also been implicated as causing CFC syndrome and Noo-

nan 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 mu-

tant proteins studied to date demonstrate increased activ-

ity. Since CFC syndrome may have a progressive, evolving

phenotype, the possible use of systemic therapies to re-

duce MAPK activity may be of great benefit to this pop-

ulation 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.


In 1979, a mental retardation syndrome with dis-

tinctive craniofacial dysmorphology, ectodermal

anomalies and cardiac defects was reported by

Blumberg and colleagues at the March of Dimes

Birth Defects Conference [1]. The three report-

ed patients had characteristic facial features,

ichthyosis with abnormal hair, ocular and car-

diac abnormalities, postnatal growth failure and

mental retardation. These three patients, in addi-

tion to five others, were subsequently reported by

Reynolds and colleagues [2] who designated this

new entity cardio-facio-cutaneous (CFC) syn-

drome based on their common phenotypic fea-

tures. Since then, more than one hundred patients

have been reported in the literature. Although

the exact incidence of this rare syndrome is un-

known, a conservative estimate is approximately

three to four hundred individuals worldwide.

Overview of the Clinical Diagnosis of

CFC Syndrome

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 crite-

ria have been established. Individuals with CFC

syndrome display phenotypic variability and,

74 Tidyman � Rauen

therefore, each affected individual may not pos-

sess all the characteristic features.

Although CFC syndrome has a distinct phe-

notype, 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 syn-

drome and include macrocephaly, broad fore-

head, bitemporal narrowing, hypoplasia of the

supraorbital ridges, down-slanting palpebral

fissures with ptosis, short nose with depressed

nasal bridge and anteverted nares, low-set, pos-

teriorly rotated ears with prominent helices and

a high-arched palate (fig. 1). Ectodermal find-

ings typically consist of sparse, curly hair with

sparse eyebrows and eyelashes, hyperkeratosis,

keratosis pilaris, hemangioma, nevi and ichthyo-

sis [3, 4]. Cardiac anomalies vary with the most

prevalent being pulmonic stenosis, atrial septal

defects, and hypertrophic cardiomyopathy [5].

a b

c d

Fig. 1. Individual with CFC syndrome who has a p.G596V missense substitution in B-Raf result-

ing 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.

Molecular Causes of the Cardio-Facio-Cutaneous Syndrome 75

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 chy-

lothoraces and lymphedema have been reported

at birth [10].

Neurologic abnormalities are universally pres-

ent in CFC. In a recent study of 39 mutation-posi-

tive 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 (perivascu-

lar) spaces (19%), abnormal myelination (13%),

and structural anomalies (16%). Seizures were

present in 38% of individuals. However, no spe-

cific genotype-phenotype correlations could be

drawn.

Neoplasia, such as benign papillomas or ma-

lignancies observed in CS, NS or neurofibro-

matosis type 1, has not been reported in CFC

syndrome. Although it is unclear if individu-

als with CFC are at an increased risk to devel-

op 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 etiolo-

gy 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 consid-

ered 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 in-

vestigations revealed no alterations in this gene,

suggesting that NS and CFC are indeed distinct

genetic entities.

A subsequent study examined a well-charac-

terized 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 dis-

tinct [17].

Subsequently, a second gene in the Ras/MAPK

pathway, HRAS, was identified as being causative

for the CS [18]. Rauen and colleagues then con-

firmed that CFC syndrome was genetically dis-

tinct 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 ap-

parent 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


of Ras and is highly conserved among eukary-

otic organisms. It is critically involved in cell

proliferation, differentiation, motility, apopto-

sis, 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/threo-

nine 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 sub-

strates [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 ap-

proximately 30% of human cancers with pancre-

as, 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 mod-

ulators 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) mutation-

negative. Heterogeneous missense mutations in

BRAF were identified in 18/23 (78%) of individ-

uals with CFC syndrome. Eleven distinct mis-

sense mutations clustered in two regions: in the

cysteine-rich domain (CRD) of the conserved re-

gion 1 (CR1) and in the protein kinase domain

(fig. 2). Niihori and colleagues [23] examined 43

CFC individuals and identified eight unique mu-

tations among 16 individuals (37%). Functional

analysis of the proteins resulting from BRAF har-

boring these mutations revealed that the type of

BRAF mutations identified in CFC syndrome is

similar to the different types of mutations identi-

fied 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 fam-

ily which also includes C-Raf and the X-linked

BRAF

Q262R

�T470

�462–469

G469QG469EF468S

N581KN581D

F595LG596V

T599RG464VG464RG466RS467A

V487G

K499N

E501VE501KE501G

G534R

N580D

D638ETGAATG

Protein kinase domain

G-loop CR3 Activation segment

Ras bindingdomain

Cysteine-richdomain

CR1 CR2

K499E

L485SL485FQ257K

Q257RA246PT244P

T241P

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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).


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 ki-

nase and is one of the many direct downstream

effectors of Ras. There are three conserved re-

gions in B-Raf. Conserved region 1 (CR1), part

of the regulatory amino-terminal, contains the

Ras binding domain and the cysteine-rich do-

main 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-Raf ’s only known down-

stream effectors are MEK1 and MEK2.

Somatic mutations in BRAF have been re-

ported at a high frequency in numerous cancers

including melanoma, thyroid, colorectal and

ovarian cancer. Approximately 70 missense mu-

tations affecting 34 codons have been reported

(www.sanger.ac.uk/genetics/CGP/cosmic). The

majority of BRAF mutations are missense sub-

stitutions 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 seg-

ment is held in an inactive conformation by as-

sociation 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

T→A transversion at nt1796 substituting glutam-

ic 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 [26–29].

Interestingly, somatic B-Raf p.V600E muta-

tions are also found in benign skin lesions and

premalignant colon polyps [28–30]. The majority

of benign nevi, as well as primary and metastat-

ic melanoma, have the B-Raf p.V600E mutation.

This suggests that MAPK activation is important

in melanocytic neoplasia, but in isolation, is in-

sufficient for tumorigenesis [30]. Although the

majority of B-Raf mutations are kinase activat-

ing, 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 oc-

cur in BRAF. Unlike the mutation spectrum seen

in cancer, the BRAF mutations in CFC individu-

als 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, 31–34; Rauen, unpub-

lished data]. Thirty-one novel CFC BRAF mu-

tations affecting 23 different codons have been

identified. Of the 18 exons in BRAF, CFC mu-

tations have been found in exon 6 and in exons

11–16. The majority of missense mutations are in

exon 6 (41%) and exon 12 (21%). The most com-

mon BRAF mutations occur in exon 6 and con-

sist 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 mu-

tations reported in cancer are found exclusively

in exon 11 and 15; whereas, only 18% of CFC mu-

tations 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 in-

frame 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


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 so-

matic or constitutional mutations had ever been

described in MEK genes. Functional studies of

the proteins encoded by these novel MEK1/2 mu-

tations 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 generat-

ed 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 approximate-

ly 104 kb. MEK2 (MAP2K2) is located on chro-

mosome 19p13.3 and spans approximately 34 kb.

Each gene contains 11 exons with intervening se-

quences. The MEK1 gene encodes the mitogen-ac-

tivated 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 redun-

dant purposes [39, 40].

Unlike B-Raf, prior to the discovery of germ-

line 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 in-

dividuals in which a gene mutation has been

identified [14, 20, 23, 31–34; Rauen, unpublished

data]. Mutations in MEK1 and MEK2 are seen in

roughly equal frequency (fig. 3). The vast majori-

ty 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 de-

leting 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 in-

creased MEK1 and MEK2 activities [22, 43], but

no somatic kinase domain MEK1 and MEK2 mu-

tations have been reported in cancer [44–47; 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 phos-

phorylation [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 wild-

type 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 phe-

notype between the two syndromes. Continued

evaluation of the patient populations and func-

tional studies will resolve these issues.

Making the Molecular Diagnosis of

CFC Syndrome

CFC syndrome is one of many syndromes includ-

ing NS, LEOPARD syndrome, CS and neurofi-

bromatosis 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


phenotypic features that are seen among indi-

viduals who have these disorders. Although CFC

syndrome has a distinctive phenotype, there are

many features in common with the other re-

lated Ras pathway syndromes [34]. Because of

this developmental overlap, making the clini-

cal diagnosis of CFC syndrome can be challeng-

ing, particularly during the newborn period.

Currently, there are no specific criteria that are

routinely used to establish the clinical diagno-

sis of CFC syndrome. Once a clinical diagnosis

is considered, molecular testing can definitive-

ly confirm the clinical diagnosis. Obtaining a

molecular diagnosis is important for many rea-

sons, 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 ap-

proach 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, 11–16). If no caus-

al 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 mu-

tation is identified, consider sequencing the re-

maining 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 di-

rect sequencing of the Noonan syndrome genes

KRAS [23, 49, 51, 53] followed by SOS1 [54–56;

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 sequenc-

ing 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].

MEK1

MEK2

1

1 2 3 4 5 6 7 8 9 10 11

2 3 4 5 6 7 8 9 10 11

�K59

�46–55

T55PP124LG128VY130NY130C

F53SE44G

F57CF57IK61EK61T

A62P

P128RK273RG132V

Y134C

ATG

ATG

TAA

TGA


D67N


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].


Future Directions and Possible Therapies

for CFC

Hyperactivated ERK is found in approximate-

ly 30% of human cancers with pancreas, colon,

lung, ovary and kidney demonstrating the high-

est levels of ERK activation [22]. One significant

aspect of the identification of germline mutations

in BRAF and MEK1/2 as causative for CFC syn-

drome was the finding that activating germline

mutations within the MAPK pathway could be

compatible with human development. The de-

velopment of potential treatment for the group

of Ras/MAPK syndromes, including CFC, woe-

fully lags behind the ability to diagnose each at

the molecular level. Since CFC syndrome may

have a progressive, evolving phenotype, the pos-

sible 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 im-

paired [20, 23]. In contrast, all MEK1/2 mutants

characterized in vitro confer an increase of kinase

activity [20; Rauen, unpublished data]. As the ma-

jority of the CFC BRAF mutations are novel and

no MEK mutations have ever been identified prior

to their discovery in CFC syndrome, the biochem-

ical aspects of the novel mutants, as well as, the

roles of these MEK mutants in the MAPK signal-

ing 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, se-

lective 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 on-going support of research in genetic medicine. The au-thors apologize for not citing all relevant references due to space limitations. This work was supported in part by NIH grant HD048502 (K.A.R.).

References

Blumberg B, Shapiro L, Punnett HH, 1 Rimoin D, Kirtenmacher M: A new mental retardation syndrome with characteristic facies, ichthyosis and abnormal hair. (March of Dimes Birth Defects Conference, Chicago, Il 1979).Reynolds JF, Neri G, Herrmann JP, 2 Blumberg B, Coldwell JG, Miles PV, Opitz JM: New multiple congenital anomalies/mental retardation syn-drome with cardio-facio-cutaneous involvement – the CFC syndrome. Am J Med Genet 1986;25:413–427.Borradori L, Blanchet-Bardon C: Skin 3 manifestations of cardio-facio-cutane-ous syndrome. J Am Acad Dermatol 1993;28:815–819.

Weiss G, Confino Y, Shemer A, Trau H: 4 Cutaneous manifestations in the car-diofaciocutaneous syndrome, a variant of the classical Noonan syndrome. Re-port of a case and review of the litera-ture. J Eur Acad Dermatol Venereol 2004;18:324–327.Wieczorek D, Majewski F, Gillessen- 5 Kaesbach G: Cardio-facio-cutaneous (CFC) syndrome–a distinct entity? Report of three patients demonstrating the diagnostic difficulties in delinea-tion of CFC syndrome. Clin Genet 1997;52:37–46.

Young TL, Ziylan S, Schaffer DB: The 6 ophthalmologic manifestations of the cardio-facio-cutaneous syndrome. J Pediatr Ophthalmol Strab 1993;30:48–52.Grebe TA, Clericuzio C: Neurologic 7 and gastrointestinal dysfunction in cardio-facio-cutaneous syndrome: identification of a severe phenotype. Am J Med Genet 2000;95:135–143.Sabatino G, Verrotti A, Domizio S, An- 8 geiozzi B, Chiarelli F, Neri G: The car-dio-facio-cutaneous syndrome: a long-term follow-up of two patients, with special reference to the neurological features. Childs Nerv Syst 1997;13:238–241.


Herman TE, McAlister WH: Gastroin- 9 testinal and renal abnormalities in cardio-facio-cutaneous syndrome. Pediatr Radiol 2005;35:202–205.Chan PC, Chiu HC, Hwu WL: Sponta-10 neous chylothorax in a case of cardio-facio-cutaneous syndrome. Clin Dys-morphol 2002;11:297–298.Yoon G, Rosenberg J, Blaser S, Rauen 11 KA: Neurological complications of cardio-facio-cutaneous syndrome. Dev Med Child Neurol 2007;49:894–899.Van Den Berg H, Hennekam RCM: 12 Acute lymphoblastic leukaemia in a patient with cardiofaciocutaneous syn-drome. J Med Genet 1999;36:799–800.Makita Y, Narumi Y, Yoshida M, Nii-13 hori T, Kure S, et al: Leukemia in car-dio-facio-cutaneous (CFC) syndrome: a patient with a germline mutation in BRAF proto-oncogene. J Pediatr He-matol Oncol 2007;29:287–290.Al-Rahawan MM, Chute DJ, Sol-14 Church K, Gripp KW, Stabley DL, et al: Hepatoblastoma and heart transplan-tation in a patient with cardio-facio-cutaneous syndrome. Am J Med Genet A 2007;143:1481–1488.Tartaglia M, Kalidas K, Shaw A, Song 15 X, Musat DL, et al: PTPN11 mutations in Noonan syndrome: molecular spec-trum, genotype-phenotype correla-tion, and phenotypic heterogeneity. Am J Hum Genet 2002;70:1555–1563.Ion A, Tartaglia M, Song X, Kalidas K, 16 Van Der Burgt I, et al: Absence of PTPN11 mutations in 28 cases of car-diofaciocutaneous (CFC) syndrome. Hum Genet 2002;111:421–427.Tartaglia M, Cotter PD, Zampino G, 17 Gelb BD, Rauen KA: Exclusion of PTPN11 mutations in Costello syn-drome: further evidence for distinct genetic etiologies for Noonan, cardio-facio-cutaneous and Costello syn-dromes. Clin Genet 2003;63:423–426.Aoki Y, Niihori T, Kawame H, Kuro-18 sawa K, Ohashi H, et al: Germline mu-tations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 2005;37:1038–1040.Estep AL, Tidyman WE, Teitell MA, 19 Cotter PD, Rauen KA: HRAS muta-tions in Costello syndrome: detection of constitutional activating mutations in codon 12 and 13 and loss of wild-type allele in malignancy. Am J Med Genet A 2006;140:8–16.

Rodriguez-Viciana P, Tetsu O, Tidy-20 man WE, Estep AL, Conger BA, et al: Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome. Science 2006;311:1287–1290.Yoon S, Seger R: The extracellular sig-21 nal-regulated kinase: multiple sub-strates regulate diverse cellular func-tions. Growth Factors 2006;24:21–44.Hoshino R, Chatani Y, Yamori T, Tsu-22 ruo T, Oka H, et al: Constitutive activa-tion of the 41-/43-kDa mitogen-acti-vated protein kinase signaling pathway in human tumors. Oncogene 1999;18:813–822.Niihori T, Aoki Y, Narumi Y, Neri G, 23 Cave H, et al: Germline KRAS and BRAF mutations in cardio-facio-cuta-neous syndrome. Nat Genet 2006;38:294–296.Wan PT, Garnett MJ, Roe SM, Lee S, 24 Niculescu-Duvaz D, et al: Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004;116:855–867.Wellbrock C, Karasarides M, Marais R: 25 The RAF proteins take centre stage. Nat Rev Mol Cell Biol 2004;5:875–885.Brose MS, Volpe P, Feldman M, Kumar 26 M, Rishi I, et al: BRAF and RAS muta-tions in human lung cancer and mela-noma. Cancer Res 2002;62:6997–7000.Gorden A, Osman I, Gai W, He D, 27 Huang W, et al: Analysis of BRAF and N-RAS mutations in metastatic mela-noma tissues. Cancer Res 2003;63: 3955–3957.Rajagopalan H, Bardelli A, Lengauer C, 28 Kinzler KW, Vogelstein B, Velculescu VE: Tumorigenesis: RAF/RAS onco-genes and mismatch-repair status. Na-ture 2002;418:934.Yuen ST, Davies H, Chan TL, Ho JW, 29 Bignell GR, et al: Similarity of the phe-notypic patterns associated with BRAF and KRAS mutations in colorectal neo-plasia. Cancer Res 2002;62: 6451–6455.Pollock PM, Harper UL, Hansen KS, 30 Yudt LM, Stark M, et al: High frequen-cy of BRAF mutations in nevi. Nat Genet 2003;33:19–20.Schulz AL, Albrecht B, Arici C, van der 31 Burgt I, Buske A, et al: Mutation and phenotypic spectrum in patients with cardio-facio-cutaneous and Costello syndrome. Clin Genet 2007;73:62–70.

Narumi Y, Aoki Y, Niihori T, Neri G, 32 Cave H, et al: Molecular and clinical characterization of cardio-facio-cuta-neous (CFC) syndrome: Overlapping clinical manifestations with Costello syndrome. Am J Med Genet A 2007;143:799–807.Nava C, Hanna N, Michot C, Pereira S, 33 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;44:763–771.Rauen KA: Distinguishing Costello 34 versus cardio-facio-cutaneous syn-drome: BRAF mutations in patients with a Costello phenotype. Am J Med Genet A 2006;140:1681–1683.Cruz F, 3rd, Rubin BP, Wilson D, Town 35 A, Schroeder A, et al: Absence of BRAF and NRAS mutations in uveal melanoma. Cancer Res 2003;63: 5761–5766.Trovisco V, Soares P, Soares R, Magal-36 haes J, Sa-Couto P, Sobrinho-Simoes M: A new BRAF gene mutation detect-ed in a case of a solid variant of papil-lary thyroid carcinoma. Hum Pathol 2005;36:694–697.Pearson G, Robinson F, Beers Gibson 37 T, Xu BE, Karandikar M, Berman K, Cobb MH: Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 2001;22:153–183.Wu J, Harrison JK, Dent P, Lynch KR, 38 Weber MJ, Sturgill TW: Identification and characterization of a new mam-malian mitogen-activated protein ki-nase kinase, MKK2. Mol Cell Biol 1993;13:4539–4548.Brott BK, Alessandrini A, Largaespada 39 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;4:921–929.Alessandrini A, Brott BK, Erikson RL: 40 Differential expression of MEK1 and MEK2 during mouse development. Cell Growth Differ 1997;8:505–511.Cowley S, Paterson H, Kemp P, Mar-41 shall CJ: Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transfor-mation of NIH 3T3 cells. Cell 1994;77:841–852.


Mansour SJ, Matten WT, Hermann AS, 42 Candia JM, Rong S, et al: Transforma-tion of mammalian cells by constitu-tively active MAP kinase kinase. Sci-ence 1994;265:966–970.Sivaraman VS, Wang H, Nuovo GJ, 43 Malbon CC: Hyperexpression of mito-gen-activated protein kinase in human breast cancer. J Clin Invest 1997;99: 1478–1483.Bignell G, Smith R, Hunter C, Stephens 44 P, Davies H, et al: Sequence analysis of the protein kinase gene family in hu-man testicular germ-cell tumors of adolescents and adults. Genes Chro-mosomes Cancer 2006;45:42–46.Davies H, Hunter C, Smith R, Stephens 45 P, Greenman C, et al: Somatic muta-tions of the protein kinase gene family in human lung cancer. Cancer Res 2005;65:7591–7595.Hunter C, Smith R, Cahill DP, Ste-46 phens P, Stevens C, et al: A hypermuta-tion phenotype and somatic MSH6 mutations in recurrent human malig-nant gliomas after alkylator chemo-therapy. Cancer Res 2006;66: 3987–3991.Stephens P, Edkins S, Davies H, Green-47 man 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;37:590–592.Estep AL, Palmer C, McCormick F, 48 Rauen KA: Mutation Analysis of BRAF, MEK1 and MEK2 in 15 ovar-ian cancer cell lines: implications for therapy. PLoS ONE 2007;2:e1279.

Schubbert S, Zenker M, Rowe SL, Boll 49 S, Klein C, et al: Germline KRAS mu-tations cause Noonan syndrome. Nat Genet 2006;38:331–336.Schubbert S, Bollag G, Lyubynska N, 50 Nguyen H, Kratz CP, et al: Biochemical and functional characterization of germ line KRAS mutations. Mol Cell Biol 2007;27:7765–7770.Zenker M, Lehmann K, Schulz AL, 51 Barth H, Hansmann D, et al: Expan-sion of the genotypic and phenotypic spectrum in patients with KRAS ger-mline mutations. J Med Genet 2007;44:131–135.Bentires-Alj M, Kontaridis MI, Neel 52 BG: Stops along the RAS pathway in human genetic disease. Nat Med 2006;12:283–285.Carta C, Pantaleoni F, Bocchinfuso G, 53 Stella L, Vasta I, et al: Germline mis-sense mutations affecting KRAS iso-form B are associated with a severe Noonan syndrome phenotype. Am J Hum Genet 2006;79:129–135.Roberts AE, Araki T, Swanson KD, 54 Montgomery KT, Schiripo TA, et al: Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet 2007;39:70–74.Tartaglia M, Pennacchio LA, Zhao C, 55 Yadav KK, Fodale V, et al: Gain-of-function SOS1 mutations cause a dis-tinctive form of Noonan syndrome. Nat Genet 2007;39:75–79.

Zenker M, Horn D, Wieczorek D, Al-56 lanson J, Pauli S, et al: SOS1 is the sec-ond most common Noonan gene but plays no major role in cardio-facio-cu-taneous syndrome. J Med Genet 2007;44:651–656.Gripp KW, Lin AE, Stabley DL, Nichol-57 son L, Scott CI Jr, et al: HRAS muta-tion analysis in Costello syndrome: Genotype and phenotype correlation. Am J Med Genet A 2006;140:1–7.Rauen KA: 58 HRAS and the Costello syn-drome. Clin Genet 2007;71: 101–108.Kerr B, Allanson J, Delrue MA, Gripp 59 KW, Lacomb D, Lin AE, Rauen KA: The diagnosis of Costello syndrome: Nomenclature in Ras/MAPK pathway disorders. Am J Med Genet A 2008;146:1218–1220.Senawong T, Phuchareon J, Ohara O, 60 McCormick F, Rauen KA, Tetsu O: Germline mutations of MEK in cardio-facio-cutaneous syndrome are sensi-tive to MEK and RAF inhibition: im-plications for therapeutic options. Hum Mol Genet 2008;17:419–430.Solit DB, Garraway LA, Pratilas CA, 61 Sawai A, Getz G, et al: BRAF mutation predicts sensitivity to MEK inhibition. Nature 2006;439:358–362.

Katherine A. Rauen

UCSF Helen Diller Family Comprehensive Cancer Center

2340 Sutter Street, Room S429, Box 0128

San Francisco, CA 94115 (USA)




The Clinical Phenotype of Costello Syndrome

B. Kerr

Regional Genetic Service and Medical Genetics Research Group, Central Manchester and

Manchester Children’s Hospitals University NHS Trust, Royal Manchester Children’s Hospital,

Manchester, UK

AbstractCostello syndrome (CS) is a rare syndrome associated with

developmental disability, prenatal overgrowth, and post-

natal 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 ul-

nar deviation at the wrists. There is an increased risk of ma-

lignancy, particularly embryonal rhabdomyosarcoma and

bladder carcinoma. The demonstration of activating mis-

sense mutations in HRAS, a component of the MAPK path-

way, 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-cuta-

neous syndrome (CFC). A severe neonatal phenotype has

emerged, consisting in some cases of a multi-system dis-

ease and in others of profound hypotonia and myopathy as-

sociated 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.


Original Description

In 1977, Dr Jack Costello, a New Zealand paedi-

atrician, published in the Australian Paediatric

Journal a report of two children with similar

physical characteristics (table 1) and mild intel-

lectual 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 com-

plicated 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 promi-

nent upper lip. The baby fed poorly and had to be

tube fed frequently until 5 weeks, and did not re-

gain birth weight until 6 weeks. He remained in

hospital for 11 weeks, a cardiac murmur was not-

ed. 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 with-

in the mild range of impaired cognitive func-

tioning, with higher verbal than performance

scores. He developed nasal warts at age 6, and

84 Kerr

later similar lesions appeared on both legs and

face. He had a divergent squint and keratoco-

nus. 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 fa-

cial features were noted; heavy jowls, very short

neck, depressed bridge of nose, epicanthic folds,

large ear lobes and third fontanelle. She had a big-

gish tongue and developed curly hair. Her fin-

gers were noted to be hyperextensible, with ulnar

deviation of the little fingers. She had transient

hepatosplenomegaly. She also required tube feed-

ing, 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 devel-

oped a calcaneovalgus deformity of the feet and

clawing of the toes and required surgery for tight

Achilles tendons. At age two she had similar na-

sal lesions resembling warts; on biopsy, these were

shown to be papillomata, with acanthosis and hy-

perkeratosis 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 de-

scribed this particular pattern of physical and

developmental characteristics as Costello syn-

drome (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 pre-

vious report. The key features of the three cas-

es 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 cardio-

facio-cutaneous (CFC) syndrome was noted and

compared.

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

The Clinical Phenotype of Costello Syndrome 85

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 litera-

ture cases [4], and contribute to the definition of

the adult phenotype (see table 2). The only clini-

cal 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 swallow-

ing, 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 secre-

tions being relatively common.

After a variable time, usually around 3 years,

feeding behaviour improves and growth be-

comes 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 add-

ed to the earliest clinical descriptions of Costello

syndrome. This is not surprising, given that mu-

tation analysis has established that Costello syn-

drome 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 sporad-

ic 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 rhab-

domyosarcoma [13]. Single reports of an acoustic

neuroma in an adult [14] and an alveolar rhab-

domyosarcoma [15] rapidly followed, with no

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

86 Kerr

further reports to date of further cases of these

latter two tumours.

Other reports [16–22] demonstrated that the

commonest malignant tumours are embryo-

nal 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 in-

traductal 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 as-

say until age 5, and annual urinalysis for haema-

turia 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.

Cardiac Involvement

A review of 94 patients with CS [26] found ev-

idence of cardiac involvement in 63% overall.

Congenital heart disease occurred in 30%, with

pulmonary stenosis accounting for 46%. The oth-

er common malformations were atrial and ven-

tricular septal defects. Cardiac hypertrophy was

reported in 34%; this involved the left ventricle in

50% and was usually consistent with classic hy-

pertrophic cardiomyopathy. In 50% of patients,

this occurred in the first two years of life, but on-

set 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 find-

ing in one third of cases.

Respiratory Involvement

Stridor and excessive secretions are common in

the young child with CS. Bronchomalacia and

tracheomalacia have been reported and occa-

sionally 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].

Table 3. Distinctive features in a series of patients with Costello syndrome [6]

Seen in 100% Seen in more than 80%

Poor feeding BW and Birth OFC >50th%Poor growth

Developmental delay

Coarse facial appearance

Low set ears

Short neck

Loose skin

Hyperpigmentation

Deep creases on palms and soles

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


Endocrine Abnormalities

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 hor-

mone 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 defi-

ciency 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 hor-

mone 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 hor-

mone treatment may pose particular hazard in

Costello syndrome because of the risk of malig-

nant tumours and a possible effect on cardiac hy-

pertrophy [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 treat-

ment 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 sug-

gested mechanism being central hypogonadism.

Precocious puberty has also been described.

Neurological, Developmental and

Behavioural Manifestations

Review of the findings on neurological investiga-

tion 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% in-

cidence 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 spo-

ken between 2 and 9 years.

Although children and adults with Costello

syndrome are described as having a warm so-

ciable personality, the early years of life are often

characterised by extreme irritability with hyper-

sensitivity to sounds and tactile stimuli, sleep

disturbance and excessive shyness with strang-

ers [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 ex-

ceeded, 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 30–87) within the mild range of

88 Kerr

intellectual handicap for the majority of pa-

tients. Intellectual function was stable over two

years.

Skeletal and Orthopaedic Abnormalities

Orthopaedic problems were part of the first de-

scriptions of Costello syndrome (table 1), par-

ticularly in relationship to foot position. Review

of bone and joint manifestations in 16 examined

patients [39], aged from 3 to 23 years, demon-

strated 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 com-

mon, and most tended to hold their elbows and

wrist f lexed 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 planoval-

gus position, bilateral overriding second toes,

plantar flexed great toe and mild metatarsus ad-

ductus. Three children had had hip subluxation.

Kyphosis and scoliosis both occurred in 17%.

Severe pectus excavatum occurred on one pa-

tient. One older patient had bilateral radial head

subluxation.

The Adult Phenotype

In a review of the clinical features in 17 adult pa-

tients, aged 16 to 40 years [24], the distinctive

facial features are summarised as progressive fa-

cial 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 hyperkerato-

sis of the palms and soles in association with ex-

cess 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 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 defi-

ciency 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 pa-

tients 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 nor-

mal. 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 search for clues to the cause of Costello syn-

drome had demonstrated a variety of metabolic

abnormalities, all in a small number of cases [6].

These include non-specific generalised aminoaci-

duria, low maternal serum α-fetoprotein, sialuria

and elevation of hexosaminadase B [6].

Several studies focussed on elastin and elas-

tic 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 nor-

mal elastin mRNA expression in skin fibroblasts

[40]. Hinek et al. [41] demonstrated a deficien-

cy in elastic fibre assembly in skin fibroblasts

due to a secondary deficiency in EBP, a 67-kDa


elastin binding protein. The observed excess of

chondroitin sulfate bearing proteoglycans in fi-

broblasts from patients with CS was postulated

as the cause, due to an inhibition of EBP recy-

cling. Subsequent demonstration of excess chon-

droitin-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 syn-

drome led several authors to speculate that a tu-

mour 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 similar-

ities between Costello syndrome and Noonan

syndrome, Aoki et al. [43] discovered activat-

ing missense mutations in HRAS to be the cause

of Costello syndrome by studying genes in the

MAPK pathway, that being the pathway regulat-

ed 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 chap-

ter. It is particularly noteworthy that for the less

common mutations, some variability in pheno-

type 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 syn-

drome 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.

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,

44, 45]. Choanal atresia has been reported in one

case [44], and in two, neonatal osteoporosis with

enlargement of anterior ribs [44]. Pyloric steno-

sis has occurred in two mutation positive cases

[27, 44].

Diagnostic testing in the newborn period has

confirmed that hypoglycaemia secondary to hy-

perinsulinism, renal abnormalities, severe early

cardiomyopathy, tracheomalacia and broncho-

malacia, pleural and pericardial effusion, chy-

lous ascites and pulmonary, hepatic and splenic

lymph angiectasia are part of the clinical spec-

trum 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 pa-

tients with congenital myopathy with excess of

muscle spindles (CMEMS), hypertrophic car-

diomyopathy 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 cas-

es. 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

In CS, diagnostic criteria exhibit age dependence

and are listed in table 4. Facial and physical fea-

tures 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.

90 Kerr

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 con-

firmed 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 atri-

al septal defect, is commoner in CFC than CS.

Atrial tachycardia is commoner in CS than CFC.

Chaotic atrial rhythm, or multifocal atrial tachy-

cardia, has only been observed in CS. Abnormal

catecholamine metabolites in the absence of a

neuroblastoma have also been observed in CFC

patients.

a

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.


References

Costello JM: A new syndrome: mental 1 subnormality and nasal papillomata. Austr J Paediatr 1977;13:114–118.Costello JM: A new syndrome. NZ Med 2 J 1971;74:397.Der Kaloustian VM, Moroz B, McIn- 3 tosh N, Watters AK, Blaichman S: Cos-tello syndrome. Am J Med Genet 1991;41:69–73.Costello JM: Costello syndrome: Up- 4 date on the original cases and com-mentary. Am J Med Genet 1996;62:199–201.Zampino G, Mastroiacovo P, Ricci R, 5 Zollini M, Segni G, Martini-Neri ME, Neri G: Costello syndrome: Further clinical delineation, natural history ge-netic definition and nosology. Am J Med Genet 1993;47:176–183.

Johnson JP, Golabi M, Norton ME, 6 Rosenblatt RM, Feldman GM, et al: Costello syndrome: Phenotype, natural history, differential diagnosis and pos-sible cause. J Pediatr 1998;133:441–448.Fryns JP, Vogels A, Haegerman J, Egger- 7 mont E, Van Den Berghe H: Costello syndrome: a postnatal growth retarda-tion syndrome with distinct phenotype. Genet Counselling 1994;5:337–343.Zampino G, Pantaleoni F, Carta C, Co- 8 bellos G, Vasta I, et al: Diversity, pa-rental germline origin and phenotypic spectrum of de novo HRAS missense changes in Costello syndrome. Hum Mut 2007;28:265–272.

Van Steensel MA, Vreeburg M, Van 9 Ravenswaaiji-Arts CM, Biljsma E, Schrander-Stumpel CT, van Geel M: Recurring HRAS mutation G12S in Dutch patients with Costello syn-drome. Exp Derm 2006;15:731–734.Siwik ES, Zahka KG, Wiesner GL: Car-10 diac disease in Costello syndrome. Pediatrics 1998;101:706–709.Lurie IW: Genetics of the Costello syn-11 drome. Am J Med Genet 1994;52:358–359.Martin RA, Jones KL: Delineation of 12 the Costello syndrome. Am J Med Gen-et 1991;41:346–349.

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 muta-

tions, but remains most characteristic of CS and

HRAS mutations. A new hand sign [47, 48], dis-

tal phalangeal creases, particularly obvious on

the palmar surface of the thumb, has been seen

in patients with HRAS, KRAS and BRAF muta-

tions and suggested as a marker for disorders of

the MAPK pathway.

Conclusion

Despite the rarity of Costello syndrome, the phe-

notype has been extensively studied. The recent

demonstration of mutations in HRAS as caus-

ative 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 Française du Syndrome de Costello, to increasing knowledge about Costello syndrome, and their generosity in sharing their experiences.

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

92 Kerr

Kerr B, Eden OM, Dandamudi R, 13 Shannon N, Quarrell O, et al: Costello syndrome: two cases with embryonal rhabdomyosarcoma. J Med Genet 1998;35:1036–1039.Suri M, Garrett C: Costello syndrome 14 with acoustic neuroma and cataract. Clin Dysmorphol 1998;7:149–151.Feingold M: Costello syndrome and 15 rhabdomyosarcoma, J Med Genet 1999;36:582–583.Franceschini P, Licata D, Di Cara G, 16 Guala A, Bianchi M, Ingrosso G, Fran-ceschini D: Bladder carcinoma in Cos-tello syndrome: report on a patient born to consanguineous parents and review. Am J Med Genet 1999;86: 174–179.Flores-Nava G, Canun-Serrano S, Moy-17 sen-Ramirez SG, Parraguirre-Marti-nez S, Escobedo-Chavez E: Costello syndrome associated to a neuroblas-toma. Presentation of a case. Gac Med Mex 2000;136:605–609.Sigaudy S, Vittu G, David A, Vigeron J, 18 Lacombe D, et al: Costello syndrome: Report of six patients including one with an embryonal rhabdomyosarco-ma. Eur J Pediatr 2000;159:139–142.Moroni I, Bedeschi F, Luksch R, Casa-19 nova M, D’Incerti L, Uziel G, Selicorni A: Costello syndrome: a cancer predis-posing syndrome? Clin Dysmorphol 2000;9:265–268.Gripp KW, Scott CI Jr, Nicholson L, 20 Figueroa TE: Second case of bladder carcinoma in a patient with Costello syndrome. Am J Med Genet 2000;90:256–259.Urakami S, Igawa M, Shiina H, Shig-21 eno K, Kikuno N, Yoshino T: Recurrent transitional cell carcinoma in a child with the Costello syndrome. J Urol 2002;168:1133–1134.Gripp KW, Scott CI, Nicholson L, Mc-22 Donald-McGinn DM, Ozeran JD, et al: Five additional Costello syndrome pa-tients with rhabdomyosarcoma: pro-posal for a tumour screening protocol. Am J Med Genet 2002;108:80–87.Di Rocco M, Dodero P: Concerning 23 ‘Five additional Costello syndrome patients with rhabdomyosarcoma: Proposal for a tumour screening pro-tocol’. Am J Med Genet A 2003;118:199.White S, Graham JM, Kerr B, Gripp K, 24 Weksberg R, et al: The adult pheno-type in Costello syndrome. Am J Med Genet A 2005;136;128–135.

Bowron A, Scott JG, Brewer C, Weir P: 25 Increased HVA detected on organic acid analysis in a patient with Costello syndrome. J Inher Metab Dis 2005;28;1155–1156.Lin AE, Grossfeld PD, Hamilton R, 26 Smoot L, Proud V, et al: Further delin-eation of cardiac anomalies in Costello syndrome. Am J Med Genet 2002;111:115–129.Lo I, Brewer C, Shannon N, Shorto J, 27 Tang B, et al: Severe neonatal manifes-tations of Costello syndrome. J Med Genet 2008;45:167–171.Della Marca G, Vasta I, Scarano E, Rig-28 ante M, DeFeo E, et al: Obstructive sleep apnoea in Costello syndrome. Am J Med Genet A 2006;140:257–262.Stein RL, Legault L, Daneman D, 29 Weksberg R, Hamilton J: Growth hor-mone deficiency in Costello syndrome. Am J Med Genet A 2004;129:166–170.Okamoto N, Chiyo H, Imai K, Otani K, 30 Futagi Y: A Japanese patient with the Costello syndrome. Hum Genet 1994;93:605–606.Gregersen N, Viljoen D: Costello syn-31 drome with growth hormone deficien-cy and hypoglycaemia: A new report and review of the endocrine associa-tions. Am J Med Genet A 2004;129:171–175.Kerr B, Delrue M-A, Sigaudy S, Per-32 veen R, Marche M, et al: Genotype-phenotype correlation in Costello syn-drome; HRAS mutation analysis in 43 cases. J Med Genet 2006;43:401–405.Alexander S, Ramadan D, Alkhayyat 33 H, Al-Sharkawi I, Backer KC, El-Sab-ban F, Hussain K: Costello syndrome and hyperinsulinemic hypoglycaemia. Am J Med Genet A 2005;139:227–230.Kerr B, Einaudi MA, Clayton P, Glad-34 man G, Eden T, et al: Is growth hor-mone treatment beneficial or harmful in Costello syndrome? J Med Genet 2003;40:e74.Delrue M-A, Chateil J-F, Arveiler B, 35 Lacombe D: Costello syndrome and neurological abnormalities. Am J Med Genet A 2003;123:301–305.Kawame H, Matsui M, Kurosawa K, 36 Matsuo M, Masuno M, et al: Further delineation of the behavioural and neurological feature in Costello syn-drome. Am J Med Genet A 2003;118:8–14.

Axelrad ME, Glidden R, Nicholson L, 37 Gripp KW: Adaptive skills, cognitive and behavioral characteristics of Cos-tello syndrome. Am J Med Genet A 2004;128:396–400.Axelrad ME, Nicholson L, Stabley DL, 38 Sol-Church K, Gripp KW: Longitudi-nal assessment of cognitive character-istics in Costello syndrome. Am J Med Genet A 2007;143:3185–3193.Yassir WK, Grottkau BE, Goldberg MJ: 39 Costello syndrome: Orthopaedic man-ifestations and functional health. J Pediatr Orthop 2003;23:94–98.Mori M, Yamagata T, Mori Y, Nokubi 40 M, Saito K, Fukushima Y, Momoi M: Elastic fiber degeneration in Costello syndrome. Am J Med Genet 1996;61:304–309.Hinek A, Rabinovitch M, Keeley F, 41 Okamura-Oho Y, Callahan J: The 67-kD elastin/laminin-binding protein is related to an enzymatically inactive, alternatively spliced form of beta-ga-lactosidase. J Clin Invest 1993;91:1198–1205.Hinek A, Teitell MA, Schoyer L, Allen 42 W, Gripp K, et al: Myocardial storage of chondroitin sulfate-containing moi-eties in Costello syndrome patients with severe hypertrophic cardiomyo-pathy. Am J Med Genet A 2005;133:1–12.Aoki Y, Niihori T, Kawame H, Kuro-43 sawa K, Ohashi H, et al: Germline mu-tations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 2005;37:1038–1040.Digilio M, Sarkozy A, Capolino R, Tes-44 ta M, Esposito G, et al: Costello syn-drome: clinical diagnosis in the first year of life. Eur J Pediatr 2008;167:621–628.Van der Burgt I, Kupsky W, Stassou S, 45 Nadroo A, Barroso C, et al: Myopathy caused by HRAS germline mutations-implications for disturbed myogenic differentiation in the presence of con-stitutive H-Ras activation. J Med Genet 2007;44:459–462.Gripp K, Lin A, Stabley DL, Nicholson 46 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;143:1472–1480.


Ørstavik K, Tangeraas T, Molven A, 47 Prescott TE: Distal phalangeal creases – a distinctive dysmorphic feature in disorders of the RAS signalling path-way. Eur J Med Genet 2007;50:155–158.

Allanson J, Kavamura I, Neri G, Noo-48 nan J, Poss A, Kerr B: Distal phalan-geal creases: More evidence of this fea-ture in disorders of the Ras signaling pathway. Eur J Med Genet 2007;50:482–483.

Bronwyn Kerr

Regional Genetic Service and Medical Genetics Research Group, Central Manchester and

Manchester Children’s Hospitals University NHS Trust, Royal Manchester Children’s Hospital

Hospital Rd

Manchester M274HA (UK)

Tel. +44 1619222335, Fax +44 1619222329, E-Mail [email protected]



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 Children’s Clinic, Wilmington, Del., USA

AbstractCostello syndrome is a rare tumor predisposition syn-

drome 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 onco-

gene, 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. Sub-

stitutions 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 ac-

ids other than G12 or G13 occurred in one individual each

(Q22K, T58I, E63K, K117R, A146T, A146V), and may be asso-

ciated 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 muta-

tion was seen in one individual. While germline mosaicism

is the likely cause for reported Costello syndrome sibling

pairs, this has not been molecularly confirmed.


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,

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 mu-

tation 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 informa-

tion, Aoki et al. (2005) analyzed other genes en-

coding MAPK pathway proteins and discovered

mutations in HRAS in patients with Costello syn-

drome [3].

HRAS Mutations in Costello Syndrome

Aoki et al. (2005) identified the genetic cause of

Costello syndrome by sequencing the entire cod-

ing region of the RAS genes from 13 Japanese and

Italian individuals with Costello syndrome [3]. In

12 individuals, they found a heterozygous muta-

tion 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

HRAS Mutations and Costello Syndrome 95

of germline origin [3]. Strikingly, all mutations af-

fected 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 gly12-

to-ser (G12S) amino acid substitution. A germ-

line 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 tran-

sition 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 com-

mon mutation in human cancers [4].

The genetic etiology of Costello syndrome

was soon confirmed in three larger studies of

American and European patients [5–7]. Gripp et

al. (2006) performed mutation analysis in 40 pa-

tients 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 ami-

no acid substitutions affect protein regions di-

rectly linked to its function as a regulator of the

MAPK pathway. In addition to the common mu-

tations affecting either residue G12 or G13 of the

Ras protein, six patients with DNA variants caus-

ing changes of amino acids 22, 58, 63, 117 or 146

were identified.

Table 1. HRAS mutations reported in 139 Costello syndrome patients

HRAS point mutation AA change Frequency in percent References

(number of patients)

c.34G>A G12S 81.3 (113) 3, 5–7, 9, 11, 12, this work

c.34G>T G12C 2 (3) 7, this work

c.35G>C G12A 7.2 (10) 3, 5–7, 15

c.35_36GC>TT G12V 1.4 (2) 3, 12

c.35_36GC>AA G12E <1 (1) 7

c.37G>T G13C 1.4 (2) 5, 6, this work

c.38G>A G13D 1.4 (2) 3

c.64C>A Q22K <1 (1) 12

c.173C>T T58I <1 (1) 18

c.187G>A E63K <1 (1) 12

c.350A>G K117R <1 (1) 7

c.436G>A A146T <1 (1) 11

c.437C>T A146V <1 (1) 18

96 Sol-Church � Gripp

G12S Variants

G12S is the most common mutation found in

Costello syndrome patients, accounting for

about 82% of reported cases [3, 5–7]. Additional

patients carrying this change were reported

by Sol-Church et al. (2006), Van Steensel et al.

(2006), and Zampino et al. (2007) [9–11]. Not

surprisingly, because the majority of patients

carry this particular mutation, the phenotype

associated with the G12S mutation encompass-

es all findings recognized as typical for Costello

syndrome prior to the gene identification [3,

5–7, 9–11]. In addition to these typical clinical-

ly 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 addi-

tional, 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 mu-

tations can occur at these sites, especially G>A

Splice variants

Intron6 Intron 1 Intron 2 Intron 3 Intron 4 Intron5

Variant 1ATG

p21

Stop Variant 2ATG

p19

Stop

mRNAs

gDNA

Proteins

E1

A

B

C

E2 E3 E4 E6 E5 (IDX) E7

GGG Ef G

N C

A146TK117RT58IQ22KG12SA146VE63KG13C

Switch I Switch II

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).


transitions resulting in the G12S change seen in

the majority of Costello syndrome patients.

G12A Variants

The second most common HRAS mutation as-

sociated with Costello syndrome in 10 unrelat-

ed patients is a c.35G>C transversion resulting

in a gly12-to-ala (G12A) amino acid substitu-

tion. 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 pa-

tients carrying this DNA variant [3, 5–7]. More

recently, Søvik et al. (2007) reported a fascinat-

ing family with two sisters initially clinically

diagnosed with Costello syndrome [15]. While

one of the sisters carries a germline HRAS muta-

tion (G12A), a germline KRAS mutation (F156L)

was identified in her sibling. The G12A muta-

tion is seen in less than 1% of sporadic malig-

nancies 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 rhabdomyosar-

coma [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 sporad-

ic 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 pa-

tient with congenital myopathy with excess of

muscle spindles [12]. This Ras mutant has a low

GTPase activity and high transformation poten-

tial [16, 17]. Based on several unpublished cases

the G12V change may be associated with a severe,

early lethal phenotype. The G12C variant result-

ing from a c.34G>T mutation was present in three

patients, one of whom developed rhabdomyosar-

coma [7, and this work]. In the same cohort Kerr

et al. (2006) identified a rare variant G12E in a pa-

tient who died at age 6 months [7].

Two patients with a gly13-to-cys (G13C) sub-

stitution caused by a c.37G>A mutation have

been identified [5, 6 and this work]. The first pa-

tient 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 sec-

ond patient was previously unpublished, she had

a history of typical feeding difficulties but nev-

er 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 be-

low the mean. She has not developed papillomata.

These two cases may suggest that G13C harbors a

slightly milder phenotype, compared to the com-

mon G12S change. Despite the fact that the HRAS

G13C mutation has been identified in three blad-

der 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 myo-

pathy with an excess of muscle spindle fibers on

biopsy was reported in four patients with HRAS

mutations. While two changes were novel, two oth-

ers, 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 path-

way’s effect on striated muscle development, rather

than a finding unique to rare HRAS mutations.


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 find-

ings 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 lys117-

to-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) sub-

stitution. 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 syn-

drome included microcephaly, sparse and thin,

but not curly hair, and ears lacking the ‘distinc-

tive 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 syn-

drome, including failure to thrive and severe hy-

pertrophic 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 impor-

tant 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 malignan-

cies, or if it is located at a crucial position within

the protein (fig. 1C). The identification of addi-

tional patients with changes of the same amino

acid, as reported for alanine 146, also supports

clinical relevance. It is impossible to draw phe-

notype-genotype conclusions based on single pa-

tients 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 as-

sociated phenotype is not easily recognized as

Costello syndrome and the patients remain un-

diagnosed. It is noteworthy that several patients

with rare mutations lack the strikingly coarse fa-

cial features and very deep palmar creases. Once

more patients with these mutations are studied it

will become clear if their phenotype varies sig-

nificantly from that associated with the G12S or

G12A changes.

Parental Origin of Germline HRAS Mutations

Heterozygous missense mutations causing con-

stitutive activation of the protein product often

occur in the paternal germline, as suggested by

Penrose (1955) who proposed that mitotic repli-

cation errors accumulate in male germ cells [19].

The paternal age effect in Costello syndrome

[20], in combination with the nature of the mis-

sense 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), proxi-

mal to the mutation site, that could be used to

trace the parental origin of the germline muta-

tions [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) inher-

ited from the fathers and there was an advanced

age at conception in fathers transmitting the mu-

tation. The Costello syndrome sibling studied by

Søvik et al. (2007) carries a G12A mutation of pa-

ternal 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


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 mu-

tations encoding gain-of-function, such as the

paternally inherited S252W in FGFR2 causing

Apert syndrome, might confer a selective ad-

vantage 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 fer-

tilization. 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 in-

herited [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 methyla-

tion in spermatogonia compared to oogonia may

allow for a higher mutation rate at CpG dinucle-

otides [14]. Alternatively, Ras proteins are found

in the acrosomal regions of sperm cells and it is

possible that sperm carrying certain HRAS mu-

tations 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 sug-

gest an autosomal recessive inheritance pattern.

However, germline mosaicism is a well known

mechanism in the recurrence of autosomal dom-

inant disorders, and, while not proven, is likely

to have caused the recurrence in these families.

Bodkin et al. (1999) postulated somatic mosa-

icism 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 au-

thors suggested that this apparent male-to-male

transmission was consistent with an autosomal

dominant inheritance pattern [27].

A single molecularly confirmed case of so-

matic mosaicism in Costello syndrome has been

reported [28]. This female presented with find-

ings suggestive of Costello syndrome, including

developmental delay, short stature, sparse hair,

coarse facial features and thickened toenails.

She had tight Achilles tendons requiring surgi-

cal lengthening. The characteristic nasal papil-

lomata of Costello syndrome developed at age 15

years, leaving no doubt about the clinical diag-

nosis. Findings not typical for Costello syndrome

included irregular skin hyper- and hypopigmen-

tation, often associated with mosaicism for chro-

mosome abnormalities. Assays performed in this

patient with standard techniques on white blood

cell derived DNA did not show an HRAS muta-

tion. However, testing of multiple samples of buc-

cal cell derived DNA revealed a sequence change

qualitatively consistent with the G12S mutation.

Allelic quantitation demonstrated the presence

of this mutation in ~25–30% of buccal cells. In

this patient, standard techniques failed to identi-

fy 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 mo-

saicism for the HRAS mutation had her menar-

che 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 af-

fected in this respect, but she is at risk for having

a child with Costello syndrome. Since the degree


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 depict-

ed in figure 1B. Splice variant 1, in which IDX

is spliced out, encodes a 21-kDa membrane an-

chored 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

10–14, 57–63, 116–119 and 144–147, that are mo-

tifs conserved amongst members of the Ras fam-

ily. A C-terminal hypervariable region includes

a plasma membrane targeting CAAX motif and

secondary signals for palmitoylation, proteolysis,

and carboxyl methylation [32]. p21ras binds gua-

nine nucleotides (GDP and GTP) through a nu-

cleotide binding pocket formed by the interacting

G-domains. Crystallographic information indi-

cates that residues 28, 116, 117, 118, 119, 144 and

146 interact specifically with the guanine ring of

the nucleotides, while residues 11, 12, 32, 58–63

are in close proximity of the γ-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 phosphoryla-

tion cascade. The signal transduction is stopped

by conversion of Ras from the GTP- to GDP-

bound form mediated by the concerted action of

Ras intrinsic GTPase activity, and GTPase acti-

vating proteins (GAPs) such as NF1. X-Ray crys-

tallography further revealed that Ras-GTP/GDP

cycling induces changes in the conformation of

two ‘switch regions’ (fig. 1C), formed by residues

30–38 (switch I) and residues 60–72 (switch II).

Proper positioning of the switch regions, notably

switch II, is required for GTP hydrolysis [34, 35].

Figure 2 illustrates the conformational changes

Switch II Switch I Switch II Switch I

Ras-GEF

Ras-GAP

Ras-GDP inactive Ras-GTP active

N

C

GTPGDP

N

C

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 hydro-

lysis of bound GTP to GDP resulting

in inactivation of Ras. The 3D struc-

tures were generated using Deep

View Swiss Pdb viewer v3.7 available

as free download from http://www.

Expasy.org/spdbv. Residue 12 (locat-

ed 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.


between the two states with switch II moving

away from the nucleotide binding pocket, and re-

positioning of the side chains of the Q61, there-

by facilitating GAP binding and GTP hydrolysis

[33–35].

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 ac-

tive GTP-bound conformation, thus disrupting

the normal biochemical Ras function and caus-

ing aberrant downstream signaling [17, 36–38].

Proteins containing mutated glycine residues

at position 12 or 13 have reduced intrinsic and

GAP-mediated GTPase activity leading to sus-

tained MAPK activation [36, 38]. An activating

gly12-to-val (G12V) mutation in the HRAS gene

present in a bladder carcinoma was the first so-

matic 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 inactiva-

tion 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 cor-

relating 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 gain-

of-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 muta-

tions are likely associated with either a more se-

vere 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 includ-

ing rhabdomyosarcoma and transitional cell car-

cinoma 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 anoth-

er MAPK pathway gene, should not be diagnosed

with Costello syndrome, but rather with the syn-

drome more consistent with the respective gene

mutation. Such a change from the original clini-

cal 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 in-

structive are the sisters reported by Søvik 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 expect-

ed, their clinical course differed as the patients


References

Tartaglia M, Mehler EL, Goldberg R, 1 Zampino G, Brunner HG, et al: Muta-tions in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 2001;29:465–468.Tartaglia M, Cotter PD, Zampino G, 2 Gelb BD, Rauen KA: Exclusion of PTPN11 mutations in Costello syn-drome: Further evidence for distinct etiologies for Noonan, cardio-facio-cutaneous and Costello syn-drome. Clin Genet 2003;63:423–426.Aoki Y, Niihori T, Kawame H, Kuro- 3 sawa K, Ohashi H, et al: Germline mu-tations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 2005;37:1038–1040.Forbes S, Clements J, Dawson E, Bam- 4 ford S, Webb T, et al: COSMIC 2005. Br J Cancer 2006;94:318–322.Gripp KW, Lin AE, Stabley DL, 5 Nicholson L, Charles I, et al: HRAS mutation analysis in Costello syn-drome: genotype and phenotype cor-relation. Am J Med Genet A 2006; 140:1–7.

Estep AL, Tidyman WE, Teitell MA, 6 Cotter PD, Rauen KA: HRAS muta-tions in Costello syndrome: Detection of constitutional activating mutations in codon 12 and 13 and loss of wild-type allele in malignancy. Am J Med Genet A 2006;140:8–16.Kerr B, Delrue MA, Sigaudy S, Perveen 7 R, Marche M, et al: Genotype-pheno-type correlation in Costello syndrome; HRAS mutation analysis in 43 cases. J Med Genet 2006;43:401–405.Lin AE, Rauen KA, Gripp KW, Carey 8 JC: Clarification of previously reported Costello syndrome patients. Am J Med Genet A 2008;146:940–943.Sol-Church K, Stabley DL, Nicholson 9 L, Gonzalez IL, Gripp KW: Paternal bias in parental origin of HRAS muta-tions in Costello syndrome. Hum Mu-tat 2006;27:736–741.van Steensel MA, Vreeburg M, Peels C, 10 van Ravenswaaij-Arts CM, Bijlsma E, et al: Recurring HRAS mutation G12S in Dutch patients with Costello syn-drome. Exp Dermatol 2006;15: 731–734.

Zampino G, Pantaleoni F, Carta C, Co-11 bellis G, Vasta I, et al: Diversity, paren-tal germline origin, and phenotypic spectrum of de novo HRAS missense changes in Costello syndrome. Hum Mutat 2007;28:265–272.van der Burgt I, Kupsky W, Stassou S, 12 Nadroo A, Barroso C, et al: Myopathy caused by HRAS germline mutations – implications on disturbed myogenic differentiation in the presence of con-stitutive H-Ras activation. J Med Genet 2007;44:459–462.Selcen D, Kupsky WJ, Benjamins D, 13 Nigro MA: Myopathy with muscle spindle excess: A new congenital neu-romuscular syndrome? Muscle Nerve 2001;24:138–143.Pfeifer GP: p53 mutational spectra and 14 the role of methylated CpG sequences. Mutat Res 2000;450:155–166.Søvik O, Schubbert S, Houge G, Steine 15 SJ, Norgård G, et al: De novo HRAS and KRAS mutations in two siblings with short stature and neuro-cardio-facio-cutaneous features. J Med Genet 2007;44:e84.Colby WW, Hayflick JS, Clark SG, 16 Levinson AD: Biochemical character-ization of polypeptides encoded by mutated human Ha-ras1 genes. Mol Cell Biol 1986;6:730–734.

grew older and the Costello syndrome individ-

ual developed acanthosis nigricans, papilloma-

ta and four asymptomatic bladder carcinomas,

whereas the individual with the KRAS muta-

tion associated phenotype had severe epilepsy

with hippocampal sclerosis and atrophy. These

sisters illustrate the overlap of clinical presenta-

tion in infancy, and the more distinctive pheno-

type of the MAPK pathway disorders thereafter.

Molecular testing should be helpful in clarifying

the diagnosis, and it is in the patient’s best inter-

est to reconsider a clinical diagnosis if the mo-

lecular test result is inconsistent. Consistency of

molecular and clinical diagnosis allows for clar-

ity in communication amongst patients and ad-

vocates, health care providers, and researchers.

In the future, drug treatment may be tailored to

counteract the specific effects of certain muta-

tions, and may vary by gene.

Acknowledgements

We thank all patients and their families for their par-ticipation in our research studies, our collaborators and numerous clinicians who referred patients and contrib-uted 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.


Fasano O, Aldrich T, Tamanoi F, Tap-17 arowsky E, Furth M, Wigler M: Analy-sis of the transforming potential of the human H-ras gene by random muta-genesis. Proc Natl Acad Sci USA 1984;81:4008–4012.Gripp KW, Innes AM, Axelrad ME, 18 Gillan TL, Parboosingh JS, et al: Cos-tello syndrome associated with novel germline HRAS mutations: An attenu-ated phenotype? Am J Med Genet A 2008;146:683–690.Penrose LS: Parental age and mutation. 19 Lancet 1955;2:312–313.Lurie IW: Genetics of the Costello syn-20 drome. Am J Med Genet 1994;52: 358–359.Goriely A, McVean GA, Rojmyr M, 21 Ingermarsson B, Wilkie AO: Evidence for selective advantage of pathogenic FGFR2 mutations in the male germ line. Science 2003;301:606–607.Goriely A, McVean GA, van Pelt AM, 22 O’Rourke AW, Wall SA, et al: Gain-of-function amino acid substitutions drive positive selection of FGFR2 mu-tations in human spermatogonia. Proc Natl Acad Sci USA 2005;102: 6051–6056.Lazaro C, Gaona A, Ainsworth P, Ten-23 coni R, Vidaud D, et al: Sex differences in mutational rate and mutational mechanism in the NF1 gene in neuro-fibromatosis type 1 patients. Hum Genet 1996;98:696–699.Lopez-Correa C, Dorschner M, Brems 24 H, Lazaro C, Clementi M, et al: Recom-bination hotspot in NF1 microdeletion patients. Hum Mol Genet 2001;10: 1387–1392.Zampino G, Mastroiacovo P, Ricci R, 25 Zollino M, Segni G, et al: Costello syn-drome: Further clinical delineation, natural history, genetic definition, and nosology. Am J Med Genet 1993;47:176–183.

Johnson JP, Golabi M, Norton ME, 26 Rosenblatt RM, Feldman GM, et al: Costello syndrome: phenotype, natural history, differential diagnosis, and possible cause. J Pediatr 1998;133:441–448.Bodkin NM, Mortimer ES, Demmer 27 LA: Male to male transmission of Cos-tello syndrome consistent with auto-somal dominant inheritance. Am J Hum Genet 1999;65(Suppl):A143.Gripp KW, Stabley DL, Nicholson L, 28 Hoffman JD, Sol-Church K: Somatic mosaicism for an HRAS mutation causes Costello syndrome. Am J Med Genet A 2006;140:2163–2169.White SM, Graham JM Jr, Kerr B, 29 Gripp K, Weksberg R, et al: The adult phenotype in Costello syndrome. Am J Med Genet A 2005;136:128–135.Cohen JB, Broz SD, Levinson AD: Ex-30 pression of the H-ras proto-oncogene is controlled by alternative splicing. Cell 1989;58:461–472.Huang MY, Cohen JB: The alternative 31 H-ras protein p19 displays properties of a negative regulator of p21Ras. On-col Res 1997;9:611–621.Bourne HR, Sanders DA, McCormick 32 F: The GTPase superfamily: conserved structure and molecular mechanism. Nature 1991;349:117–127.Pai EF, Kabsch W, Krengel U, Holmes 33 KC, John J, Wittinghofer A: Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene prod-uct p21 in the triphosphate conforma-tion. Nature 1989;341:209–214.Pai EF, Krengel U, Petsko GA, Goody 34 RS, Kabsch W, Wittinghofer A: Re-fined crystal structure of the triphos-phate conformation of H-ras p21 at 1.35 Å resolution: implications for the mechanism of GTP hydrolysis. EMBO J 1990;9:2351–2359.

Vetter IR, Wittinghofer A: The gua-35 nine nucleotide-binding switch in three dimensions. Science 2001;294:1299–1304.Oliva JL, Zarich N, Martinez N, Jorge 36 R, Castrillo A, et al: The P34G muta-tion reduces the transforming activity of K-Ras and N-Ras in NIH 3T3 cells but not of H-Ras. J Bio Chem 2004;279:33480–33489.Lowy DR, Willumsen BM: Function 37 and regulation of Ras. Annu Rev Bio-chem 1993;62:851–891.Ahmadian MR, Zor T, Vogt D, Kabsch 38 W, Selinger Z, et al: Guanosine triphosphatase stimulation of onco-genic Ras mutants. Proc Natl Acad Sci USA 1999;96:7065–7070.Tabin CJ, Bradley SM, Bargmann CI, 39 Weinberg RA, Papageorge AG, et al: Mechanism of activation of a human oncogene. Nature 1982;300:143–149.Reddy EP, Reynolds RK, Santos E, Bar-40 bacid M: A point mutation is respon-sible for the acquisition of transform-ing properties by the T24 human bladder carcinoma oncogene. Nature 1982;300:149–152.Gripp KW, Lin AE, Nicholson L, Allen 41 W, Cramer A, et al: Further delinea-tion of the phenotype resulting from BRAF or MEK1 germline mutations helps differentiate cardio-facio-cuta-neous syndrome from Costello syn-drome. Am J Med Genet A 2007;143:1472–1480.Rauen KA: Distinguishing Costello 42 versus cardio-facio-cutaneous syn-drome: BRAF mutations in patients with a Costello phenotype. Am J Med Genet A 2006;140:1681–1683.

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)




Endocrine Regulation of Growth and Short Stature in Noonan Syndrome

G. Binder

University-Children’s Hospital Tuebingen, Pediatric Endocrinology Section,

Tübingen, Germany

AbstractShort 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 chil-

dren with Noonan syndrome. SHP2 in vitro binds and

dephosphorylates signaling molecules that are positive

regulators of the cellular response to GH. Gain-of-func-

tion mutations of PTPN11, the gene encoding SHP2, in

Noonan syndrome were therefore predicted to regulate

the cellular response to GH negatively. Growth in chil-

dren 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 in-

sulin-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 nor-

mal in children with Noonan syndrome and no muta-

tion 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 influ-

ence longitudinal growth in Noonan syndrome. These

deleterious mechanisms are likely to affect the intrinsic

regulation of the bone development itself.


Short Stature

Short stature is a major feature of Noonan syn-

drome. Adults with Noonan syndrome reach a

mean final height around 1.9 SD below the nor-

mal mean (–1.9 SDS), 166 cm in men and 153 cm

in women [1–3]. As a consequence, 40% of adults

with Noonan syndrome have short stature

(height below –1.88 SDS) and about 15% have se-

vere short stature (height below –2.5 SDS). Birth

length and weight are normal in Noonan syn-

drome [1]. In the growth of a child three phases

can be distinguished: the infancy phase (main-

ly 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

[1–3]. 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].

Noonan Syndrome and Growth Failure 105

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 GH-

regulated insulin-like growth factor (IGF)-I pro-

duction in the liver. Both hormones, GH and

IGF-I act in concert at the epiphyseal plate ac-

cording to the dual effector theory. While GH

promotes differentiation and sensitivity to IGF-I

of the precursor cells, IGF-I causes the clonal pro-

liferation. 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 com-

parison to normal individuals, a mild increase of

spontaneous GH secretion with high trough GH

concentrations was described [4, 5]. Such a con-

stellation would be compatible with a mild form

of GH insensitivity.

GH Therapy

GH substitution was started about 50 years ago in

children with severe GH deficiency with satisfy-

ing 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 pro-

mote growth in short children who have a distur-

bance of their target organ, the epiphyseal plate.

This is the case in children with Turner syn-

drome [6]. It is also used in children with a com-

bination of both, disturbed endocrine regulation

and target organ resistance, such as the hetero-

geneous group of short children who are born

small-for-gestational-age and do not show catch-

up growth [7]. Pharmacological GH therapy has

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 re-

vealed a mean effect of 7 cm height gain in girls

with Turner syndrome [9].

As Noonan syndrome shares many dysmor-

phic 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 pro-

nounced than in Turner syndrome [10] or similar

[11]. Randomized controlled studies on the effi-

cacy of this treatment are still missing. The in-

terpretation of effects on growth is difficult due

to the natural course of prolonged growth period

in Noonan syndrome. Calculations should im-

plicate the retardation of bone age and puberty,

which may enable spontaneous catch-up growth

after the time of normal growth arrest [1–3].

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 lev-

el of GH signaling. The mechanisms implicated

in GH signaling are as follows: after binding of

GH, the GH receptor (GHR) dimer initially ac-

tivates JAK2, which includes autophosphory-

lation of the kinase domain of JAK2. Activated

JAK2 then phosphorylates itself at additional ty-

rosine residues as well as tyrosines within the cy-

toplasmic domain of the GHR and many other

signaling molecules including STAT5b. This fac-

tor 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

106 Binder

demonstrated that SHP2 acts as a cytosolic phos-

phatase of STAT5 downregulating its activity [15,

16]. Accordingly, a mutation of the SHP2 bind-

ing site in GHR has been shown to cause prolon-

gation of tyrosyl phosphorylation of GHR, JAK2

and STAT5b in response to GH in vitro [17]. In

summary, SHP2 binds and dephosphorylates sig-

naling molecules which are positive regulators

of the cellular response to GH (fig. 1). Therefore,

gain-of-function mutations of PTPN11 may be as-

sumed to negatively regulate the cellular response

to GH in children with Noonan syndrome.

Short Stature in Noonan Syndrome:


Short stature is considered to be a general char-

acteristic 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 syn-

drome that are clinically relevant.

Notably, the growth in children with Noonan

syndrome due to SOS1 mutations is less compro-

mised than in the presence of PTPN11 mutations

[18]. SOS1 encodes a RAS-specific guanine nu-

cleotide 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 ob-

vious genotype-phenotype correlation regarding

growth in patients with PTPN11 and SOS1 muta-

tions, respectively, suggests that mutant SHP2 has

stronger impacts on growth than mutant SOS1.

It is tempting to speculate that the negative ef-

fect of mutant SHP2 on longitudinal growth is in

part mediated by an interference with GH signal-

ing. However, this suggestion is challenged by the

fact that patients affected by germline mutations

of other downstream components of the RAS-

MAPK 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 RAS-

MAPK 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 pos-

sible 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

Fig. 1. Schematic drawing illustrating known inter-

actions of SHP2 with components of the GH signaling

pathway.

STAT5b

STAT5b

JAK2JAK2

P

Cytoplasm

Nucleus

P

P

P P

STATs

P

P STAT5b

IGF-I transcription

GH

STATs

P

STAT5b

P

P

P

JAK2

PSHP2

SHP2

SHP2

Noonan Syndrome and Growth Failure 107

References

Ranke MB, Heidemann P, Knupfer C, 1 Enders H, Schmaltz AA, Bierich JR: Noonan syndrome: growth and clini-cal manifestations in 144 cases. Eur J Pediatr 1988;148:220–227.Witt DR, Keena BA, Hall JG, Allanson 2 JE: Growth curves for height in Noo-nan syndrome. Clin Genet 1986;30:150–153.

Shaw AC, Kalidas K, Crosby AH, Jeffery 3 S, Patton MA: The natural history of Noonan syndrome: a long-term follow-up study. Arch Dis Child 2007;92:128–132.Ahmed ML, Foot AB, Edge JA, Lamkin 4 VA, Savage MO, Dunger DB: Noonan’s syndrome: abnormalities of the growth hormone/IGF-I axis and the response to treatment with human biosynthetic growth hormone. Acta Paediatr Scand 1991;80:446–450.

Noordam C, van der Burgt I, Sweep 5 CG, Delemarre-van de Waal HA, Sen-gers RC, Otten BJ: Growth hormone (GH) secretion in children with Noo-nan syndrome: frequently abnormal without consequences for growth or response to GH treatment. Clin Endo-crinol 2001;54:53–59.

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 muta-

tion in PTPN11 [19–21]. 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 mu-

tations in other components of the RAS-MAPK

pathway.

In the presence of GH resistance due to

PTPN11 mutations, responsiveness to a phar-

macological GH therapy would be suspected to

be low. The above mentioned studies have ad-

dressed this issue as well: the design was pro-

spective in two studies and retrospective in

one. Overall, there was a trend to a better first-

year growth velocity of Noonan children with-

out 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 fi-

nal 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 per-

spective from the endocrinological point of view,

since SHP2 is implicated in the downregulation

of GH receptor signaling. Current data show de-

creased IGF-I and IGFBP-3 levels, which are mark-

ers of the biological effect of GH, in those children

with Noonan syndrome who carry a PTPN11 mu-

tation. 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 admin-

istered in high doses to short children to promote

growth and improve final height. GH respon-

siveness 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 mu-

tations 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 mech-

anisms that influence longitudinal growth. These

deleterious mechanisms are likely to affect the in-

trinsic regulation of the bone development itself.

108 Binder

Marchini A, Rappold G, Schneider KU: 6 SHOX at a glance: from gene to pro-tein. Arch Physiol Biochem 2007;113:116–123.Saenger P, Czernichow P, Hughes I, 7 Reiter EO: Small for gestational age: short stature and beyond. Endocr Rev 2007;28:219–251.Cutfield WS, Lindberg A, Rapaport R, 8 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):153–159.Stephure DK, Canadian Growth Hor- 9 mone Advisory Committee: Impact of growth hormone supplementation on adult height in Turner syndrome: re-sults of the Canadian randomized con-trolled trial. J Clin Endocrinol Metab 2005;90:3360–2266.Noordam C, van der Burgt I, Sengers 10 RCA, Delemarre-van de Waal HA, Ot-ten BJ: Growth hormone treatment in children with Noonan’s syndrome: four year results of a partly controlled trial. Acta Paediatr 2001;90:889–894.

Osio D, Dahlgren J, Albertsson Wik-11 land K, Westphal O: Improved final height with long-term growth hor-mone treatment in Noonan syndrome. Acta Paediatr 2005;94:1232–1237.Woelfle J, Chia DJ, Massart-Schlesing-12 er MB, Moyano P, Rotwein P: Molecu-lar physiology, pathology, and regula-tion of the growth hormone/insulin-like growth factor-I system. Pediatr Nephrol 2005;20:295–302.Argetsinger LS, Carter-Su C: Mecha-13 nism of signaling by growth hormone receptor. Physiol Rev 1996;76: 1089–1107.Kim SO, Jiang J, Yi W, Feng GS, Frank 14 SJ: Involvement of the Src homology 2-containing tyrosine phosphatase SHP2 in growth hormone signaling. J Biol Chem 1998;273:2344–2354.Yu C-L, Yong-Jiu J, Burakoff SJ: Cyto-15 solic tyrosine dephosphorylation of STAT5. J Biol Chem 2000;275:599–604.Chen Y, Wen R, Yang S, Schuman J, 16 Zhang EE, et al: Identification of Shp-2 as a Stat5A phosphatase. J Biol Chem 2003;278:16520–16527.

Stofega MR, Herrington J, Billestrup 17 N, Carter-Su C: Mutation of the SHP2 binding site in growth hormone (GH) receptor prolongs GH-promoted ty-rosyl phosphorylation of GH receptor, JAK2, and STAT5B. Mol Endocrinol 2000;14:1338–1350.Tartaglia M, Pennacchio LA, Zhao C, 18 Yadav KK, Fodale V, et al: Gain-of-function SOS1 mutations cause a dis-tinctive form of Noonan syndrome. Nat Genet 2007;39:75–79.Binder G, Neuer K, Ranke MB, Witte-19 kindt NE: PTPN11 mutations are as-sociated with mild growth hormone resistance in individuals with Noonan syndrome. J Clin Endocrinol Metab 2005;90:5377–5381.Ferreira LV, Souza SAL, Arnhold IJP, 20 Mendonca BB, Jorge AAL: PTPN11 mutations and response to growth hor-mone therapy in children with Noonan syndrome. J Clin Endocrinol Metab 2005;90:5156–5160.Limal J-M, Parfait B, Cabrol S, Bonnet 21 D, Leheup B, et al: Noonan syndrome: relationships between genotype, growth, and growth factors. J Clin En-docrinol Metab 2006;91:300–306.

Gerhard Binder

University-Children’s Hospital Tuebingen, Pediatric Endocrinology Section

Hoppe-Seyler-Strasse 1

DE–72076 Tübingen (Germany)

Tel. +49 7071 2983781, Fax +49 7071 294157, E-Mail [email protected]



The Heart in Ras-MAPK Pathway Disorders

M.C. Digilioa � B. Marinob � A. Sarkozyc �

P. Versaccib � B. Dallapiccolac

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

AbstractNoonan syndrome (NS) and related disorders are due to

mutations in the genes of the Ras-MAPK pathway. Congeni-

tal heart defect (CHD) is diagnosed in 60–85% of patients af-

fected 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 anom-

alies (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 an-

terior motion of the mitral valve. The prevalence of specific

CHDs in the different clinical conditions is varying depend-

ing 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.


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 character-

ized by pulmonary valvular and/or supravalvular

stenosis with dysplastic and thickened valves was

then confirmed by several other reports [2–7].

‘Eccentric left ventricular hypertrophy’ (asym-

metrical 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 de-

fects are not specific of NS, but they also occur

in the ‘cardio-cutaneous syndromes’ (LEOPARD

and Watson syndromes) and in neurofibromato-

sis type 1 (NF1) (also called ‘Noonan-like’ syn-

dromes). At the same time, Dr. Noonan argued

that ‘with further clinical studies the group of pa-

tients 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 phe-

notypes resulting from mutations in the genes

110 Digilio � Marino � Sarkozy � Versacci � Dallapiccola

belonging to the same pathogenetic pathway

[12, 13]. Nevertheless, the prevalence of specific

CHDs in the different clinical conditions is vary-

ing depending on the mutated gene, and at time

also on the different allelic mutations [14].

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 con-

ditions [7, 15]. Nevertheless, the spectrum of car-

diac malformations in NS has been progressively

widened, to include also atrioventricular canal de-

fect (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 as-

sociated with PVS [17].

The anatomic pattern is quite distinct, with

dysplastic pulmonary valve and fibrous thicken-

ing of the annulus and the leaflets [7, 15, 16, 18].

Stenosis results from the fibrous thickening of the

valvular leaflets, which appear deformed, glisten-

ing and edematous, eventually without fusion of

commissures [19, 20]. In some cases pulmonary

Table 1. Prevalence of congenital heart defects (CHDs) in the different syndromes linked to

mutations in genes belonging to the Ras/MAPK pathway

Syndrome Prevalence of CHD (%)

Noonan syndrome

PTPN11 80

SOS1 80

RAF1 90

KRAS 65

LEOPARD syndrome

PTPN11 60

RAF1 100

Cardio-Facio-Cutaneous syndrome

BRAF 85

MEK1/MEK2 45

Costello syndrome

HRAS 65

Neurofibromatosis 1

NF1 2–6

The Heart in Ras-MAPK Pathway Disorders 111

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 non-

syndromic patients with PVS [21].

Histological characteristics of PVS in NS in-

clude 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, percu-

taneous 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, con-

sisting in asymmetric septal thickening, frequent

systolic anterior motion of the mitral valve, and

decreased left ventricular compliance (fig. 2)

[16, 22–25]. The association with structural mi-

tral anomalies, as prolapsing, myxomatous, re-

dundant 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

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

NS LS CFCS CS NF1

PTPN11 SOS1 RAF1 KRAS PTPN11 RAF1 BRAF MEK1-2 HRAS NF1

Pulmonary valve

stenosis

70

73

15

45

25

50

45

20

45

3

Hypertrophic

cardiomyopathy

10 10 75 20 75 100 30 20 40 –

Atrial septal defect 25 20 30 15 – – 20 20 7 1

Atrioventricular

canal defect

3 – – – 4 – – – – –

Aortic coarctation 1 – – – – – – – – 1

Ventricular septal

defect

7 7 – – 4 – – – – –

Tetralogy of Fallot – – 4 – – – – – – –

Mitral anomalies 5 – 40 – 40 100 – – 30 1

Arrhythmia – – – – 25 – – – 30 –

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, 49–53, 55, 56, 62]; LS [25, 27, 50, 57, 58, 67–69]; CFCS [73, 74]; CS [22, 24, 75–78]; NF1

[79–82].


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 dis-

ease, the NS patients show a more severe type of

HCM with reduced diastolic function and high-

er prevalence of left ventricular outflow tract ob-

struction [25]. It has also been shown that HCM

associated with malformation syndromes (78%

of these cases are NS) have a worse clinical out-

come [28].

Atrioventricular Canal Defect (AVCD)

AVCD is a quite common defect in NS individu-

als [15, 16, 29–32]. The partial type is prevailing,

and may be associated with subaortic stenosis, as

left ventricular outflow obstruction due to anom-

alous 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 poly-

valvular disease [16, 18, 26, 27, 33]. An isolated

anomalous insertion of the mitral valve on the

ventricular septum causing obstruction of the

left ventricular outflow tract has been rarely ob-

served in patients [26]. The leaflets of the mitral

valve can be dysplastic with short cords and nod-

ular myxomatous tissue.

A defect of the cardiac jelly and extracellular

matrix has been suggested as a likely pathoge-

netic mechanism for some CHDs in NS, includ-

ing MVA associated with HCM and AVCD

[34–36].

Atrial Septal Defect (ASD)

Patients with NS can display the ‘ostium secun-

dum’ type of ASD, often in association with PVS

[14, 16, 37, 38].

Aortic Coarctation (AC)

AC is diagnosed in a subgroup of NS patients

[39–41], mainly without any detectable mutation

in some of the known genes [14, 38]. These pa-

tients 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 occasional-

ly diagnosed in NS patients include tetralogy of

Fallot [16, 42], ventricular septal defect [16], and

aortic root dilatation [16, 43–45].

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 pa-

tients evaluated before the molecular testing be-

came available was estimated in the range of 50

to 90% [16, 46–48]. PVS was the most common

defect (20–50%), followed by HCM (20–30%),

AVCD (15%), ASD (10%), and aortic coarctation

(10%).

Fig. 1. Two-dimensional echocardiography: apical 5-

chamber 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 ar-

tery; PV: pulmonary valve.


Following the discovery of PTPN11 as the

most common affected gene, PVS was con-

firmed as the prevailing CHD in NS [14, 49–54].

It was also proven that HCM was less com-

mon 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 developmen-

tal 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 mu-

tations are considered a mutational hot spot re-

lated 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 relat-

ed 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 cul-

ture 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 associ-

ated 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 as-

sociated 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 pa-

tients, 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

[64–66].

a b

Fig. 2. Two-dimensional echocardiography: (a) Parasternal long-axis view shows an asymmet-

ric 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.


LEOPARD Syndrome

PTPN11: The most common CHD in LS patients

with PTPN11 gene mutations is HCM, which is

usually manifesting with left ventricular hyper-

trophy. 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 as-

sociation with left ventricular hypertrophy and

PVS.

The prevailing PTPN11 mutations in LS af-

fect exons 7 and 12 [50, 57, 67–69]. A subset of

cases display mutations in exon 13, while the

Gln510Glu mutation is associated with a dis-

tinct cardiac phenotype, with rapidly progres-

sive 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 fail-

ure symptoms can manifest in the first years

of life, and can even require cardiac surgery

(myectomy).

Interestingly, PTPN11 gene LS-related mu-

tations appear to dysregulate the Ras-MAPK

pathway in a different way in respect to those oc-

curring in NS. In fact, LS mutations seem to have

a negative effect onto the catalytic activity, im-

plying 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 de-

velop significant symptoms and arrhythmias dur-

ing 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 con-

duction defects [25]. Additional cardiac findings

in these patients include coronary artery anoma-

lies [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 wild-

type protein, in a manner similar to those of NS-

associated 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, MEK1–2: 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 MEK1–2

mutations, raising the question of whether ASD

could represent a physiologic response to the

frequent downstream pulmonary f low obstruc-

tion, 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 distur-

bances [22, 24, 75–78]. 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 man-

ifesting as atrial tachycardia, also referred to

as chaotic, multifocal, and ectopic tachycardia


[24], possibly secondary to dysplasia of the con-

duction system. The occurrence of both HCM

and atrial tachycardia in a dysmorphic neonate

is a striking feature of CS, prompting the mo-

lecular search of HRAS gene mutations.

The onset of HCM in CS usually occurs in the

first two years of life [24]. Also rhythm distur-

bances 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 pre-

cede arrhythmias in many cases; (3) histological

evidence of congenital dysplasia of the conduc-

tion 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 pa-

tients 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 ana-

tomically 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 pa-

tients without NF1.

The involvement of the neurofibromin gene

in the cardiac development is supported by Nf1

‘knockout’ mouse models. In fact, homozy-

gous Nf1 mutant embryos display CHD, includ-

ing double outlet right ventricle and associated

anomalies of the cardiac outflow tract formation,

endocardial cushion development, and myocar-

dial structure [83].

Similarly, the vascular characteristic of NF1

may result from abnormal neurofibromin func-

tion, 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 di-

agnosis, including clinical evaluation, electrocar-

diography, and two-dimensional color-Doppler

echocardiography. Four extremity blood pres-

sure measurements should be controlled, due to

possible occurrence of aortic coarctation, partic-

ularly in patients with NF1.

Cardiac hypertrophy may develop with time.

Therefore, an echocardiographic follow-up ev-

ery 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 monitor-

ing, due to the possible occurrence of rhythm

disturbances.

Patients with a diagnosed CHD should be fol-

lowed by a pediatric cardiologist, and the timing

of follow-up should be programmed based on

the results of periodical investigations and clini-

cal course.

Treatment

PVS

Correction of pulmonary outflow obstruction

caused by valvular dysplasia is indicated. The

initial choice can be transcatheter balloon val-

vuloplasty, although this procedure is frequent-

ly ineffective in the patients with NS, due to the

peculiar dysplasia of the pulmonary valve leaf-

lets. For this reason, surgical valvotomy is often

required.

Prophylaxis for bacterial endocarditis is re-

quired in all patients with a CHD for dental treat-

ments, surgery, or catheterization.


References

Noonan JA, Ehmke DA: Associated 1 noncardiac malformations in children with congenital heart disease. J Pediatr 1963;63:468–470.Caralis DG, Char F, Graber JD, Voigt 2 GC: Delineation of multiple cardiac anomalies associated with the Noonan syndrome in an adult and review of the literature. Johns Hopkins Med J 1974;134:346–355.Noonan JA: Hypertelorism with Turn- 3 er phenotype. A new syndrome with associated congenital heart disease. Am J Dis Child 1968;116:373–380.Siggers DC, Polani PE: Congenital 4 heart disease in male and female sub-jects with somatic features of Turner’s syndrome and normal sex chromo-somes (Ullrich’s and related syn-dromes). Br Heart J 1972;34:41–46.Nora JJ, Nora AH, Sinha AK, Spangler 5 RD, Lubs HA: The Ullrich-Noonan syndrome (Turner phenotype). Am J Dis Child 1974;127:48–55.Pearl W: Cardiovascular anomalies in 6 Noonan’s syndrome. Chest 1977;71:677–679.van der Hauwaert LG, Fryns JP, Du- 7 moulin M, Logghe N: Cardiovascular malformations in Turner’s and Noo-nan’s syndrome. Br Heart J 1978;40:500–509.Engle MA, Ehlers KH: Cardiovascular 8 malformations in the syndrome of Turner phenotype with normal karyo-type. BD:OAS 1972;8:104–109.Pernot C, Hoeffel JC, Worms AM, 9 Ravault MC, Contet-Audonneau N: Les sténoses pulmonaires atypiques au cours de certains syndromes polymal-formatifs: Fréquence de la myocar-diopathie hypertrophique associée. Arch Mal Cœur 1977;70:391–398.Hoeffel JC, Ravault MC, Worms AM, 10 Pernot C: Atypical pulmonary steno-sis: Radiological features. Am Heart J 1979;98:315–320.

Noonan JA: Association of congenital 11 heart disease with syndromes or other defects. Pediatr Clin North Am 1978;25:797–816.Tartaglia M, Gelb BD: Noonan syn-12 drome and related disorders: Genetics and pathogenesis. Ann Rev Genomics Hum Genet 2005;6:45–68.Denayer E, Legius E: What’s new in the 13 neuro-cardio-facial-cutaneous syn-dromes? Eur J Pediatr 2007;166: 1091–1098.Sarkozy A, Conti E, Seripa D, Digilio 14 MC, Grifone N, et al: Correlation be-tween PTPN11 gene mutations and congenital heart defects in Noonan and LEOPARD syndromes. J Med Gen-et 2003;40:704–708.Burch M, Sharland M, Shinebourne E, 15 Smith G, Patton M, McKenna W: Car-diologic abnormalities in Noonan syn-drome: phenotypic diagnosis and echocardiographic assessment in 118 patients. J Am Coll Cardiol 1993;22: 1189–1192.Marino B, Digilio MC, Toscano A, Gi-16 annotti A, Dallapiccola B: Congenital heart diseases in children with Noo-nan syndrome: an expanded cardiac spectrum with high prevalence of atrioventricular canal. J Pediatr 1999;135:703–706.Ferencz C, Loffredo CA, Correa-Villa-17 senor A, Wilson PD: Genetic and envi-ronmental risk factors of major cardio-vascular malformations. The Baltimore-Washington Infant Study 1981–1989 (Futura Publishing Com-pany Inc., Armonk, New York 1997).Narayanswami S, Kitchiner D, Smith 18 A: Spectrum of valvar abnormalities in Noonan’s syndrome. A pathologic study. Cardiol Young 1994;4:62–66.

Koretzki ED, Moller JH, Korus ME, 19 Schwartz CJ, Eduards JE: Congenital pulmonary stenosis resulting from dysplasia of the valve. Circulation 1969;40:43–45.Neill CA: Congenital cardiac malfor-20 mations and syndromes, in Pierpont MEM, Moller JH (eds): Genetics of Car-diovascular Disease, pp 95–112 (Marti-nus-Nijoff Publishing, Boston, 1987).Sarkozy A, Conti E, Esposito G, Pizzuti 21 A, Dallapiccola B, et al: Nonsyndromic pulmonary valve stenosis and the PTPN11 gene. Am J Med Genet A 2003;116:389–390.Siwik ES, Zahka KG, Wiesner GL, Lim-22 wongse C: Cardiac disease in Costello syndrome. Pediatrics 1998;101: 706–709.Tomita H, Fuse S, Ikeda K, Matsuda K, 23 Chiba S: An infant with Costello syn-drome complicated by fatal hypertro-phic obstructive cardiomyopathy. Acta Paediatr Jap 1998;40:608–611.Lin AE, Grossfeld PD, Hamilton RM, 24 Smoot L, Gripp KW, et al: Further de-lineation of cardiac abnormalities in Costello syndrome. Am J Med Genet 2002;111:115–129.Limongelli G, Pacileo G, Marino B, 25 Digilio MC, Sarkozy A, et al: Preva-lence and clinical significance of car-diovascular abnormalities in patients with the LEOPARD syndrome. Am J Cardiol 2007;100:736–741.Marino B, Gagliardi MG, Digilio MC, 26 Polletta B, Grazioli S, et al: Noonan syndrome: structural abnormalities of the mitral valve causing subaortic ob-struction. Eur J Pediatr 1995;154: 949–952.Digilio MC, Sarkozy A, Pacileo G, Li-27 mongelli G, Marino B, Dallapiccola B: PTPN11 gene mutations: linking the Gln510Glu mutation to the ‘LEOPARD syndrome phenotype’. Eur J Pediatr 2006;165:803–805.

HCM

Pharmacological treatment of obstructive HCM

includes β-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 pro-

phylaxis against sudden death with implantable

cardioverter defibrillators in some cases. Severe

HCM can also be treated by surgical myomec-

tomy or transplantation.


Colan SD, Lipshultz SE, Lowe AM, 28 Sleeper LA, Messere J, et al: Epidemiol-ogy and cause-specific outcome of hy-pertrophic cardiomyopathy in chil-dren. Finding from the pediatric cardiomyopathy registry. Circulation 2007;115:776–781.Nora JJ, Lortscher RH, Spangler RD: 29 Echocardiographic studies of left ven-tricular disease in Ullrich-Noonan syndrome. Am J Dis Child 1975;129:1417–1420.Starr A, Hovaguimian H: Surgical re-30 pair of subaortic stenosis in atrioven-tricular canal defects. J Thorac Cardio-vasc Surg 1994;108:373–376.Feit LR, Hansen K, Oyer CE, Werner 31 JC: Unusual combination of congenital heart defects in an infant with Noonan syndrome. Pediatr Cardiol 1995;16:95–99.Digilio MC, Marino B, Toscano A, Gi-32 annotti A, Dallapiccola B: Atrioventric-ular canal defect without Down syn-drome: a heterogeneous malformation. Am J Med Genet 1999;85:140–146.Hirsch HD, Gelband H, Garcia O, Got-33 tlieb S, Tamer DM: Rapidly progressive obstructive cardiomyopathy in infants with Noonan’s syndrome. Reports of two cases. Circulation 1975;52: 1161–1165.Klues HG, Roberts WC, Maron BJ: 34 Anomalous insertion of papillary mus-cle directly into anterior mitral leaflet in hypertrophic cardiomyopathy. Sig-nificance in producing left ventricular outflow obstruction. Circulation 1991;84:1188–1197.Klues HG, Maron BJ, Dollar AL, Rob-35 erts WC: Diversity of structural mitral valve alterations in hypertrophic car-diomyopathy. Circulation 1992;85:1651–1660.Clark EB: Mechanisms in the patho-36 genesis of congenital heart defects; in Pierpont ME, Moller JM (eds): The Genetics of Cardiovascular Disease, pp 3–11 (Martinus-Nijoff, Boston, 1986).Tartaglia M, Pennacchio LA, Zhao C, 37 Yadav KK, Fodale V, et al: Gain-of-function SOS1 mutations cause a dis-tinctive form of Noonan syndrome. Nat Genet 2007;39:75–79.Sznajer Y, Keren B, Baumann C, Perei-38 ra S, Alberti C, et al: The spectrum of cardiac anomalies in Noonan syn-drome as a result of mutations in the PTPN11 gene. Pediatrics 2007;119:1325–1331.

Hasegawa T, Ogata T, Hasegawa Y, 39 Honda M, Nagai T, et al: Coarctation of the aorta and renal hypoplasia in a boy with Turner-Noonan surface anoma-lies and a 46,XY karyotype: A clinical model for the possible impairment of a putative lymphogenic gene(s) for Turn-er somatic stigmata. Hum Genet 1996;97:564–567.Digilio MC, Marino B, Picchio F, 40 Prandstraller D, Toscano A, Giannotti A, Dallapiccola B: Noonan syndrome and aortic coarctation. Am J Med Gen-et 1998;80:160–162.Lemire EG: Noonan syndrome or new 41 autosomal dominant condition with coarctation of the aorta, hypertrophic cardiomyopathy, and minor anoma-lies. Am J Med Genet 2002;113: 286–290.Digilio MC, Marino B, Giannotti A, 42 Dallapiccola B: Exclusion of 22q11 de-letion in Noonan syndrome with te-tralogy of Fallot. Am J Med Genet 1996;62:413–414.Shachter N, Perloff JK, Mulder DG: 43 Aortic dissection in Noonan’s syn-drome (46 XY Turner). Am J Cardiol 1984;54:464–465.Lin AE, Garver KL, Allanson J: Aortic-44 root dilatation in Noonan’s syndrome. N Engl J Med 1987;317:1668–1669.Power PD, Lewin MB, Hannibal MC, 45 Glass IA: Aortic root dilatation is a rare complication of Noonan syn-drome. Pediatr Cardiol 2006;27:478–480.Allanson JE: Noonan syndrome. J Med 46 Genet 1987;24:9–13.Patton MA: Noonan syndrome: A re-47 view. Growth Genet Horm 1994;33:1–3.Sharland M, Burch M, McKenna WM, 48 Patton MA: A clinical study of Noonan syndrome. Arch Dis Child 1992;67:178–183.Tartaglia M, Kalidas K, Shaw A, Song 49 X, Musat DL, et al: PTPN11 mutations in Noonan syndrome: Molecular spec-trum, genotype-phenotype correla-tion, and phenotypic heterogeneity. Am J Hum Genet 2002;70:1555–1563.Sarkozy A, Conti E, Digilio MC, Ma-50 rino B, Morini E, et al: Clinical and molecular analysis of 30 patients with multiple lentigines LEOPARD syn-drome. J Med Genet 2004;41:e68.Zenker M, Buheitel G, Rauch R, Koen-51 ig R, Bosse K, et al: Genotype-pheno-type correlations in Noonan syndro-me. J Pediatr 2004;144:368–374.

Jongmans M, Sistermans EA, Rikken 52 A, Nillessen WM, Tamminga R, et al: Genotypic and phenotypic character-ization of Noonan syndrome: New data and review of the literature. Am J Med Genet A 2005;134:165–170.Musante L, Kehl HG, Majewski F, Mei-53 necke P, Schweiger S, et al: Spectrum of mutations in PTPN11 and geno-type-phenotype correlation in 96 pa-tients with Noonan syndrome and five patients with cardio-facio-cutaneous syndrome. Eur J Hum Genet 2002;11:201–206.Kamisago M, Hirayama-Yamad K, 54 Kato T, Imamura S, Joo K, et al: Mis-sense 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. Mo-lecular and Genetic Mechanisms, pp 273–276 (Blackwell Futura Publishing, Malden, Massachusetts, 2005).Pandit B, Sarkozy A, Pennacchio LA, 55 Carta C, Oishi K, et al: Gain-of-func-tion RAF1 mutations cause Noonan and LEOPARD syndromes with hyper-trophic cardiomyopathy. Nat Genet 2007;39:1007–1012.Razzaque MA, Nishizawa T, Komoike 56 Y, Yagi H, Furutani M, et al: Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet 2007;39:1013–1017.Digilio MC, Conti E, Sarkozy A, Min-57 garelli R, Dottorini T, et al: Grouping of Multiple Lentigines/LEOPARD and Noonan syndrome on the PTPN11 gene. Am J Hum Genet 2002;71:389–394.Digilio MC, Sarkozy A, de Zorzi A, 58 Pacileo G, Limongelli G, et al: LEOP-ARD syndrome: Clinical diagnosis in the first year of life. Am J Med Genet A 2006;140:740–746.Chen B, Bronson RT, Klaman RD, 59 Hampton TG, Wang J-F, et al: Mice mutant for Egfr and Shp2 have defec-tive cardiac semilunar valvulogenesis. Nat Genet 2000;24:296–299.Marino B, Digilio MC: Congenital 60 heart disease and genetic syndromes: specific correlation between cardiac phenotype and genotype. Cardiovasc Pathol 2000;9:303–315.


Krenz M, Yutzey KE, Robbins J: Noo-61 nan syndrome mutation Q79R in Shp2 increases proliferation of valve pri-mordia mesenchymal cells via extra-cellular signal regulated kinase 1/2 sig-naling. Circ Res 2005;97:813–820.Roberts AE, Araki T, Swanson KD, 62 Montgomery KT, Schiripo TA, et al: Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet 2007;39:70–74.Zenker M, Horn D, Wieczorek D, Al-63 lanson J, Pauli S, et al: SOS1 is the sec-ond most common Noonan gene but plays no major role in cardio-facio-cu-taneous syndrome. J Med Genet 2007;44:651–656.Schubbert S, Zenker M, Rowe SL, Boll 64 S, Klein C, et al: Germline KRAS mu-tations cause Noonan syndrome. Nat Genet 2006;38:331–336.Carta C, Pantaleoni F, Bocchinfuso G, 65 Stella L, Vasta I, et al: Germline mis-sense mutations affecting KRAS iso-form B are associated with a severe Noonan syndrome phenotype. Am J Hum Genet 2006;79:129–135.Zenker M, Lehmann K, Schulz AL, 66 Barth H, Hansmann D, et al: Expan-sion of the genotypic and phenotypic spectrum in patients with KRAS ger-mline mutations. J Med Genet 2007;44:131–135.Legius E, Schrander-Stumpel C, Schol-67 len E, Pulles-Heintzberger C, Gewillig M, Fryns JP: PTPN11 mutations in LEOPARD syndrome. J Med Genet 2002;39:571–574.Keren B, Hadchouel A, Saba S, Sznajer 68 Y, Bonneau D, et al, for the French Col-laborative Noonan Study Group: PTPN11 mutations in patients with LEOPARD syndrome: a French multi-centric experience. J Med Genet 2004;41:e117.

Yoshida R, Nagai T, Hasegawa T, 69 Kinoshita E, Tanaka T, Ogata T: Two novel and one recurrent PTPN11 mu-tations in LEOPARD syndrome. Am J Med Genet A 2004;130:432–434.Tartaglia M, Martinelli S, Stella L, 70 Bocchinfuso G, Flex E, et al: Diversity and functional consequences of ger-mline and somatic PTPN11 mutations in human disease. Am J Hum Genet 2006;78:279–290.Yagubyan M, Panneton JM, Lindor 71 NM, Conti E, Sarkozy A, Pizzuti A: LEOPARD syndrome: a new polyaneu-rism association and an update on the molecular genetics of the disease. J Vasc Surg Apr 2004;39:897–900.Pacileo G, Calabrò P, Limongelli G, 72 Santoro G, Digilio MC, Sarkozy A, et al: Diffuse coronary artery dilation in a young patient with LEOPARD syn-drome. Int J Cardiol 2006;112:e35–e37.Roberts A, Allanson J, Jadico SK, Ka-73 vamura MI, Noonan J, et al: The car-dio-facio-cutaneous (CFC) syndrome: a review. J Med Genet 2006;43: 833–842.Gripp KW, Lin AE, Nicholson L, Allen 74 W, Cramer A, et al: Further delinea-tion of the phenotype resulting from BRAF or MEK1 germline mutations helps differentiate Cardio-Facio-Cuta-neous syndrome from Costello syn-drome. Am J Med Genet A 2007;143:1472–1480.van Eeghen AM, van Gelderen I, Hen-75 nekam RCM: Costello syndrome: Re-port and review. Am J Med Genet 1999;82:187–193.Lin AE, Gripp KW, Kerr B: Costello 76 syndrome; in Cassidy SB, Allanson JE (eds): Management of Genetic Syn-dromes, ed. 2, pp 151–161 (John Wiley & Sons, Inc., Hoboken, New Jersey 2005).

Gripp KW, Lin AE, Stabley DL, Nichol-77 son L, Scott CI Jr, et al: HRAS muta-tion analysis in Costello syndrome: Genotype and phenotype correlation. Am J Med Genet A 2006;140:1–7.Digilio MC, Sarkozy A, Capolino R, 78 Chiarini Testa MB, Esposito G, et al: Costello syndrome: clinical diagnosis in the first year of life. Eur J Pediatr 2008;167:621–628.Lin AE, Birch PH, Korf BR, Tenconi R, 79 Niimura M, et al: Cardiovascular mal-formations and other cardiac abnor-malities in neurofibromatosis 1 (NF1). Am J Med Genet 2000;95:108–117.Friedman JM, Arbiser J, Epstein JA, 80 Gutmann DH, Huot SJ, et al: Cardio-vascular disease in neurofibromatosis 1: Report of the NF1 Cardiovascular Task Force. Genet Med 2002;4:105–111.De Luca A, Bottillo I, Sarkozy A, Carta 81 C, Neri C, et al: NF1 gene mutations represent the major molecular event underlying Neurofibromatosis-Noo-nan syndrome. Am J Hum Genet 2005;77:1092–1101.Venturin M, Guarnieri P, Natacci F, 82 Stabile M, Tenconi R, et al: Mental re-tardation and cardiovascular malfor-mations in NF1 microdeleted patients point to candidate genes in 17q11.2. J Med Genet 2004;41:35–41.Brannan CI, Perkins AS, Vogel KS, 83 Ratner N, Nordlund ML, et al: Target-ed disruption of the Neurofibromatosis type-1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues. Genes Dev 1994;8:1019–1029.Hamilton SJ, Friedman JM: Insights 84 into the pathogenesis of neurofibroma-tosis 1 vasculopathy. Clin Genet 2000;58:341–344.

M. Cristina Digilio

Medical Genetics, Bambino Gesù Hospital

Piazza S. Onofrio 4

IT–00165 Rome (Italy)




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

AbstractThe Ras signaling pathway has been extensively studied

because of its fundamental role in cancer. This review dis-

cusses 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. Approxi-

mately 50% of reported NS/MPD cases are caused by ger-

mline 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 can-

cer. 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.


To date, 88 cases of myeloproliferative disease

(MPD) or cancer have been reported in individ-

uals with Noonan syndrome (NS), Costello syn-

drome (CS), cardio-facio-cutaneous syndrome

(CFC), or LEOPARD syndrome (LS) (table 1)

[1–59]. 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 neo-

plasms, 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 re-

views the occurrence of MPD and cancer in NS

and related syndromes and discusses clinical and

scientific implications.

Myeloproliferative Disease in Infants with

Noonan Syndrome

Twenty-six cases of NS with infantile MPD (NS/

MPD) have been reported previously [17–28, 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

120 Kratz

NS, which is explained by the observation that

NS/MPD is associated with specific PTPN11 mu-

tations (see below). Patients with NS/MPD usual-

ly present with thrombocytopenia (which can be

present at birth [25, 27]), increased myelomono-

cytic cells, and splenomegaly in the first months of

life [17]. Typically, hematologic abnormalities im-

prove 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 myelo dysplastic 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 juve-

nile myelomonocytic leukemia (JMML), a rare,

acquired MPD of early childhood characterized

by the proliferation of myelomonocytic cells

causing splenomegaly, monocytosis, and throm-

bocytopenia (reviewed in [61]). The disease is

usually rapidly lethal if patients are left untreat-

ed, 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, re-

spectively [21, 22], and another 11% of cases of

JMML have a clinical diagnosis of neurofibro-

matosis type 1 (NF1) (reviewed in [63]). Patients

with NF1 and JMML show acquired uniparen-

tal disomy (UPD) at the NF1 gene locus lead-

ing 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 illustrat-

ed in table 2, the germline PTPN11 mutations

Table 1. Eighty-eight cases of myeloproliferative disease or cancer in patients with Noonan syndrome and related

disorders reported by October 2007

Diseasea Noonan syndrome Costello syndrome CFC syndrome LEOPARD syndrome

RMS 3 [1–3] 20 [4–16]

MPD 26 [17–28] 3 [22, 26, 29]

ALL/NHL 10 [30–37] 2 [38–40]

AML 3 [34, 41, 42] 1 [43]

NBL 4 [42, 44–46] 4 [14, 47–50] 1 [39] 1 [35]

UC 4 [5, 16, 51–53]

HB 1 [54, 55]

CMML 1 [56]

TC 2 [57, 58]

CLL 1 [59]

WT 1 [35]

a ALL/NHL: Acute lymphoblastic leukemia/non-Hodgkin’s lymphoma; AML: Acute myeloid leukemia; CLL: Chronic lympho-

cytic leukemia; CMML: Chronic myelomonocytic leukemia; HB: Hepatoblastoma; MPD: Myeloproliferative disease; NBL:

Neuroblastoma; RMS: Rhabdomyosarcoma; TC: Testical cancer; UC: Carcinoma of the urinary tract; WT: Wilm’s tumor.

Myeloproliferative Disease and Cancer 121

Table 2. Germline PTPN11 mutations in patients with Noonan syndrome and myeloproliferative

disease differ from somatic PTPN11 mutations in juvenile myelomonocytic leukemia

PTPN11 mutation

[21–23, 25, 26, 28, 74]

Germline: NS/MPD

n = 22

Somatic: JMML

n = 107

Exon 3

T52S – 1

G60R – 2

G60V – 9

delG60 1 –

D61Y – 12

D61V – 7

D61G 2 1

D61N 1 –

Y62D 1 –

A72T – 9

A72V – 7

A72G 1 –

T73I 10 –

E76K – 29

E76G – 10

E76V – 2

E76A – 2

E76M – 1

Exon 13

R498Wa 1 –

S502A 1 –

S502T 1 –

G503R 1 –

G503A – 6

G503V – 4

Q506Pa 2 –

aMutation associated with LEOPARD syndrome.

122 Kratz

in patients with NS/MPD (PTPN11 T73I most

common) differ from somatic PTPN11 muta-

tions observed in sporadic JMML (PTPN11 E76K

most common) [26]. Although tempting, the

mutations identified in cases of NS/MPD cur-

rently do not allow for predicting the course of

the hematologic disease, because (1) many mu-

tations 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 cor-

relations 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 muta-

tions 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 re-

viewed by Gripp [13]. The most common malig-

nancy in patients with CS is rhabdomyosarcoma

(RMS). The annual incidence of sporadic RMS in

children younger than 19 years of age is 4–5 cases

per million children (reviewed in [69]). Twenty

cases of RMS have been previously reported in

CS [4–16]. Considering the low incidence of CS,

this relatively high number of reports indicates

that patients with CS are predisposed to devel-

op RMS [13]. ERMS is the most common histo-

logic 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 develop-

ment in this region is unknown. Interestingly,

somatic HRAS mutations in sporadic ERMS suc-

ceed the emergence of UPD [70]. In contrast,

HRAS germ line 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 illustrat-

ing the presumed pathogenesis of CS/ERMS and

sporadic ERMS.

Neuroblastoma has been reported in four

cases of CS [14, 47–50]. Ganglioneuroblastoma

is the most frequently reported histologic sub-

type [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 predispo-

sition syndrome associated with childhood car-

cinoma of the urinary tract [13] and four cases

have been reported [7, 17, 51–53]. 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], infer-

ring 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 germ line

mutations [5].


Neoplasms Occurring at Lower Frequencies in

Patients with Noonan Syndrome and Related

Disorders

There is considerable overlap in the tumor spec-

trum observed in patients with NS and related

syndromes (table 1). This observation is likely

due to the fact that these syndromes share a com-

mon pathway. The relatively low number of re-

ported cases of MPD or cancer beside NS/MPD,

CS/ERMS, CS/carcinoma of the urinary tract,

and CS/neuroblastoma may be due to several rea-

sons: (1) the features of NS can be subtle and over-

looked 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 so-

matic gene fusions have been described [37, 42,

56]. Notably, recent reports describe an indi-

vidual with CFC due to a germline mutation of

MEK1 who developed hepatoblastoma [54, 55]. It

remains to be determined if somatic MEK1 mu-

tations are involved in the pathogenesis of non-

syndromic 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 num-

ber of genetic factors: (1) The disease causative

gene and the transforming potential of the mu-

tant protein. Patients with HRAS mutations have

the highest tumor risk. The respective involved

Remission post HSCT

Spontaneous remission

T73IT73I

Bone marrow and blood

Cell ofmyelomonocytic

lineage


SomaticPTPN11

mutation

E76K

JMML

E76KE76K

E76KE76K

Transient proliferation

T73IT73I

T73IT73I


Cell ofmyelomono-cytic lineage

GermlinePTPN11

mutation

T73I T73I

Fetal period or early infancya

b

Later life

Fig. 1. Model of transient myeloproliferation in association with Noonan syndrome and non-syndromic juvenile my-

elomonocytic 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 addi-

tional 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).

124 Kratz

HRAS proteins are highly potent and the same

mutations also occur as somatic mutations in

cancer, although the most common cancer asso-

ciated mutant HRAS G12V is rare in CS [5, 14].

By contrast, there is very little overlap between

germline and cancer associated somatic muta-

tions 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 germ-

line 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 addi-

tional 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 re-

lated syndromes. Recently, a small hairpin RNA

targeting the dominant mutant form of Fgfr2 pre-

vented Apert-like syndrome in mice. Moreover,

treatment of the mutant mice with U0126, an in-

hibitor of MEK1/2 that blocks phosphorylation

G12S

Soft tissue

GermlineHRAS

mutation

Infancya

b

Soft tissue

UPD

UPD chrom. 11p15.5

G12SUPD

Somatic HRAS mutation

G12SUPD

UPD chrom. 11p15.5

G12SUPD

G12SUPD

G12SUPD

Embryonalrhabdomyosarcoma

G12SUPD

G12SUPD

G12SUPD

Embryonalrhabdomyosarcoma

Fig. 2. Model of embryonal rhabdomyosarcoma in association with Costello syndrome and non-syndromic embryo-

nal 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].


References

Khan S, McDowell H, Upadhyaya M, 1 Fryer A: Vaginal rhabdomyosarcoma in a patient with Noonan syndrome. J Med Genet 1995;32:743–745.Jung A, Bechthold S, Pfluger T, Renner 2 C, Ehrt O: Orbital rhabdomyosarcoma in Noonan syndrome. J Pediatr Hema-tol Oncol 2003;25:330–332.Moschovi M, Vassiliki T, Anna P, Ma- 3 ria-Alexandra M, Polyxeni NK, Kit-siou-Tzeli S: Rhabdomyosarcoma in a patient with Noonan syndrome pheno-type and review of the literature. J Pe-diatr Hematol Oncol 2007;29:341–344.Kerr B, Eden OB, Dandamudi R, Shan- 4 non N, Quarrell O, et al: Costello syn-drome: two cases with embryonal rhabdomyosarcoma. J Med Genet 1998;35:1036–1039.Gripp KW, Lin AE, Stabley DL, Nichol- 5 son L, Scott CI Jr, et al: HRAS muta-tion analysis in Costello syndrome: genotype and phenotype correlation. Am J Med Genet A 2006;140:1–7.Feingold M: Costello syndrome and 6 rhabdomyosarcoma. J Med Genet 1999;36:582–583.Bisogno G, Murgia A, Mammi I, Stra- 7 fella MS, Carli M: Rhabdomyosarcoma in a patient with cardio-facio-cutane-ous syndrome. J Pediatr Hematol On-col 1999;21:424–427.Sigaudy S, Vittu G, David A, Vigneron 8 J, Lacombe D, et al: Costello syndrome: report of six patients including one with an embryonal rhabdomyosarco-ma. Eur J Pediatr 2000;159:139–142.Kawame H: [Costello syndrome]. Ryo- 9 ikibetsu Shokogun Shirizu 2001;33: 497–498.

Gripp KW, Scott CI Jr, Nicholson L, 10 McDonald-McGinn DM, Ozeran JD, et al: Five additional Costello syndrome patients with rhabdomyosarcoma: pro-posal for a tumor screening protocol. Am J Med Genet 2002;108:80–87.Kerr B, Einaudi MA, Clayton P, Glad-11 man G, Eden T, et al: Is growth hor-mone treatment beneficial or harmful in Costello syndrome? J Med Genet 2003;40:e74.O’Neal JP, Ramdas J, Wood WE, Pellit-12 teri PK: Parameningeal rhabdomyo-sarcoma in a patient with Costello syn-drome. J Pediatr Hematol Oncol 2004;26:389–392.Gripp KW: Tumor predisposition in 13 Costello syndrome. Am J Med Genet C Semin Med Genet 2005;137:72–77.Aoki Y, Niihori T, Kawame H, Kuro-14 sawa K, Ohashi H, et al: Germline mu-tations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 2005;37:1038–1040.Kerr B, Delrue MA, Sigaudy S, Perveen 15 R, Marche M, et al: Genotype-pheno-type correlation in Costello syndrome: HRAS mutation analysis in 43 cases. J Med Genet 2006;43:401–405.Estep AL, Tidyman WE, Teitell MA, 16 Cotter PD, Rauen KA: HRAS muta-tions in Costello syndrome: detection of constitutional activating mutations in codon 12 and 13 and loss of wild-type allele in malignancy. Am J Med Genet A 2006;140:8–16.Bader-Meunier B, Tchernia G, Mielot 17 F, Fontaine JL, Thomas C, et al: Occur-rence of myeloproliferative disorder in patients with Noonan syndrome. J Pe-diatr 1997;130:885–889.

Fukuda M, Horibe K, Miyajima Y, Mat-18 sumoto K, Nagashima M: Spontaneous remission of juvenile chronic myelo-monocytic leukemia in an infant with Noonan syndrome. J Pediatr Hematol Oncol 1997;19:177–179.Choong K, Freedman MH, Chitayat D, 19 Kelly EN, Taylor G, Zipursky A: Juve-nile myelomonocytic leukemia and Noonan syndrome. J Pediatr Hematol Oncol 1999;21:523–527.Silvio F, Carlo L, Elena B, Nicoletta B, 20 Daniela F, Roberto M: Transient ab-normal myelopoiesis in Noonan syn-drome. J Pediatr Hematol Oncol 2002;24:763–764.Tartaglia M, Niemeyer CM, Fragale A, 21 Song X, Buechner J, et al: Somatic mu-tations in PTPN11 in juvenile myelo-monocytic leukemia, myelodysplastic syndromes and acute myeloid leuke-mia. Nat Genet 2003;34:148–150.Loh ML, Vattikuti S, Schubbert S, 22 Reynolds MG, Carlson E, et al: Muta-tions in PTPN11 implicate the SHP-2 phosphatase in leukemogenesis. Blood 2004;103:2325–2331.Jongmans M, Otten B, Noordam K, 23 van der Burgt I: Genetics and variation in phenotype in Noonan syndrome. Horm Res 2004;62 (Suppl) 3:56–59.Yoshida R, Miyata M, Nagai T, Ya-24 mazaki T, Ogata T: A 3-bp deletion mutation of PTPN11 in an infant with severe Noonan syndrome including hydrops fetalis and juvenile myelo-monocytic leukemia. Am J Med Genet A 2004;128:63–66.Giovannini L, Cave H, Ferrero-Vacher 25 C, Boutte P, Sirvent N: A new PTPN11 mutation in juvenile myelomonocytic leukaemia associated with Noonan syndrome. Acta Paediatr 2005;94:636–637.

and activation of ERK1/2, inhibits craniosynos-

tosis [72, 73]. Thus, potentially, it will be possible

to prevent tumors in persons with CS in the fu-

ture. These and related issues will need to be ad-

dressed 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.

126 Kratz

Kratz CP, Niemeyer CM, Castleberry 26 RP, Cetin M, Bergstrasser E, et al: The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and Noonan syndrome/myeloproliferative disease. Blood 2005;106: 2183–2185.Schubbert S, Zenker M, Rowe SL, Boll 27 S, Klein C, et al: Germline KRAS mu-tations cause Noonan syndrome. Nat Genet 2006;38:331–336.Cheong JL, Moorkamp MH: Respira-28 tory failure, juvenile myelomonocytic leukemia, and neonatal Noonan syn-drome. J Pediatr Hematol Oncol 2007;29:262–264.Kratz CP, Nathrath M, Freisinger P, 29 Dressel P, Assmuss HP, et al: Lethal proliferation of erythroid precursors in a neonate with a germline PTPN11 mutation. Eur J Pediatr 2006;165:182–185.Merino AH, Etxeandia IA, Borja BB, 30 Olivera IA: Sindrome de Noonan aso-ciado a leucosis aguda linfoblastica. An Esp Pediatr 1982;17:78–80.Hernandez Merino A, Asolo Etxeandia 31 I, Bernacer Borja M, Azcona Olivera I: Noonan syndrome associated with acute lymphoblastic leukosis. An Esp Pediatr 1982;17:78–80.Piombo M, Rosanda C, Pasino M, 32 Marasini M, Cerruti P, Comelli A: Acute lymphoblastic leukemia in Noo-nan syndrome: report of two cases. Med Pediatr Oncol 1993;21:454–455.Attard-Montalto SP, Kingston JE, Eden 33 T: Noonan’s syndrome and acute lym-phoblastic leukaemia. Med Pediatr On-col 1994;23:391–392.Johannes JM, Garcia ER, De Vaan GA, 34 Weening RS: Noonan’s syndrome in association with acute leukemia. Pe-diatr Hematol Oncol 1995;12:571–575.Merks JH, Caron HN, Hennekam RC: 35 High incidence of malformation syn-dromes in a series of 1,073 children with cancer. Am J Med Genet A 2005;134:132–143.Roti G, La Starza R, Ballanti S, 36 Crescenzi B, Romoli S, et al: Acute lymphoblastic leukaemia in Noonan syndrome. Br J Haematol 2006;133:448–450.Karow A, Steinemann D, Gohring G, 37 Hasle H, Greiner J, et al: Clonal dupli-cation of a germline PTPN11 mutation due to acquired uniparental disomy in acute lymphoblastic leukemia blasts from a patient with Noonan syndrome. Leukemia 2007;21:1303–1305.

van Den Berg H, Hennekam RC: Acute 38 lymphoblastic leukaemia in a patient with cardiofaciocutaneous syndrome. J Med Genet 1999;36:799–800.Niihori T, Aoki Y, Narumi Y, Neri G, 39 Cave H, et al: Germline KRAS and BRAF mutations in cardio-facio-cuta-neous syndrome. Nat Genet 2006;38:294–296.Makita Y, Narumi Y, Yoshida M, Nii-40 hori T, Kure S, et al: Leukemia in Car-dio-facio-cutaneous (CFC) syndrome: a patient with a germline mutation in BRAF proto-oncogene. J Pediatr He-matol Oncol 2007;29:287–290.Matsubara K, Yabe H, Ogata T, Yoshida 41 R, Fukaya T: Acute myeloid leukemia in an adult Noonan syndrome patient with PTPN11 mutation. Am J Hematol 2005;79:171–172.Chantrain CF, Jijon P, De Raedt T, Ver-42 mylen C, Poirel HA, Legius E, Brichard B: Therapy-related acute myeloid leu-kemia in a child with Noonan syn-drome and clonal duplication of the germline PTPN11 mutation. Pediatr Blood Cancer 2007;48:101–104.Ucar C, Calyskan U, Martini S, Hein-43 ritz W: Acute myelomonocytic leuke-mia in a boy with LEOPARD syndrome (PTPN11 gene mutation positive). J Pediatr Hematol Oncol 2006;28: 123–125.Lopez-Miranda B, Westra SJ, Yazdani 44 S, Boechat MI: Noonan syndrome as-sociated with neuroblastoma: a case report. Pediatr Radiol 1997;27: 324–326.Cotton JL, Williams RG: Noonan syn-45 drome and neuroblastoma. Arch Pe-diatr Adolesc Med 1995;149: 1280–1281.Ijiri R, Tanaka Y, Keisuke K, Masuno 46 M, Imaizumi K: A case of Noonan’s syndrome with possible associated neuroblastoma. Pediatr Radiol 2000;30:432–433.Zampino G, Mastroiacovo P, Ricci R, 47 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;47:176–183.Moroni I, Bedeschi F, Luksch R, Casa-48 nova M, D’Incerti L, Uziel G, Selicorni A: Costello syndrome: a cancer predis-posing syndrome? Clin Dysmorphol 2000;9:265–268.

Flores-Nava G, Canun-Serrano S, Moy-49 sen-Ramirez SG, Parraguirre-Marti-nez S, Escobedo-Chavez E: [Costello syndrome associated to a neuroblas-toma. Presentation of a case]. Gac Med Mex 2000;136:605–609.Zampino G, Pantaleoni F, Carta C, Co-50 bellis G, Vasta I, et al: Diversity, paren-tal germline origin, and phenotypic spectrum of de novo HRAS missense changes in Costello syndrome. Hum Mutat 2007;28:265–272.Franceschini P, Licata D, Di Cara G, 51 Guala A, Bianchi M, Ingrosso G, Fran-ceschini D: Bladder carcinoma in Cos-tello syndrome: report on a patient born to consanguineous parents and review. Am J Med Genet 1999;86:174–179.Gripp KW, Scott CI Jr, Nicholson L, 52 Figueroa TE: Second case of bladder carcinoma in a patient with Costello syndrome. Am J Med Genet 2000;90:256–259.Urakami S, Igawa M, Shiina H, Shig-53 eno K, Kikuno N, Yoshino T: Recurrent transitional cell carcinoma in a child with the Costello syndrome. J Urol 2002;168:1133–1134.Gripp KW, Lin AE, Nicholson L, Allen 54 W, Cramer A, et al: Further delinea-tion of the phenotype resulting from BRAF or MEK1 germline mutations helps differentiate cardio-facio-cuta-neous syndrome from Costello syn-drome. Am J Med Genet A 2007;143:1472–1480.Al-Rahawan MM, Chute DJ, Sol-55 Church K, Gripp KW, Stabley DL, et al: Hepatoblastoma and heart transplan-tation in a patient with cardio-facio-cutaneous syndrome. Am J Med Genet A 2007;143:1481–1488.La Starza R, Rosati R, Roti G, Gorello 56 P, Bardi A, et al: A new NDE1/PDGFRB fusion transcript underlying chronic myelomonocytic leukaemia in Noonan Syndrome. Leukemia 2007;21:830–833.Aggarwal A, Krishnan J, Kwart A, Per-57 ry D: Noonan’s syndrome and semi-noma of undescended testicle. South Med J 2001;94:432–434.Sriram K, Thomas K, Barnes R: Noo-58 nan’s syndrome. With carcinoma of undescended testis. IMJ Ill Med J 1987;171:294–296.Riederer J: [‘Benign’ monoclonal gam-59 mopathy and chronic lymphatic leuke-mia in a patient with Noonan syn-drome]. Med Klin (Munich) 1998;93:433–437.


Wilcox WD: Occurrence of myelopro-60 liferative disorder in patients with Noonan syndrome. J Pediatr 1998;132:189–190.Niemeyer CM, Kratz C: Juvenile my-61 elomonocytic leukemia. Curr Oncol Rep 2003;5:510–515.Locatelli F, Nollke P, Zecca M, Korthof 62 E, Lanino E, et al: Hematopoietic stem cell transplantation (HSCT) in chil-dren with juvenile myelomonocytic leukemia (JMML): results of the EWOG-MDS/EBMT trial. Blood 2005;105:410–419.Kratz CP, Niemeyer CM, Zenker M: An 63 unexpected new role of mutant Ras: perturbation of human embryonic de-velopment. J Mol Med 2007;85: 223–231.Flotho C, Steinemann D, Mullighan 64 CG, Neale G, Mayer K, et al: Genome-wide single-nucleotide polymorphism analysis in juvenile myelomonocytic leukemia identifies uniparental dis-omy surrounding the NF1 locus in cas-es associated with neurofibromatosis but not in cases with mutant RAS or PTPN11. Oncogene 2007;26: 5816–5821.

Stephens K, Weaver M, Leppig KA, 65 Maruyama K, Emanuel PD, Le Beau MM, Shannon KM: Interstitial unipa-rental isodisomy at clustered break-point intervals is a frequent mecha-nism of NF1 inactivation in myeloid malignancies. Blood 2006;108: 1684–1689.Braun BS, Tuveson DA, Kong N, Le DT, 66 Kogan SC, et al: Somatic activation of oncogenic Kras in hematopoietic cells initiates a rapidly fatal myeloprolifera-tive disorder. Proc Natl Acad Sci USA 2004;101:597–602.Le DT, Kong N, Zhu Y, Lauchle JO, Ai-67 yigari A, et al: Somatic inactivation of Nf1 in hematopoietic cells results in a progressive myeloproliferative disor-der. Blood 2004;103:4243–4250.Kratz CP, Niemeyer CM, Thomas C, 68 Bauhuber S, Matejas V, et al: Mutation analysis of Son of Sevenless in juvenile myelomonocytic leukemia. Leukemia 2007;21:1108–1109.Wexler LH, Helman LJ: Pediatric soft 69 tissue sarcomas. CA Cancer J Clin 1994;44:211–247.

Kratz CP, Steinemann D, Niemeyer 70 CM, Schlegelberger B, Koscielniak E, Kontny U, Zenker M: Uniparental dis-omy at chromosome 11p15.5 followed by HRAS mutations in embryonal rhabdomyosarcoma: lessons from Cos-tello syndrome. Hum Mol Genet 2007;16:374–379.Schubbert S, Shannon K, Bollag G: Hy-71 peractive Ras in developmental disor-ders and cancer. Nat Rev Cancer 2007;7:295–308.Shukla V, Coumoul X, Wang RH, Kim 72 HS, Deng CX: RNA interference and inhibition of MEK-ERK signaling pre-vent abnormal skeletal phenotypes in a mouse model of craniosynostosis. Nat Genet 2007;39:1145–1150.Wilkie AO: Cancer drugs to treat birth 73 defects. Nat Genet 2007;39:1057–1059.Loh ML, Reynolds MG, Vattikuti S, 74 Gerbing RB, Alonzo TA, et al: PTPN11 mutations in pediatric patients with acute myeloid leukemia: results from the Children’s Cancer Group. Leuke-mia 2004;18:1831–1834.

Christian Kratz

Division of Pediatric Hematology/Oncology, Department of Pediatrics and Adolescent Medicine, University of Freiburg

Mathildenstrasse 1

DE–79104 Freiburg (Germany)




Neurofibromatosis Type 1-Noonan Syndrome: What’s the Link?

E. Denayer � E. Legius

Department of Human Genetics, Catholic University of Leuven, Leuven, Belgium

AbstractNeurofibromatosis type 1 is an autosomal dominant condi-

tion characterized by café-au-lait spots, axillary and ingui-

nal 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 retarda-

tion and facial dysmorphism. Watson syndrome and Neu-

rofibromatosis-Noonan syndrome have been described as

separate entities combining features of both conditions.

During recent years Watson and Neurofibromatosis-Noo-

nan syndrome have been shown to result from mutations

in the NF1 gene. Neurofibromatosis type 1 and Noonan syn-

drome also share some phenotypical features with other

syndromes like Costello, cardio-facio-cutaneous, LEOPARD,

and the SPRED1 syndrome. These ‘neuro-cardio-facial-cuta-

neous 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 fea-

tures of these syndromes.


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 pa-

tients. Some café-au-lait spots might be present at

birth, but increase in size, number and pigmenta-

tion during the first years of life. Axillary or in-

guinal freckling is commonly present by the age

of seven. Lisch nodules are hamartomas of the

iris and can best be identified with split-lamp ex-

amination. 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 with-

out 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-def-

icit disorder [4, 5] occur in about 50–75%. The

NF1 neuropsychological profile is characterized

by deficits in visuo-spatial performance, execu-

tive functioning (planning and abstract concept

formation), and attention [6]. Cardiovascular

complications of NF1 are congenital heart de-

fects (2.1%) [7], most commonly pulmonary

valve stenosis [8], and hypertension. The latter

can either be essential or secondary to NF1 vas-

culopathy which can for example produce renal

Neurofibromatosis Type 1-Noonan Syndrome: What’s the Link? 129

artery stenosis. Neurofibromas are a key hall-

mark of NF1. Numerous benign neurofibromas

are usually present in adults, but they rarely de-

velop before late childhood. Plexiform neurofi-

bromas 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 devel-

ops during the first 2–3 years of life. Other brain

tumours, rhabdomyosarcoma and leukaemia, es-

pecially 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 fre-

quent malignant neoplasm associated with NF1

with a life time risk of 8–13% [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 clini-

cal base using the NIH diagnostic criteria [12].

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 un-

related families with pulmonary valve steno-

sis, borderline intelligence, multiple café-au-lait

spots and freckling [13]. Allanson et al. expand-

ed 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 com-

mon to WS and NF1, only axillary freckling and

café-au-lait spots show equal incidence in the

two conditions. Other shared clinical features

show marked differences in frequency in the two

conditions and this appears to provide the ma-

jor factor differentiating the two conditions. For

example, pulmonary valve stenosis, low intelli-

gence 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 in-

frame 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)

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 man-

ifestations of Noonan syndrome including short

stature, ptosis, ‘midface hypoplasia’, apparent-

ly 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 ap-

pearance resembling Noonan syndrome in chil-

dren 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 vari-

ant of either NF1 or Noonan syndrome. Since

both NF1 – with a prevalence of about 1/3,000

130 Denayer � Legius

Table 1. Overview of clinical and molecular findings in patients with WS or NFNS and a mutation in NF1

Reference Clinical findings Molecular findings

Upadhyaya et al. [15] One individual with Watson syndrome 80-kb deletion

Tassabehji et al. [16] 3 members of a family with Watson

syndrome: 26-year-old mother has multiple

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

in-frame tandem duplication of 42 bases in

exon 28

Bahuau et al. [22] 8 family members with NFNS, 2 with NF1

only

Nonsense mutation exon 16 (c.2446C>T;

p.R816X)

Carey et al. [23] Family with NFNS 3-bp deletion in exon 17

Baralle et al. [24] 6-year-old boy, more than six CALM, ptosis,

epicanthic folds, low posterior hairline, low-

set ears, pulmonic stenosis, severe feeding

problems in infancy

3-bp deletion in exon 25 (c.4312 del GAA)

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,

optic glioma, learning difficulties,

macrocephaly, hypertelorism, downslanting

palpebral fissures, ptosis, epicanthic folds,

low-set ears, thoracic anormality

Splice-site mutation exon 11 (c.1721+3A>G)

6-year-old boy, CALM, learning difficulties,

short stature, hypertelorism, downslanting

palpebral fissures, malar hypoplasia, low

posterior hairline, webbed neck, low-set

ears, hypotonia

4-bp deletion exon 12a (c.1756 del ACTA)

2.2-year-old girl, CALM, macrocephaly, PS,

hypertelorism, downslanting palpebral

fissures, ptosis, epicanthic folds, low


ears

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




De Luca et al. [25] 8-year-old boy, CALM, axillary freckling,

mild MR, short stature, macrocephaly,


fissures, malar hypoplasia, low posterior

hairline, webbed neck, low-set ears,

thoracic abnormality, cubitus valgus

1-bp deletion exon 13 (c.2153 del A)

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-bp inframe deletion (c.2970 del AAT; p.

991del M)

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

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

E1415X)

2.2-year-old boy, CALM, macrocephaly, PS,


fissures, ptosis, epicanthic folds, low

posterior hairline, low-set ears, webbed neck

Missense mutation exon 24 (c. 4267A>G; p.

K1423E)

6-year-old girl, CALM, Lisch nodules, short

stature, hypertelorism, downslanting

palpebral fissures, ptosis, malar hypoplasia,

epicanthic folds, low posterior hairline, webbed

neck, low-set ears, thoracic abnormality

Missense mutation exon 24 (c. 4267A>G; p.

K1423E)

4-year-old girl, CALM, macrocephaly, ASD,

hypertelorism, ptosis, epicanthic folds, low-

set ears

Missense mutation exon 25 (c.4289A>C; p.

N1430T)

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

Missense mutation exon 25 (c.4294G>C; p.

V1432L)

9.5-year-old girl, CALM, axillary freckling,

scoliosis, short stature, macrocephaly, PS,


fissures, ptosis, low posterior hairline,


abnormality, keratosis pilaris,

sensorineural deafness

3-bp inframe deletion exon 25 (c.4312 del

GAA; p.1438 del E)




De Luca et al. [25] 38-year-old female, CALM,

neurofibromas, 1 plexiform, axillary

freckling, short stature, mitral valve

thickening, downslanting palpebral fissures,

epicanthic folds, low posterior hairline,


abnormality

Nonsense mutation exon 29 (c.5339T>G; p.

L1780X)

10-year-old boy, CALM, axillary freckling,

Lisch nodules, macrocephaly, arrhythmia

(right-branch block), ptosis, low-set ears,

thoracic abnormality, renal cyst

Splice-site mutation exon 35 (c.6641+1G>A)

2 family members, CALM, neurofibromas,

axillary freckling, Lisch nodules in 1,

learning difficulties, scoliosis, macrocephaly,


fissures, ptosis, low-set ears

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

7-year-old boy, CALM, axillary freckling,

Lisch nodules, optic glioma, MR,


fissures, ptosis, low-set ears, thoracic

abnormality, cubitus valgus

Partial NF1 gene deletion

Hüffmeier et al. [26] 4-year-old, 5 CALM, delayed psychomotor

development, PS, relative macrocephaly,


fissures, ptosis, low posterior hairline,

webbed neck, thoracic abnormality

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

p.L1196R)

2 family members, CALM, neurofibromas in

1, aortic insufficiency in 1, hypertelorism,

downslanting palpebral fissures, low


ears, thoracic abnormality

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

2 family members, CALM, neurofibromas,

axillary freckling, hypertelorism,


posterior hairline, webbed neck, thoracic

abnormality

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

42-year-old, CALM, neurofibromas, axillary

freckling, Lisch nodules, short stature,

macrocephaly, hypertelorism,

downslanting palpebral fissures, ptosis,

low posterior hairline, webbed neck, low-

set ears, thoracic abnormality, scoliosis

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


– and Noonan syndrome – with a prevalence

of 1/1,000 to 1/2,500 – are rather common dis-

orders chance association is a possibility. There

has been one report of a female patient with typi-

cal 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 fami-

lies 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 individu-

als with NFNS, a 3-bp deletion in exon 25 and a

2-bp insertion in exon 23–2. In four other indi-

viduals no mutations were found in NF1 nor in

PTPN11 [24]. De Luca et al. identified heterozy-

gous NF1 mutations in 16 of 17 unrelated sub-

jects 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 dis-

orders by excluding mutations in exons 11–27 of

NF1 in 100 PTPN11-negative Noonan syndrome

patients [25]. Hüffmeier 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 syn-

drome and neurofibromatosis type 1 [26].

Some Tumour Types and Leukaemias Occur in

Both NF1 and Noonan Syndrome

Haematological malignancies occur with in-

creased frequency in both NF1 and Noonan syn-

drome. 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 abnor-

malities has been described including isolated

monocytosis, a CMML-like condition that remits

spontaneously [29] and JMML [30]. JMML is a

rare (annual incidence of 1–2 per million) myelo-

proliferative disorder characterized by leukocyto-

sis with tissue infiltration and it has a severe and

often lethal course. The incidence of JMML is in-

creased 200- to 500-fold in children with NF1. Loss



Hüffmeier et al. [26] 20-year-old, CALM, neurofibromas, axillary

freckling, Lisch nodules, learning difficulties,

short stature, macrocephaly, hypertelorism,



ears, thoracic abnormality, scoliosis

Whole gene deletion


of the normal NF1 allele (LOH, loss of heterozy-

gosity) 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 syn-

drome who develop JMML have specific germline

PTPN11 mutations, most frequently T73I.

Apart from the leukaemias there is also over-

lap in the type of solid tumours observed in NF1

and Noonan syndrome. Rhabdomyosarcoma [34,

35] and neuroblastoma [36–38] have been report-

ed in association with both syndromes. Giant cell

tumours of the bone are usually benign, but local-

ly aggressive tumours characterized by the pres-

ence of multinucleated giant cells. They have been

associated with NF1 [39–41], Noonan syndrome

[42–44] 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].

The NF1 Gene

The NF1 gene located on chromosome 17q11.2 is

a large gene (~350 kilobases) containing 60 ex-

ons. 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

generation of intracellular cyclic-AMP. However

most important is its function as a RAS GTPase

Activating Protein (GAP). By means of its GAP-

related domain (exons 20–27a) neurofibromin

functions as a negative regulator of the RAS-

MAPKinase cascade. It stimulates the hydroly-

sis 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 result-

ing in more active RAS and increased signalling

through the RAS-MAPK pathway.

In 2001 Costa et al. linked the learning dif-

ficulties in NF1 individuals to the GAP-activity

of neurofibromin by showing that Nf1 mice lack-

ing the alternatively spliced exon 23a exhibited

learning difficulties but no apparent develop-

mental abnormalities or tumour predisposition

[51]. They proposed that hyperactive RAS result-

ing from Nf1 haploinsufficiency leads to over-

activity of inhibitory GABAergic neurons. The

increased inhibition by GABAergic neurons

would then lead to the learning disorder pheno-

type in these animals. To prove this hypothesis

they showed that genetically (by crossing Nf1+/–

mice with Kras or Nras+/– mice) as well as phar-

macologically (by use of the farnesyltransferase

inhibitor lovastatin) diminished RAS function

rescued the learning difficulties observed in Nf1

heterozygous animals [52]. It can be hypothe-

sized that the learning problems observed in the

other NCFC syndromes are also the result of an

increased RAS activation in certain brain cells.

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-cutane-

ous (CFC) syndrome. Recently the term ‘neuro-

cardio-facial-cutaneous (NCFC) syndromes’ has


References

Friedman JM: Epidemiology of neuro- 1 fibromatosis type 1. Am J Med Genet 1999;89:1–6.Ozonoff S: Cognitive impairment in 2 neurofibromatosis type 1. Am J Med Genet 1999;89:45–52.

Rosser TL, Packer RJ: Neurocognitive 3 dysfunction in children with neurofi-bromatosis type 1. Curr Neurol Neuro-sci Rep 2003;3:129–136.

Kayl AE, Moore BD, III: Behavioral 4 phenotype of neurofibromatosis, type 1. Ment Retard Dev Disabil Res Rev 2000;6:117–124.

been coined to group these syndromes. These

conditions all share a variable degree of learn-

ing disabilities or mental retardation, congenital

heart defects, facial dysmorphy and skin abnor-

malities. 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 syn-

drome. The PTPN11 gene encodes the non-recep-

tor 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) [57–59] and also in non-

PTPN11 associated Noonan syndrome (KRAS mu-

tations in less than 2% [59], SOS1 in 10% [60, 61]

and RAF1 in 3–17% [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 respon-

sible as a mechanism for several of the overlap-

ping symptoms in the different NCFC syndromes

such as specific facial features, learning difficul-

ties 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 illus-

tration in 1996 a young woman with a prior diag-

nosis of LEOPARD syndrome and hypertrophic

cardiomyopathy who had a de novo missense mu-

tation 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 muta-

tions in the SPRED1 gene [65]. Affected individ-

uals presented with café-au-lait spots, skinfold

freckling and macrocephaly. Most of the indi-

viduals fulfilled the NIH diagnostic criteria for

NF1. Some typical features of NF1 were system-

atically absent in the reported patients such as

Lisch nodules, neurofibromas and central ner-

vous system tumours. In adults multiple lipomas

were observed. A Noonan-like facial morphology

has been observed in some patients. One individ-

ual had a pulmonary valve stenosis. The SPRED1

gene is, like NF1, a negative regulator of the RAS-

MAPK 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

café-au-lait spot of an affected individual, sup-

porting 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 re-

sponsible for symptoms seen in either NF1 or

Noonan syndrome.


Mautner VF, Kluwe L, Thakker SD, 5 Leark RA: Treatment of ADHD in neu-rofibromatosis type 1. Dev Med Child Neurol 2002;44:164–170.Hyman SL, Shores A, North KN: The 6 nature and frequency of cognitive defi-cits in children with neurofibromatosis type 1. Neurology 2005;65: 1037–1044.Friedman JM, Birch PH: Type 1 neuro- 7 fibromatosis: a descriptive analysis of the disorder in 1,728 patients. Am J Med Genet 1997;70:138–143.Lin AE, Birch PH, Korf BR, Tenconi R, 8 Niimura M, et al: Cardiovascular mal-formations and other cardiovascular abnormalities in neurofibromatosis 1. Am J Med Genet 2000;95:108–117.Evans DG, Baser ME, McGaughran J, 9 Sharif S, Howard E, Moran A: Malig-nant peripheral nerve sheath tumours in neurofibromatosis 1. J Med Genet 2002;39:311–314.Maertens O, Prenen H, Debiec-Rychter 10 M, Wozniak A, Sciot R, et al: Molecu-lar pathogenesis of multiple gastroin-testinal stromal tumors in NF1 pa-tients. Hum Mol Genet 2006;15: 1015–1023.De Smet L, Sciot R, Legius E: Multifo-11 cal glomus tumours of the fingers in two patients with neurofibromatosis type 1. J Med Genet 2002;39:e45.Neurofibromatosis. Conference state-12 ment. National Institutes of Health Consensus Development Conference. Arch Neurol 1988;45:575–578.Watson GH: Pulmonary stenosis, café-13 au-lait spots, and dull intelligence. Arch Dis Child 1967;42:303–307.Allanson JE, Upadhyaya M, Watson 14 GH, Partington M, MacKenzie A, et al: Watson syndrome: is it a subtype of type 1 neurofibromatosis? J Med Genet 1991;28:752–756.Upadhyaya M, Shen M, Cherryson A, 15 Farnham J, Maynard J, Huson SM, Harper PS: Analysis of mutations at the neurofibromatosis 1 (NF1) locus. Hum Mol Genet 1992;1:735–740.Tassabehji M, Strachan T, Sharland M, 16 Colley A, Donnai D, Harris R, Thakker N: Tandem duplication within a neuro-fibromatosis type 1 (NF1) gene exon in a family with features of Watson syn-drome and Noonan syndrome. Am J Hum Genet 1993;53:90–95.Allanson JE, Hall JG, Van Allen MI: 17 Noonan phenotype associated with neurofibromatosis. Am J Med Genet 1985;21:457–462.

Kaplan P, Rosenblatt B: A distinctive 18 facial appearance in neurofibromatosis von Recklinghausen. Am J Med Genet 1985;21:463–470.Colley A, Donnai D, Evans DG: Neuro-19 fibromatosis/Noonan phenotype: a variable feature of type 1 neurofibro-matosis. Clin Genet 1996;49:59–64.Bertola DR, Pereira AC, Passetti F, de 20 Oliveira PS, Messiaen L, et al: Neurofi-bromatosis-Noonan syndrome: molec-ular evidence of the concurrence of both disorders in a patient. Am J Med Genet A 2005;136:242–245.Bahuau M, Flintoff W, Assouline B, 21 Lyonnet S, Le Merrer M, et al: Exclu-sion of allelism of Noonan syndrome and neurofibromatosis-type 1 in a large family with Noonan syndrome-neurofibromatosis association. Am J Med Genet 1996;66:347–355.Bahuau M, Houdayer C, Assouline B, 22 Blanchet-Bardon C, Le Merrer M, et al: Novel recurrent nonsense mutation causing neurofibromatosis type 1 (NF1) in a family segregating both NF1 and Noonan syndrome. Am J Med Genet 1998;75:265–272.Carey JC, Stevenson DA, Ota M, Neil S, 23 Viskochil DH: Is there a Noonan syn-drome: Part 2:Documentation of the clinical and molecular aspects of an important family. Proc Greenwood Genet Center 2007;17:52–53.Baralle D, Mattocks C, Kalidas K, 24 Elmslie F, Whittaker J, et al: Different mutations in the NF1 gene are associ-ated with Neurofibromatosis-Noonan syndrome (NFNS). Am J Med Genet A 2003;119:1–8.De Luca A, Bottillo I, Sarkozy A, Carta 25 C, Neri C, et al: NF1 gene mutations represent the major molecular event underlying neurofibromatosis-Noonan syndrome. Am J Hum Genet 2005;77:1092–1101.Huffmeier U, Zenker M, Hoyer J, Fah-26 sold R, Rauch A: A variable combina-tion of features of Noonan syndrome and neurofibromatosis type I are caused by mutations in the NF1 gene. Am J Med Genet A 2006;140: 2749–2756.Stiller CA, Chessells JM, Fitchett M: 27 Neurofibromatosis and childhood leu-kaemia/lymphoma: a population-based UKCCSG study. Br J Cancer 1994;70:969–972.

Bader JL, Miller RW: Neurofibromato-28 sis and childhood leukemia. J Pediatr 1978;92:925–929.Bader-Meunier B, Tchernia G, Mielot 29 F, Fontaine JL, Thomas C, et al: Occur-rence of myeloproliferative disorder in patients with Noonan syndrome. J Pe-diatr 1997;130:885–889.Choong K, Freedman MH, Chitayat D, 30 Kelly EN, Taylor G, Zipursky A: Juve-nile myelomonocytic leukemia and Noonan syndrome. J Pediatr Hematol Oncol 1999;21:523–527.Shannon KM, O’Connell P, Martin GA, 31 Paderanga D, Olson K, Dinndorf P, McCormick F: Loss of the normal NF1 allele from the bone marrow of chil-dren with type 1 neurofibromatosis and malignant myeloid disorders. N Engl J Med 1994;330:597–601.Flotho C, Valcamonica S, Mach-Pas-32 cual S, Schmahl G, Corral L, et al: RAS mutations and clonality analysis in children with juvenile myelomonocyt-ic leukemia (JMML). Leukemia 1999;13:32–37.Kratz CP, Niemeyer CM, Castleberry 33 RP, Cetin M, Bergstrasser E, et al: The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and Noonan syndrome/myeloprolif-erative disease. Blood 2005;106: 2183–2185.Matsui I, Tanimura M, Kobayashi N, 34 Sawada T, Nagahara N, Akatsuka J: Neurofibromatosis type 1 and child-hood cancer. Cancer 1993;72: 2746–2754.Moschovi M, Vassiliki T, Anna P, Ma-35 ria-Alexandra M, Polyxeni NK, Kit-siou-Tzeli S: Rhabdomyosarcoma in a patient with Noonan syndrome pheno-type and review of the literature. J Pe-diatr Hematol Oncol 2007;29:341–344.Ijiri R, Tanaka Y, Keisuke K, Masuno 36 M, Imaizumi K: A case of Noonan’s syndrome with possible associated neuroblastoma. Pediatr Radiol 2000;30:432–433.Lopez-Miranda B, Westra SJ, Yazdani 37 S, Boechat MI: Noonan syndrome as-sociated with neuroblastoma: a case report. Pediatr Radiol 1997;27: 324–326.Origone P, Defferrari R, Mazzocco K, 38 Lo CC, De Bernardi B, Tonini GP: Ho-mozygous inactivation of NF1 gene in a patient with familial NF1 and dis-seminated neuroblastoma. Am J Med Genet A 2003;118:309–313.


Ardekian L, Manor R, Peled M, Laufer 39 D: Bilateral central giant cell granulo-mas in a patient with neurofibromato-sis: report of a case and review of the literature. J Oral Maxillofac Surg 1999;57:869–872.Krammer U, Wimmer K, Wiesbauer P, 40 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;18:371–373.Ruggieri M, Pavone V, Polizzi A, Alba-41 nese S, Magro G, Merino M, Duray PH: Unusual form of recurrent giant cell granuloma of the mandible and lower extremities in a patient with neurofi-bromatosis type 1. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:67–72.Bertola DR, Kim CA, Pereira AC, Mota 42 GF, Krieger JE, et al: Are Noonan syn-drome and Noonan-like/multiple giant cell lesion syndrome distinct entities? Am J Med Genet 2001;98:230–234.Cohen MM Jr, Gorlin RJ: Noonan-like/43 multiple giant cell lesion syndrome. Am J Med Genet 1991;40:159–166.Ucar B, Okten A, Mocan H, Ercin C: 44 Noonan syndrome associated with central giant cell granuloma. Clin Genet 1998;53:411–414.Posligua L, McDonald DJ, Dehner LP: 45 Diffuse-type tenosynovial giant cell tumor in association with neurofibro-matosis type 1-Noonan syndrome: possibly more than a chance relation-ship. Am J Surg Pathol 2006;30: 734–738.Yazdizadeh M, Tapia JL, Baharvand M, 46 Radfar L: A case of neurofibromatosis-Noonan syndrome with a central giant cell granuloma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:316–320.Sahn EE, Dunlavey ES, Parsons JL: 47 Multiple cutaneous granular cell tu-mors in a child with possible neurofi-bromatosis. J Am Acad Dermatol 1997;36:327–330.

Lohmann DR, Gillessen-Kaesbach G: 48 Multiple subcutaneous granular-cell tumours in a patient with Noonan syn-drome. Clin Dysmorphol 2000;9:301–302.Legius E, Marchuk DA, Collins FS, 49 Glover TW: Somatic deletion of the neurofibromatosis type 1 gene in a neurofibrosarcoma supports a tumour suppressor gene hypothesis. Nat Genet 1993;3:122–126.Maertens O, Brems H, Vandesompele J, 50 De Raedt T, Heyns I, et al: Comprehen-sive NF1 screening on cultured Schwann cells from neurofibromas. Hum Mutat 2006;27:1030–1040.Costa RM, Yang T, Huynh DP, Pulst 51 SM, Viskochil DH, Silva AJ, Brannan CI: Learning deficits, but normal de-velopment and tumor predisposition, in mice lacking exon 23a of Nf1. Nat Genet 2001;27:399–405.Costa RM, Federov NB, Kogan JH, 52 Murphy GG, Stern J, et al: Mechanism for the learning deficits in a mouse model of neurofibromatosis type 1. Nature 2002;415:526–530.Tartaglia M, Mehler EL, Goldberg R, 53 Zampino G, Brunner HG, et al: Muta-tions in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 2001;29:465–468.Digilio MC, Conti E, Sarkozy A, Min-54 garelli 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.Legius E, Schrander-Stumpel C, Schol-55 len E, Pulles-Heintzberger C, Gewillig M, Fryns JP: PTPN11 mutations in LEOPARD syndrome. J Med Genet 2002;39:571–574.Aoki Y, Niihori T, Kawame H, Kuro-56 sawa K, Ohashi H, et al: Germline mu-tations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 2005;37:1038–1040.

Niihori T, Aoki Y, Narumi Y, Neri G, 57 Cave H, et al: Germline KRAS and BRAF mutations in cardio-facio-cuta-neous syndrome. Nat Genet 2006;38:294–296.Rodriguez-Viciana P, Tetsu O, Tidy-58 man WE, Estep AL, Conger BA, et al: Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome. Science 2006;311:1287–1290.Schubbert S, Zenker M, Rowe SL, Boll 59 S, Klein C, et al: Germline KRAS mu-tations cause Noonan syndrome. Nat Genet 2006;38:331–336.Roberts AE, Araki T, Swanson KD, 60 Montgomery KT, Schiripo TA, et al: Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet 2007;39:70–74.Tartaglia M, Pennacchio LA, Zhao C, 61 Yadav KK, Fodale V, et al: Gain-of-function SOS1 mutations cause a dis-tinctive form of Noonan syndrome. Nat Genet 2007;39:75–79.Pandit B, Sarkozy A, Pennacchio LA, 62 Carta C, Oishi K, et al: Gain-of-func-tion RAF1 mutations cause Noonan and LEOPARD syndromes with hyper-trophic cardiomyopathy. Nat Genet 2007;39:1007–1012.Razzaque MA, Nishizawa T, Komoike 63 Y, Yagi H, Furutani M, et al: Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet 2007;39:1013–1017.Wu R, Legius E, Robberecht W, Du-64 moulin M, Cassiman JJ, Fryns JP: Neu-rofibromatosis type I gene mutation in a patient with features of LEOPARD syndrome. Hum Mutat 1996;8:51–56.Brems H, Chmara M, Sahbatou M, De-65 nayer E, Taniguchi K, et al: Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1-like phe-notype. Nat Genet 2007;39:1120–1126.

Eric Legius

Department of Human Genetics, Catholic University of Leuven

Herestraat 49

BE–3000 Leuven (Belgium)




Animal Models for Noonan Syndrome and Related Disorders

T. Araki � B.G. Neel

Ontario Cancer Institute, University Health Network, TMDT8–355, Toronto, Ont., Canada

AbstractNoonan syndrome (NS) and related disorders, including LEOP-

ARD syndrome (LS), cardio-facial-cutaneous (CFC) syndrome,

Costello syndrome (CS) and neurofibromatosis Type-I (NF1)

can be grouped together as the ‘neuro-cardio-facio-cuta-

neous (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 (hereaf-

ter, ‘RAS/ERK’) cascade. Presumably, the common features of

these syndromes reflect abnormal function of this pathway

during development and postnatally. Nevertheless, the de-

tailed mechanism by which abnormal ERK activation results

in shared syndromic phenotypes remains unclear. Moreover,

although clearly related, specific NCFC syndromes are clini-

cally distinguishable. How mutations within the same signal-

ing pathway have such distinct phenotypic consequences is

unknown. NCFC syndromes predominantly affect events that

occur during embryogenesis and affect complex develop-

mental/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 extensive-

ly outbred, it is difficult to determine whether allele-specific

phenotypic differences exist and/or whether there are key ge-

netic 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.


It is now clear that germline mutations in mem-

bers of the RAS/ERK cascade cause NCFC syn-

dromes [1]. Individuals with these syndromes

typically display some combination of facial ab-

normalities, cardiac defects and proportional

short stature, although skin, lymphatic and geni-

tal 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 dis-

tinguishable. For example, multiple neurofibro-

mas and café 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 hemato-

logical malignancies, particularly the rare disor-

der juvenile myelomonocytic leukemia (JMML),

are associated with NF1. JMML, possibly other

Animal Models for Noonan Syndrome and Related Disorders 139

hematological (e.g., acute lymphoblastic leuke-

mia (ALL)) malignancies, and potentially neu-

roblastoma, are associated with NS (caused by

PTPN11, KRAS, SOS1 or RAF1 mutations) [5–10].

Somatic PTPN11 mutations are also common in

sporadic JMML [11, 12], and occur less frequent-

ly in a variety of other leukemias and myelopro-

liferative 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 spo-

radic versions of these malignancies [14].

Although much progress has been made in de-

fining the genetic basis for the NCFC syndromes,

several fundamental issues remain to be ad-

dressed. First, the molecular and cellular mech-

anisms responsible for the defects seen in these

syndromes remain poorly understood. Also un-

clear is how mutations in components of the same

signaling pathway can cause the different features

that allow individual NCFC syndromes to be dis-

tinguished clinically. Even within the same syn-

drome, 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 be-

cause 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 deter-

mination. 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, in-

stead of BRAF, can cause CFC) [2, 3]. Certain

RAF1 mutations may predispose to hypertro-

phic 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-asso-

ciated mutations is similar remains largely un-

known. 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, gain-

and 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 re-

view 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 do-

main-containing protein tyrosine phosphatase

SHP2. A key component of the RAS/ERK path-

way, SHP2 and, in particular, SHP2 catalytic ac-

tivity, is required for normal RAS activation by

most, if not all, growth factors and cytokines [re-

viewed 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 catalyt-

ic activity is suppressed until it is needed at the

140 Araki � Neel

right time and place. In the basal (closed) state,

the amino terminal SH2 (N-SH2) domain inter-

acts with the PTP domain to suppress catalytic

activity [21]. Binding of a phosphotyrosyl pep-

tide (e.g., from an appropriate tyrosyl phospho-

rylated receptor or adapter protein) to the N-SH2

domain disrupts this inhibitory interaction, re-

sulting 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 activ-

ity ([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 inhibi-

tion. Not surprisingly, these mutants almost in-

variably show enhanced catalytic (PTP) activity

in vitro [15], indicating a shift towards the ‘open’

state of the enzyme. Consistent with this inter-

pretation, NS-associated SHP2 mutants show in-

creased binding to the adapter Gab1 and, when

co-expressed with Gab1, enhance Erk activation

in transfection assays [23].

SOS1, which encodes a major guanine-nucle-

otide 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 ef-

fects 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 wheth-

er 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-associat-

ed, 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 conclu-

sion that RAF1/BRAF heterodimers comprise

the bona fide kinase that activates MEK in vivo

[27, 28]. Conceivably, NS RAF1 mutants with ‘de-

creased’ 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 report-

ed recently that loss-of-function SPRED1 mutants

cause a variant NF1-like syndrome [29]; conceiv-

ably, some NS-associated RAF1 proteins could

be resistant to the inhibitory effects of SPRED1.

Such complexities and potential complications in

interpretation of heterologous expression exper-

iments emphasize the need to assess the effects

(both biochemically and biologically) or disease-

associated mutants expressed at more physiologi-

cal levels.

LS, which shares multiple phenotypic features

with NS, also is caused by PTPN11 mutations [5].

Yet surprisingly, LS alleles exhibit markedly de-

creased catalytic activity and act as dominant

negative mutants to inhibit growth factor-evoked

ERK activation [30, 31]. Some mutations report-

edly cause both NS and LS [5]. It is unclear whether

this represents misdiagnosis, similar phenotypes

caused by SHP2 gain-of-function and partial de-

ficiency, artifacts of the in vitro and ex vivo SHP2

assays, or as yet unknown PTP activity-indepen-

dent effects of SHP2.

Most CFC syndrome-associated mutants of

BRAF and all MEK1/2 alleles cause increased

ERK activation of downstream pathway in tran-

sient transfection experiments. Again, however,

there are some exceptions [2, 3].

These cell line studies have provided a rela-

tively easy way in which to test the effects of hu-

man disease-associated mutations on selected

signaling pathways, and to provide initial in-

sights into potential biochemical mechanisms of

pathogenesis. Yet, as detailed above, some of the


results obtained have been confusing, as they do

not quite fit with the simple model that NCFC

syndromes are diseases of RAS/ERK pathway hy-

permorphism. Even more importantly, heterolo-

gous cell lines provide limited insight into the cell

biological basis of the type of cell- and tissue-spe-

cific 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-associ-

ated PTPN11 mutants on primary hematopoietic

cells to gain insights into their role in leukemo-

genesis. Retroviral transduction of somatic leu-

kemia-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 colony-

stimulating factor (GM-CSF) [32–34]. Unlike in

human JMML, mouse myeloid progenitors ex-

pressing PTPN11 mutants also show increased

sensitivity to IL3; such differences likely reflect

intrinsic differences between murine and hu-

man 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-as-

sociated 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 lat-

ter finding suggests that even constitutively ac-

tivated SHP2 must be targeted correctly via its

SH2 domains to promote malignant transforma-

tion. 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 re-

maining mice succumb to T cell leukemia/lym-

phoma, 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 po-

tent leukemogenic mutants [33].

Such experiments have also provided some

insight into the biochemical effects of leuke-

mogenic mutants. Macrophages derived from

BM transduced with such mutants display en-

hanced GM-CSF-evoked ERK activation [33,

34], whereas bone marrow mast cells from mice

with SHP2-evoked MPD show increased activa-

tion 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 differen-

tial expression of important SHP2-binding pro-

teins may be one reason for the tissue-specific

effects of disease-associated PTPN11 mutants.

Taken together, these retroviral gene transduc-

tion studies have provided firm evidence for

the causal role of PTPN11 mutants in leukemo-

genesis, and identified key structure/function

relationships (most notably, the PTP domain

requirement) and abnormal biochemical conse-

quences 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 affect-

ed individuals. Compared to normal fibroblasts,

these have increased proliferation (as assayed by

142 Araki � Neel

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 abnor-

malities and cardiac defects, there have been few

studies of the effects of NCFC mutations on the

cell types relevant to these defects (in part, be-

cause 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 de-

velopment, 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, spe-

cialized endothelial cells (termed cushion en-

dothelium or cushion endocardium), which rest

upon a specialized extracellular matrix (the ‘car-

diac jelly’), respond to signals from the subjacent

myocardium and undergo an endothelial to mes-

enchymal transition (EMT). Once transformed,

cushion mesenchymal cells invade the cardiac

jelly and proliferate. A complex morphogenetic

process ensues, which entails cessation of cush-

ion 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 ef-

fects 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 mes-

enchymal 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 hy-

pertrophic valves seen in many NS patients.

However, these results are somewhat incon-

sistent 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 pulmon-

ic stenosis more often than in the general pop-

ulation [44], suggesting that, contingent upon

the genetic background, NF1 may be haploin-

sufficient for vavulogenesis. Consequently, the

cardiac phenotype of Nf1–/– embryos may repre-

sent a more severe version of the consequences

of NF1 heterozygosity in humans. Such embry-

os exhibit pan-valvular stenosis, atrial and ven-

tricular septal defects, and double outlet right

ventricle. Studies using a conditional Nf1 allele

indicate that these result selectively from the ab-

sence of Nf1 in endothelial cells [43, 45]. Similar

to the phenotype of NS mice (see below), Nf1–/–

embryos show increased mesenchymal cell pro-

liferation, but in contrast to the above chicken

explant studies, Nf1–/– cushion explants report-

edly 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 dis-

ease-associated NCFC mutants in model genetic


organisms. When such mutants evoke relevant

phenotypes, such organisms as Drosophila mela-

nogaster provide the potentially big advantage of

rapidly deciphering key pathways using genetic

analysis and the large number of hyper- or hypo-

morphic lines already established. Flies also have

some clear limitations: obviously, key syndromic

features such as cardiac, facial and stature abnor-

malities cannot be modeled accurately.

Drosophila does, however, have a rudimen-

tary hematopoietic system, and earlier work

established that expression of activated RAS mu-

tants causes excessive hemocyte production [46].

Similarly, expression of the leukemia-associat-

ed 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 dif-

ferentiation. These effects are qualitatively sim-

ilar, although considerably weaker, than those

evoked by activated RAS. It is unclear if this re-

flects 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 potent-

ly 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 de-

grees 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 mu-

tation 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., manu-

script 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 mu-

tants do not reflect universal abnormality of

Drosophila tyrosine kinase signaling. For exam-

ple, the N308D mutant causes a wing vein pheno-

type similar to that evoked by gain-of-function

mutants in the Drosophila EGFR, and this phe-

notype 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 path-

ways, respectively, which may have important

implications for the pathogenesis of key NS phe-

notypes. 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 sug-

gest impaired gastrulation, and indeed, cell trac-

ing experiments indicate that both extension and

convergence movements are reduced significant-

ly 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 mu-

tants were indistinguishable, which is similar to

the human situation. This is, of course, surpris-

ing, given that NS and LS alleles have opposite

effects on SHP2 catalytic activity. However, the

effects of the two mutants were neither additive

144 Araki � Neel

nor synergistic, which the authors interpreted as

indicating opposing actions on the same path-

way. 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 de-

ficiency 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 mor-

phants, 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 differen-

tial ability of NS and LS mutants to mediate these

pathways may help explain the phenotypic simi-

larities and differences between LS and NS.

Mouse Models

Over the past decade, several groups have devel-

oped mouse models of NF1, by means of conven-

tional and conditional Nf1 gene inactivation. As

noted above, Nf1–/– mice are not viable. In con-

trast, 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 15–18 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 inacti-

vation in Schwann cells (peripheral nervous sys-

tem) or astrocytes (central nervous system) also

fails to cause tumors. By contrast, Nf1 deletion in

hematopoietic cells results in a progressive my-

eloproliferative disorder that resembles JMML

[55].

Parada and colleagues resolved this para-

dox by realizing that in NF1 patients, neurofi-

bromas, which are of Schwann cell origin, are

generated in the context of NF1 heterozygous

tissues. Remarkably, they found that homozy-

gous Nf1 deletion in Schwann cells, in the back-

ground of Nf1 heterozygosity results in fusiform

paraspinal masses with histological features of

plexiform neurofibroma [56]. Nf1+/– mice lack-

ing neurofibromin in astroglial precursors de-

veloped fusiform masses of the optic nerve and

chiasm, resembling optic nerve gliomas in chil-

dren 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 chemotac-

tic factors, including Kit Ligand. In response to

such factors, Nf1+/– mast cells are recruited to the

vicinity of Nf1–/– Schwann cells to facilitate neo-

plastic transformation [58].

Our group has generated and character-

ized 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 de-

fects, 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 phe-

notypes seen in NS patients (and in DG/+ mice).


Mid-gestation DG/DG embryos also are marked-

ly edematous (probably due to their severe cardiac

defects), and show evidence of hemorrhage and

liver necrosis. Bleeding and clotting abnormali-

ties are seen with variable penetrance in NS pa-

tients [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 hemor-

rhage 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), mid-

gestation hepatic necrosis is a classical manifes-

tation of defects in the NF-κB pathway (which

is required to prevent apoptosis in response to

TNFα produced at that time). There has been a

report that SHP2 regulates NFκB 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 necro-

sis 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 non-

compaction or hypoplasia has been reported in

some NS patients [64–66], although the geno-

types of these individuals have not been reported.

Transgenic expression of an NS mutant causes a

similar phenotype [67], although as discussed be-

low, 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 per-

inatally 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 em-

bryos have ventricular septal defects, double-outlet

right ventricle and increased size of all valve pri-

mordia. 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 ne-

crosis). 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 in-

creased activity of the Ras/Erk pathway. Indeed, in-

creased 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 de-

fects 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

146 Araki � Neel

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 sub-

sequently (see below) – Erk hyper-activation is not

uniform. Furthermore, primary mouse embryo fi-

broblasts 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 mu-

tants have a cell/tissue-selective ability to increase

Erk activation. The reason for this selectivity re-

mains unknown, although possible explanations

include differential levels of SHP2 binding proteins

(discussed above) or substrates or differential ac-

tivity of homeostatic feedback pathways able to di-

minish the effects of increased SHP2 activity.

Severely affected DG/+ embryos survive to at

least E18.5 (the last developmental time point an-

alyzed), 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 my-

eloid hyperplasia, and factor-independent colony

production by BM and spleen cells. Although this

MPD is well-tolerated at first, DG/+ mice eventu-

ally die much earlier (at 12–15 months) than their

WT counterparts (T.A. and B.G.N., unpublished).

Notably in this regard, the D61G allele, which ini-

tially was reported only in NS patients, was sub-

sequently 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 altera-

tions 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 pro-

viding 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 modi-

fier loci segregating in the mixed background used

for the initial studies. Second, biochemical analy-

ses show that D61G is one of, if not the, most high-

ly activated PTPN11 alleles associated with NS [15].

Given that increasing gene dosage enhances phe-

notypic severity caused by activated Shp2 in flies

and mice (see above), mutants with varying de-

grees of catalytic activation might also be expect-

ed 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 is-

sues using both our initial DG mice and sever-

al new mouse models, including knock-in mice

expressing Ptpn11N308D and inducible knock-

in mice expressing Ptpn11D61Y (T.A., G.C., and

B.G.N., manuscript in preparation). By cross-

ing the DG allele onto 129S6/SvEv, C57BL6/J

or Balb/c, we have found that the genetic back-

ground strongly influences the NS phenotype.

It is not yet clear, however, if cloneable modi-

fiers exist or if these strain differences are at-

tributable to heterosis. Our data also indicate,

however, that, as in the Drosophila studies, the

specific Ptpn11 allele (on the same genetic back-

ground) can strongly influence the NS pheno-

type. 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 re-

quired to evoke facial abnormalities, cardiac

defects and fatal MPD, respectively. Studies us-

ing tissue-specific Cre recombinase lines to ac-

tivate the Ptpn11D61Y allele selectively show that

the facial abnormalities result from mutant ex-

pression in NC-derived cells, whereas all car-

diac defects are caused by mutant expression in


endocardium/endothelium, not the NC or myo-

cardium. 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/+ em-

bryos, 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 stud-

ies [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 pro-

liferation for technical reasons. Notably, mesen-

chymal outgrowths are observed in these explants

when growth factors (e.g., PDGF, FGF, Neuregulin)

are added exogenously, suggesting that endog-

enous growth factors may be limiting in mouse

explants (but not in chicken). The emerging pic-

ture, 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 ac-

tivation translates into enhanced EMT and prolif-

eration. One attractive possibility is that NS alleles

hyper-activate the transcription factor Sox9. Sox9

is known to be an immediate-early gene depen-

dent on Erk activation [70], and Sox9-deficient

mice have hypoplastic cardiac valves due to defec-

tive 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 αMHC and

βMHC promoters, respectively [67]. Because the

βMHC promoter becomes active in early gesta-

tion, whereas αMHC turns on postnatally, mutant

Shp2 expression occurs during different time win-

dows in each line. Embryonic expression of Q79R

resulted in altered cardiomyocyte cell prolifera-

tion, ventricular non-compaction, and ventricular

septal defects. In contrast, postnatal expression

of Q79R mutant had no apparent effect. Erk ac-

tivation was increased in mutant hearts, and de-

creasing expression of Erk1 or 2 (by crossing to

Erk1+/– or Erk2+/– mice) ablated the cardiac defects

caused by embryonic Q79R expression. It is diffi-

cult to reconcile these findings with our observa-

tions 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 endothelial-

driven expression phenocopies all aspects of the

mouse NS phenotype (including ventricular thin-

ning). 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 myocar-

dial-specific DY expression. Indeed, there was a

Q79R dosage-dependent difference in penetrance

of the myocardial phenotype in different transgen-

ic lines generated by Nakamura et al. Alternatively,

owing to position effects, the Q79R allele could

have been expressed gratuitously in the develop-

ing 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 activat-

ed than the KRAS alleles found in NS patients,

the effects of G12D are likely to be qualitatively

148 Araki � Neel

References

Bentires-Alj M, Kontaridis MI, Neel 1 BG: Stops along the RAS pathway in human genetic disease. Nat Med 2006;12:283–285.Niihori T, Aoki Y, Narumi Y, Neri G, 2 Cave H, et al: Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome. Nat Genet 2006;38:294–296.Rodriguez-Viciana P, Tetsu O, Tidy- 3 man WE, Estep AL, et al: Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome. Science 2006;311: 1287–1290.Zebisch A, Troppmair J: Back to the 4 roots: the remarkable RAF oncogene story. Cell Mol Life Sci 2006;63: 1314–1330.

Tartaglia M, Gelb BD: Noonan syn- 5 drome and related disorders: genetics and pathogenesis. Annu Rev Genomics Hum Genet 2005;6:45–68.Schubbert S, Zenker M, Rowe SL, Boll 6 S, Klein C, et al: Germline KRAS muta-tions cause Noonan syndrome. Nat Genet 2006;38:331–336.Roberts AE, Araki T, Swanson KD, 7 Montgomery KT, Schiripo TA, et al: Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet 2007;39:70–74.Tartaglia M, Pennacchio LA, Zhao C, 8 Yadav KK, Fodale V, et al: Gain-of-function SOS1 mutations cause a dis-tinctive form of Noonan syndrome. Nat Genet 2007;39:75–79.

Razzaque MA, Nishizawa T, Komoike 9 Y, Yagi H, Furutani M, et al: Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet 2007;39:1013–1017.Pandit B, Sarkozy A, Pennacchio LA, 10 Carta C, Oishi K, et al: Gain-of-func-tion RAF1 mutations cause Noonan and LEOPARD syndromes with hyper-trophic cardiomyopathy. Nat Genet 2007;39:1007–1012.Loh ML, Vattikuti S, Schubbert S, 11 Reynolds MG, Carlson E, et al: Muta-tions in PTPN11 implicate the protein tyrosine phosphatase SHP-2 in leuke-mogenesis. Blood 2003;103:2325–2331.

similar (though more severe). Global G12D ex-

pression causes early embryonic lethality due to

trophoblast defects. The early lethality can be by-

passed 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 hematopoi-

etic abnormalities and a profound defect in lung

branching morphogenesis, associated with upreg-

ulation of Sprouty-2, a member of the Spry fam-

ily (Sprouty 1–4) of poorly understood feedback

inhibitors of Ras/Erk pathway. Although defec-

tive lung branching morphogenesis is not char-

acteristic of NCFC syndromes, these findings

nevertheless suggest that Spry proteins and their

relatives, the Spreds (Spred1–3) may be important

modifiers of hyper-activated RAS/ERK pathway

components, and thus may help explain pheno-

typic variation in the NCFC. Consistent with this

notion, Spred1 and Spred2 knockout mice exhib-

it 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 pathophys-

iological 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 de-

termining the precise upstream signaling path-

ways affected and how aberrant signaling by

these pathways is translated into abnormal mor-

phogenesis. 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 de-

fine 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 similari-

ties and differences between these fascinating

syndromes.


Tartaglia M, Niemeyer CM, Fragale A, 12 Song X, Buechner J, et al: Somatic mu-tations in PTPN11 in juvenile myelo-monocytic leukemia, myelodysplastic syndromes and acute myeloid leuke-mia. Nat Genet 2003;34:148–150.Aoki Y, Niihori T, Kawame H, Kurosawa 13 K, Ohashi H, et al: Germline mutations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 2005;37:1038–1040.Bos JL: ras oncogenes in human can-14 cer: a review. Cancer Res 1989;49: 4682–4689.Keilhack H, David FS, McGregor M, 15 Cantley LC, Neel BG: Diverse biochem-ical properties of Shp2 mutants. Impli-cations for disease phenotypes. J Biol Chem 2005;280:30984–30993.Kratz CP, Niemeyer CM, Thomas C, 16 Bauhuber S, Matejas V, et al: Mutation analysis of Son of Sevenless in juvenile myelomonocytic leukemia. Leukemia 2007;21:1108–1109.Swanson KD, Winter JM, Reis M, Ben-17 tires-Alj M, Greulich H, et al: SOS1 mu-tations are rare in human malignan-cies: Implications for Noonan syndrome patients. Genes Chromo-somes Cancer 2008;47:253–259.Neel BG, Gu H, Pao L: The ‘Shp’ing 18 news: SH2 domain-containing ty-rosine phosphatases in cell signaling. Trends Biochem Sci 2003;28:284–293.Feng GS: Shp-2 tyrosine phosphatase: 19 signaling one cell or many. Exp Cell Res 1999;253:47–54.Hof P, Pluskey S, Dhe-Paganon S, Eck 20 MJ, Shoelson SE: Crystal structure of the tyrosine phosphatase SHP-2. Cell 1998;92:441–450.Barford D, Neel BG: Revealing mecha-21 nisms for SH2 domain mediated regula-tion of the protein tyrosine phosphatase SHP-2. Structure 1998;6:249–254.O’Reilly AM, Pluskey S, Shoelson SE, 22 Neel BG: Activated mutants of SHP-2 preferentially induce elongation of Xe-nopus animal caps. Mol Cell Biol 2000;20:299–311.Fragale A, Tartaglia M, Wu J, Gelb BD: 23 Noonan syndrome-associated SHP2/PTPN11 mutants cause EGF-depen-dent prolonged GAB1 binding and sus-tained ERK2/MAPK1 activation. Hum Mutat 2004;23:267–277.Margarit SM, Sondermann H, Hall BE, 24 Nagar B, Hoelz A, et al: Structural evi-dence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS. Cell 2003;112:685–695.

Sondermann H, Soisson SM, Boykev-25 isch S, Yang SS, Bar-Sagi D, Kuriyan J: Structural analysis of autoinhibition in the Ras activator Son of sevenless. Cell 2004;119:393–405.Nimnual A, Bar-Sagi D: The two hats 26 of SOS. Sci STKE 2002;PE36.Wan PT, Garnett MJ, Roe SM, Lee S, 27 Niculescu-Duvaz D, et al: Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004;116:855–867.Garnett MJ, Rana S, Paterson H, Bar-28 ford D, Marais R: Wild-type and mu-tant B-RAF activate C-RAF through distinct mechanisms involving het-erodimerization. Mol Cell 2005;20:963–969.Brems H, Chmara M, Sahbatou M, De-29 nayer E, Taniguchi K, et al: Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1-like phe-notype. Nat Genet 2007;39:1120–1126.Kontaridis MI, Swanson KD, David FS, 30 Barford D, Neel BG: PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activat-ing, effects. J Biol Chem 2006;281: 6785–6792.Hanna N, Montagner A, Lee WH, 31 Miteva M, Vidal M, et al: Reduced phosphatase activity of SHP-2 in LEOPARD syndrome: consequences for PI3K binding on Gab1. FEBS Lett 2006;580:2477–2482.Mohi MG, Williams IR, Dearolf CR, 32 Chan G, Kutok JL, et al: Prognostic, therapeutic, and mechanistic implica-tions of a mouse model of leukemia evoked by Shp2 (PTPN11) mutations. Cancer Cell 2005;7:179–191.Schubbert S, Lieuw K, Rowe SL, Lee 33 CM, Li X, et al: Functional analysis of leukemia-associated PTPN11 muta-tions in primary hematopoietic cells. Blood 2005;106:311–317.Chan RJ, Leedy MB, Munugalavadla V, 34 Voorhorst CS, Li Y, Yu M, Kapur R: Human somatic PTPN11 mutations induce hematopoietic-cell hypersensi-tivity to granulocyte-macrophage col-ony-stimulating factor. Blood 2005;105:3737–3742.Schubbert S, Shannon K, Bollag G: Hy-35 peractive Ras in developmental disor-ders and cancer. Nat Rev Cancer 2007;7:295–308.Mohi MG, Neel BG: The role of Shp2 36 (PTPN11) in cancer. Curr Opin Genet Dev 2007;17:23–30.

Krenz M, Yutzey KE, Robbins J: Noo-37 nan syndrome mutation Q79R in Shp2 increases proliferation of valve pri-mordia mesenchymal cells via extra-cellular signal-regulated kinase 1/2 signaling. Circ Res 2005;97:813–820.Armstrong EJ, Bischoff J: Heart valve 38 development: endothelial cell signal-ing and differentiation. Circ Res 2004;95:459–470.Kirby ML, Gale TF, Stewart DE: Neural 39 crest cells contribute to normal aorti-copulmonary septation. Science 1983;220:1059–1061.Kirby ML, Waldo KL: Neural crest and 40 cardiovascular patterning. Circ Res 1995;77:211–215.Runyan RB, Markwald RR: Invasion of 41 mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue. Dev Biol 1983;95:108–114.Nakajima Y, Miyazono K, Kato M, 42 Takase M, Yamagishi T, Nakamura H: Extracellular fibrillar structure of la-tent TGF beta binding protein-1:role in TGF beta-dependent endothelial-mes-enchymal transformation during en-docardial cushion tissue formation in mouse embryonic heart. J Cell Biol 1997;136:193–204.Lakkis MM, Epstein JA: Neurofibro-43 min modulation of ras activity is re-quired for normal endocardial-mesen-chymal transformation in the developing heart. Development 1998;125:4359–4367.Friedman JM, Arbiser J, Epstein JA, 44 Gutmann DH, Huot SJ, et al: Cardio-vascular disease in neurofibromatosis 1: report of the NF1 Cardiovascular Task Force. Genet Med 2002;4:105–111.Gitler AD, Zhu Y, Ismat FA, Lu MM, 45 Yamauchi Y, Parada LF, Epstein JA: Nf1 has an essential role in endothelial cells. Nat Genet 2003;33:75–79.Dearolf CR: Fruit fly ‘leukemia’. Biochim 46 Biophys Acta 1998;1377:M13–M23.Oishi K, Gaengel K, Krishnamoorthy 47 S, Kamiya K, Kim IK, et al: Transgenic Drosophila models of Noonan syn-drome causing PTPN11 gain-of-func-tion mutations. Hum Mol Genet 2006;15:543–553.Araki T, Mohi MG, Ismat FA, Bronson 48 RT, Williams IR, et al: Mouse model of Noonan syndrome reveals cell type- and gene dosage-dependent effects of Ptpn11 mutation. Nat Med 2004;10:849–857.

150 Araki � Neel

Jopling C, van Geemen D, den Hertog J: 49 Shp2 Knockdown and Noonan/LEOP-ARD mutant Shp2-induced gastrula-tion defects. PLoS Genet 2007;3:e225.Tang TL, Freeman RM Jr, O’Reilly AM, 50 Neel BG, Sokol SY: The SH2-contain-ing protein-tyrosine phosphatase SH-PTP2 is required upstream of MAP kinase for early Xenopus development. Cell 1995;80:473–483.Saxton TM, Henkemeyer M, Gasca S, 51 Shen R, Rossi DJ, et al: Abnormal me-soderm patterning in mouse embryos mutant for the SH2 tyrosine phos-phatase Shp-2. EMBO J 1997;16: 2352–2364.Jacks T, Shih TS, Schmitt EM, Bronson 52 RT, Bernards A, Weinberg RA: Tu-mour predisposition in mice heterozy-gous for a targeted mutation in Nf1. Nat Genet 1994;7:353–361.Silva AJ, Frankland PW, Marowitz Z, 53 Friedman E, Laszlo GS, et al: A mouse model for the learning and memory deficits associated with neurofibroma-tosis type I. Nat Genet 1997;15:281–284.Costa RM, Federov NB, Kogan JH, 54 Murphy GG, Stern J, et al: Mechanism for the learning deficits in a mouse model of neurofibromatosis type 1. Nature 2002;415:526–530.Le DT, Kong N, Zhu Y, Lauchle JO, Ai-55 yigari A, et al: Somatic inactivation of Nf1 in hematopoietic cells results in a progressive myeloproliferative disor-der. Blood 2004;103:4243–4250.Zhu Y, Ghosh P, Charnay P, Burns DK, 56 Parada LF: Neurofibromas in NF1: Schwann cell origin and role of tumor environment. Science 2002;296:920–922.Bajenaru ML, Zhu Y, Hedrick NM, 57 Donahoe J, Parada LF, Gutmann DH: Astrocyte-specific inactivation of the neurofibromatosis 1 gene (NF1) is in-sufficient for astrocytoma formation. Mol Cell Biol 2002;22:5100–5113.Yang FC, Ingram DA, Chen S, Hingt-58 gen CM, Ratner N, et al: Neurofibro-min-deficient Schwann cells secrete a potent migratory stimulus for Nf1+/– mast cells. J Clin Invest 2003;112:1851–1861.

de Haan M, vd Kamp JJ, Briet E, Dub-59 beldam J: Noonan syndrome: partial factor XI deficiency. Am J Med Genet 1988;29:277–282.Sharland M, Patton MA, Talbot S, Chi-60 tolie A, Bevan DH: Coagulation-factor deficiencies and abnormal bleeding in Noonan’s syndrome. Lancet 1992;339:19–21.You M, Flick LM, Yu D, Feng GS: Mod-61 ulation of the nuclear factor kappa B pathway by Shp-2 tyrosine phos-phatase in mediating the induction of interleukin (IL)-6 by IL-1 or tumor necrosis factor. J Exp Med 2001;193:101–110.Marino B, Digilio MC, Toscano A, Gi-62 annotti A, Dallapiccola B: Congenital heart diseases in children with Noo-nan syndrome: An expanded cardiac spectrum with high prevalence of atrioventricular canal. J Pediatr 1999;135:703–706.Tartaglia M, Kalidas K, Shaw A, Song 63 X, Musat DL, et al: PTPN11 mutations in Noonan syndrome: molecular spec-trum, genotype-phenotype correla-tion, and phenotypic heterogeneity. Am J Hum Genet 2002;70:1555–1563.Antonelli D, Antonelli J, Rosenfeld T: 64 Noonan’s syndrome associated with hypoplastic left heart. Cardiology 1990;77:62–65.Amann G, Sherman FS: Myocardial 65 dysgenesis with persistent sinusoids in a neonate with Noonan’s phenotype. Pediatr Pathol 1992;12:83–92.Wilmshurst P, Da Costa P: Probable 66 right ventricular dysplasia and patent foramen ovale presenting with cyano-sis and clubbing in a patient with char-acteristics of Noonan syndrome. Br Heart J 1995;74:471–475.Nakamura T, Colbert M, Krenz M, 67 Molkentin JD, Hahn HS, Dorn GW, 2nd, Robbins J: Mediating ERK 1/2 signaling rescues congenital heart de-fects in a mouse model of Noonan syn-drome. J Clin Invest 2007;117: 2123–2132.

Kratz CP, Niemeyer CM, Castleberry 68 RP, Cetin M, Bergstrasser E, et al: The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and Noonan syndrome/myeloprolif-erative disease. Blood 2005;106: 2183–2185.Gauthier AS, Furstoss O, Araki T, 69 Chan R, Neel BG, Kaplan DR, Miller FD: Control of CNS cell-fate decisions by SHP-2 and its dysregulation in Noo-nan syndrome. Neuron 2007;54: 245–262.Murakami S, Kan M, McKeehan WL, 70 de Crombrugghe B: Up-regulation of the chondrogenic Sox9 gene by fibro-blast growth factors is mediated by the mitogen-activated protein kinase path-way. Proc Natl Acad Sci USA 2000;97:1113–1118.Akiyama H, Chaboissier MC, Behring-71 er 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: 6502–6507.Bi W, Deng JM, Zhang Z, Behringer 72 RR, de Crombrugghe B: Sox9 is re-quired for cartilage formation. Nat Genet 1999;22:85–89.Kulyk WM, Franklin JL, Hoffman LM: 73 Sox9 expression during chondrogen-esis in micromass cultures of embry-onic limb mesenchyme. Exp Cell Res 2000;255:327–332.Tuveson DA, Shaw AT, Willis NA, Sil-74 ver DP, Jackson EL, et al: Endogenous oncogenic K-ras(G12D) stimulates pro-liferation and widespread neoplastic and developmental defects. Cancer Cell 2004;5:375–387.Shaw AT, Meissner A, Dowdle JA, 75 Crowley D, Magendantz M, et al: Sprouty-2 regulates oncogenic K-ras in lung development and tumorigenesis. Genes Dev 2007;21:694–707.Bundschu K, Walter U, Schuh K: Get-76 ting a first clue about SPRED func-tions. Bioessays 2007;29:897–907.

Benjamin G. Neel

Ontario Cancer Institute, University Health Network

101 College Street, TMDT8–355

Toronto, ON, M5G1L7 (Canada)




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., bDepartment of Pathology, Massachusetts General Hospital, Boston, Mass., cDepartment of Cardiology, Children’s Hospital Boston, Boston, Mass., USA

AbstractThe molecular pathogenesis of Noonan, Costello, and

Cardio-facio-cutaneous syndromes has recently been de-

scribed. All of these disorders result from an abnormal ac-

tivation 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 applica-

tion of these inhibitors to individuals with RAS-MAPK devel-

opmental disorders will also be discussed.


Human genetic disorders can be classified into

monogenic and complex disorders. Monogenic

disorders, in turn, are classified based upon their

inheritance patterns: autosomal dominant, au-

tosomal recessive and sex-linked being the most

common. A large number of monogenic disor-

ders have been described (OMIM). In most cas-

es 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 in-

herited in an autosomal dominant fashion and

individuals with a mutation in one of sever-

al genes (see below) are affected. Cardio-facio-

cutaneous syndrome (CFC; MIM 115150) and

Costello syndrome (CS; MIM 218040), by con-

trast, typically occur sporadically in families.

Newborns with one of these syndromes can of-

ten 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 diag-

nosis and examination of the nature of the mu-

tations 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 rela-

tive rarity of these monogenic disorders, phar-

maceutical companies, who have the expertise to

develop new drug entities and test them in pa-

tients, are unlikely to be interested in developing

152 Joshi � Roberts � Kucherlapati

drug based therapies. However, these syndromes

have unique properties that may allow them to

take advantage of drugs that are already in de-

velopment 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 de-

tected 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 de-

veloping inhibitors that aim to block the activa-

tion 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 devel-

oping 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 clini-

cal features that result from defects in the RAS-

MAPK 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, primari-

ly pulmonary valve stenosis and hypertrophic car-

diomyopathy, which affect 20–50% and 20–30%

of individuals, respectively, are the primary cause

of morbidity and mortality. Juvenile myelomono-

cytic 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 identi-

fied in 35% of individuals. It is possible, therefore,

that this cohort is not completely representative

of the adult NS population. Additional longitu-

dinal studies should help clarify the risk of neo-

plasia 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 al-

lelic disorder of NS. The multiple lentigines and

deafness observed in these individuals are char-

acteristic and unique. CFC is distinctive in that

individuals typically have hair and skin abnor-

malities such as sparse curly hair, absent eyelash-

es, patchy alopecia, ichthyosis, hyperkeratosis,

and ulerythema ophryogenes (absent eyebrows

with hyperkeratosis). Individuals with CFC and

CS typically have more significant cognitive de-

lays 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 rhab-

domyosarcoma, although neuroblastoma and

bladder cancer have also been observed in mul-

tiple individuals [6]. Because the cancer risk is as

high as 17%, a screening protocol has been rec-

ommended for individuals with CS consisting of

ultrasound examination of the abdomen and pel-

vis, urine catecholamine metabolite analysis, and

urinalysis [7].

Towards a Treatment for RAS-MAPK Pathway Disorders 153

The medical and developmental issues seen

in these disorders are treated symptomatically

as there is no curative treatment for the under-

lying molecular perturbation of the RAS-MAPK

pathway. The treatment of cardiac manifesta-

tions is largely the same as in the general pop-

ulation. The pulmonary valve stenosis varies in

severity from mild, requiring no intervention,

to severe, requiring surgery. Dysplastic pulmo-

nary valves respond less often to balloon dilation

than pulmonary stenosis without valve dyspla-

sia [8]. Hypertrophic cardiomyopathy may be

treated pharmacologically with beta blockers or

calcium channel blockers, may require myomec-

tomy or, less commonly, progress to the point of

requiring heart transplant. Arrhythmias (usu-

ally supraventricular or paroxysmal tachycar-

dia, 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 re-

quire antibiotic prophylaxis for subacute bacte-

rial endocarditis.

Feeding problems are common and a majority

of children require treatment for gastroesopha-

geal reflux. Most children with CFC and CS re-

quire 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 mu-

tation 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 hor-

mone 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 ther-

apy. Large joint extension might be limited and

requires physical therapy and occasionally sur-

gical 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 pop-

ulation though hypoglycemia, low serum cor-

tisone, 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 ma-

lignant hyperthermia or myopathy [14].

Early developmental milestones are often de-

layed; 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 inter-

vention with occupational and physical therapy.

Articulation difficulties respond well to hearing

aids when indicated and speech therapy. In gen-

eral, IQ falls within the normal range for children

with NS and in the mild to moderate mental re-

tardation 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 evalu-

ations with an individualized education plan.


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 infec-

tions 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 papillo-

mata of CS can cause irritation or inflammation

and can be removed surgically or, in the facial

region, treated with cryotherapy. Local medica-

tions 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 defini-

tively understood.

Children with NS are at increased risk for a

bleeding disorder. Platelet aggregation abnor-

malities, 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-contain-

ing medications should be avoided. The need for

surgical pre-treatment should be assessed by a

hematologist.

Lymphatic abnormalities including periph-

eral edema, pulmonary lymphangiectasia, and

intestinal lymphangiectasia are reported in a mi-

nority of cases but when present can cause signif-

icant morbidity and mortality. Support stockings

and careful foot hygiene are important for lower

extremity edema. Chylothorax may require sur-

gical drainage and/or respond to low-fat diet.

Treatment with prednisone has also been report-

ed 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 de-

fect 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 indi-

viduals with CFC or CS [17–20]. 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 3–17% of cases of NS respectively

[21–26]. A fraction of cases of LS also have muta-

tions in RAF1 [22]. Mutations in BRAF, KRAS,

MEK1, and MEK2 cause 37–78, 7, 9, and 4%, re-

spectively, 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 de-

velopment of therapies that can be used to treat

affected individuals.

All of these genes encode proteins that are

components of the RAS-MAPK signal transduc-

tion pathway. This pathway is responsible for the

communication of extracellular growth signals

to the nucleus through a complex phosphoryla-

tion cascade. Activation of this pathway initiates

transcription of genes involved in cell prolifera-

tion, inhibition of apoptosis, and metastasis. It has

been recognized for some time that upregulation

of this pathway is a common feature of tumori-

genesis. Somatic activating BRAF mutations, for

example, are present in 66% of malignant mela-

nomas 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 typi-

cally 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


surfaces, an interaction which is critical for the bas-

al 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 muta-

tions have also been reported to cause LS [22].

Some RAF1 mutations showed an increase in ki-

nase 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 in-

creased kinase activity as compared to wild type

[22]. In addition, unlike LS-associated muta-

tions 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 iden-

tified in up to 19% of tumors examined and is

found in up to 12% of tumors of the large intes-

tine [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 re-

sult 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 tumorigen-

esis, 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 anti-

body or small molecule inhibitors that target re-

ceptors, 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 can-

didates 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 rare-

ly 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 fre-

quently mutated (20%, n = 23,269, [31]).


Sorafenib (BAY 43–9006, Nexavar®, Bayer

Pharmaceuticals) is a RAF inhibitor that is FDA-

approved for use in advanced renal cell carci-

noma. This drug is generally well-tolerated with

skin rash and diarrhea being the most common

adverse events. Sorafenib was developed as an in-

hibitor of RAF1; however, inhibition by this drug

is not specific and it has proven to be capable of in-

hibiting both wild-type and mutant BRAF, as well

as receptor tyrosine kinases such as VEGFR-2,

PDGFR-β, Flt-3, c-Kit, and FGFR-1 [36]. As such,

the mechanism of anti-tumor activity is not cer-

tain. Sorafenib treatment does inhibit mitogen-

stimulated RAF activity in vivo, as measured by

ERK phosphorylation [37]. In addition, RAF1 is

activated in up to 55% (6/11) of RCC tumors, sup-

porting 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 tar-

gets is equally important in this drug’s 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 devel-

opment [39]. However, a vast amount of in vitro

data suggests that MEK inhibition could be an

effective treatment strategy, and two second-

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 Brand Name Company Mechanism Stage of development

Sorafenib Nexavar® Bayer Pharmaceuticals small molecule multi-

kinase inhibitor

FDA-approved for

advanced renal cell

carcinoma

PD-325901 Pfizer small molecule MEK

inhibitor

phase I trial in breast,

colon cancer, and mela-

noma

AZD6244 Astra Zeneca/Array small molecule MEK

inhibitor

phase I/II trials in pan-

creas, biliary cancer,

liver cancer, advanced

solid tumors

Tipifarnib Zarnestra™ Johnson & Johnson farnesyltransferase

inhibitor

phase I/II trials in breast

cancer, AML, CML, lym-

phoma, brain and CNS

cancer, melanoma, NF1

Lonafarnib Sarasar® Schering Plough farnesyltransferase

inhibitor

phase I/II/III trials in pro-

geria, brain and CNS

cancer, advanced breast

cancer, MDS, CMML


generation MEK inhibitors, PD325901, a deriv-

ative of CI-1040, and AZD6244 (ARRY-142886)

are in development. PD325901 suppressed the

growth of tumor xenografts that carried an acti-

vating 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 ad-

verse events included rash, fatigue, diarrhea, nau-

sea, 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 stud-

ies 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 spe-

cifically supports the idea that inhibition is func-

tioning 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 com-

plicated by the observation that in the presence of

Grb2

SOS1PTPN11

MEK1/2

ERK1/2

GDP

KRASHRAS

GDP

Receptor

Cellmembrane

Transcription

Growth factoror cytokine

KRASHRAS

GTPGTP

MEK inhibitors

RAF inhibitorsBRAFRAF1

FTase inhibitors

Fig. 1. RAS-MAPK Pathway. Components of the RAS-MAPK pathway in which germline muta-

tions 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.


FTase inhibitors, RAS proteins undergo alterna-

tive lipid modification: prenylation by geranylger-

anyltransferase I (GGTaseI), which is sufficient to

target RAS to the plasma membrane. GGTase in-

hibitors also exist and may complement the inhi-

bition of FTase. FTase inhibitors (FTI), like RAF

inhibitors, are non-specific and inhibit farnesyla-

tion 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 undergo-

ing clinical development. Several others, includ-

ing 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 myelo-

dysplastic 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 de-

crease in MAPK phosphorylation did not corre-

late with response, suggesting that RAS inhibition

may not be a major mechanism of response [45].

Lonafarnib monotherapy or combination ther-

apy has been shown to have activity in the treat-

ment of several solid tumors, including NSCLC

and advanced breast cancer [47–49]. Both lona-

farnib monotherapy and imatinib combination

therapy have demonstrated activity in individu-

als with CML [50, 51]. In advanced urothelial can-

cer, metastatic colorectal cancer, and advanced

head and neck squamous cell carcinoma, no tu-

mor responses were observed [44]. The primary

toxicities included diarrhea, neutropenia, nau-

sea, 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 cur-

rently 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 tip-

ifarnib treatment in children and young adults

with NF1 and progressive plexiform neurofibro-

mas [53]. One phase II clinical trial of tipifarn-

ib 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 pe-

diatric cases of advanced CNS tumors has been

reported [55]. Of 48 assessable patients, one ex-

hibited partial response and nine had stable dis-

ease. Toxicities included grade 4 neutropenia,

alterations in electrolytes, respiratory tract com-

plaints, neuropathy, fatigue, and pain. Diarrhea

was treatable with loperamide. Four of six as-

sessable 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


improved bone health through a number of pa-

rameters [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 charac-

teristics in the model are positively affected by in-

hibition, 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 fea-

tures (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 inhibi-

tors. Additional trials of other individuals that do

not have cancer can also be considered.

Several mouse models of NS have been gener-

ated. Mice heterozygous for the D61G mutation

in PTPN11 exhibit several of the clinical features

of NS [59]. They have short stature (proportion-

al growth failure), craniofacial features, and my-

eloproliferative disease. In addition, about 50%

of mice have ventricular septal defects, double-

outlet right ventricle, and enlarged valve primor-

dia; however, the mice do not develop cardiac

hypertrophy. Mice that express the Q79R mu-

tation in PTPN11 during development similarly

showed ventricular non-compaction, ventricu-

lar 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 as-

pects 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 RAS-

MAPK 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 individual’s specific

causative mutation. One challenge could be that

target inhibition is circumvented by an activat-

ed 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 acti-

vating 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 effec-

tive. One preliminary study evaluating the use of

RAS-MAPK inhibitors in cultured cells express-

ing 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, how-

ever, did prevent pathway activation by mutated

MEK1 (F53S and Y130C) and MEK2 (F57C).

Inhibition of MEK, the most downstream ef-

fector, may be a strategy that will be effective in

the majority of individuals. An added benefit of

MEK inhibitors could be that, unlike RAF inhi-

bition by sorafenib or FTase inhibition by lona-

farnib or tipifarnib, inhibition is highly specific.

A remaining challenge will be the small but sig-

nificant 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


development of inhibitor therapy are those who

develop cancer, whether due to germline or so-

matic 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% so-

matic mutations in NF1, and 15% germline mu-

tations in NF1. JMML in individuals without NS

is often rapidly fatal; left untreated, the mortal-

ity rate nears 100% within the first year. In indi-

viduals 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 HLA-

matched donor can be difficult, and the side ef-

fects 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 9–10% of pediatric tu-

mors 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 ma-

lignancies, including hepatoblastoma and ALL,

have been observed [67–69]. 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 trans-

plant but since hepatoblastoma can be seen in CS,

it could also be related to the common perturba-

tion of the RAS-MAPK pathway in CFC and CS

[67]. Given the small numbers of clinically diag-

nosed CFC individuals and the lack of longitudi-

nal studies in this patient group, the frequency of

cancer may be larger than originally appreciated.

It is difficult to predict what impact such inhibi-

tor treatment may have on the congenital features

of NS, CFC, or CS such as developmental delay, car-

diac 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 [71–73]. A phase I clinical trial

evaluating the use of lovastatin in adults with NF1 is

currently underway [74]. The finding that pharma-

cologically reducing RAS-MAPK pathway activi-

ty could improve cognitive features in individuals

with aberrant pathway activation is encouraging,

although it is not clear what if any benefit post-

natal 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, expres-

sion 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 eliminat-

ing 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


complete may have no impact on heart structure

or function. The exception to this could possibly

be in individuals that have hypertrophic cardio-

myopathy or valve stenosis, where RAS-MAPK in-

hibition 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 addition-

al medical complications including congenital

heart defects. Cardiotoxicity, such as acute cor-

onary syndromes including myocardial infarc-

tion 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, monitor-

ing cardiac outcomes has not typically been built

into clinical trial design of most of these inhibi-

tors. As the molecular mechanism of these cardi-

ac effects is not well understood, caution should

be used in selecting inhibitors for treatment and

consideration for evaluating adverse cardiovas-

cular 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 mu-

tations are somatic and often strongly activate the

pathway. The activating mutations found in tu-

mors and those found in NS, LS, CFC, or CS pa-

tients are not typically the same. Studies in mice

have revealed that the germline presence of can-

cer-associated activating mutations are often not

compatible with life. Experimental evidence sug-

gests that the mutations that lead to developmental

disorders result in the activation of the RAS-

MAPK pathway, but that the level of activation is

often milder than that seen in tumors. This obser-

vation 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 clini-

cal benefit for the patients.

Conclusions

If successful treatments for cancers caused by so-

matic 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 hy-

pertrophy, short stature, or cognitive delay and

then, as methods of delivery improve, attempt-

ing prenatal treatment to prevent or slow devel-

opment of congenital malformation of the heart,

skeleton, kidneys, brain, or face. RAS-MAPK

inhibitors hold promise that today’s treatments

could inform tomorrow’s preventions.

References

Bos JL: 1 ras oncogenes in human cancer: a review. Cancer Res 1989;49:4682–4689.Piombo M, Rosanda C, Pasino M, 2 Marasini M, Cerruti P, Comelli A: Acute lymphoblastic leukemia in Noo-nan syndrome: report of two cases. Med Pediatr Oncol 1993;21:454–455.

Shaw AC, Kalidas K, Crosby AH, Jef- 3 fery S, Patton MA: The natural history of Noonan syndrome: a long-term fol-low-up study. Arch Dis Child 2007;92:128–132.Tartaglia M, Gelb BD: Noonan syn- 4 drome and related disorders: genetics and pathogenesis. Annu Rev Genomics Hum Genet 2005;6:45–68.

Allanson JE: Noonan syndrome. Am J 5 Med Genet C Semin Med Genet 2007;145:274–279.Gripp KW: Tumor predisposition in 6 Costello syndrome. Am J Med Genet C Semin Med Genet 2005;137:72–77.


Gripp KW, Scott CI, Nicholson L, Mc- 7 Donald-McGinn DM, Ozeran JD, et al: Five additional Costello syndrome pa-tients with rhabdomyosarcoma: pro-posal for a tumor screening protocol. Am J Med Genet 2002;108:80–87.Ishizawa A, Oho S, Dodo H, Katori T, 8 Homma SI: Cardiovascular abnormali-ties in Noonan syndrome: the clinical findings and treatments. Acta Paediatr Jpn 1996;38:84–90.Lin AE, Grossfeld PD, Hamilton RM, 9 Smoot L, Gripp KW, et al: Further de-lineation of cardiac abnormalities in Costello syndrome. Am J Med Genet 2002;111:115–129.Limal JM, Parfait B, Cabrol S, Bonnet 10 D, Leheup B, et al: Noonan syndrome: relationships between genotype, growth, and growth factors. J Clin En-docrinol Metab 2006;91:300–306.Ogawa M, Moriya N, Ikeda H, Tanae A, 11 Tanaka T, et al: Clinical evaluation of recombinant human growth hormone in Noonan syndrome. Endocr J 2004;51:61–68.Gripp KW, Scott CI, Nicholson L, 12 Figueroa TE: Second case of bladder carcinoma in a patient with Costello syndrome. Am J Med Genet 2000;90:256–259.Gregersen N, Viljoen D: Costello syn-13 drome with growth hormone deficien-cy and hypoglycemia: a new report and review of the endocrine associations. Am J Med Genet A 2004;129:171–175.Allanson J: Noonan Syndrome, in 14 Cassidy AJ (ed): Management of Ge-netic Syndromes (Wiley-Liss, New York 2001).Young TL, Ziylan S, Schaffer DB: The 15 ophthalmologic manifestations of the cardio-facio-cutaneous syndrome. J Pediatr Ophthalmol Strabismus 1993;30:48–52.Rauen KA: Cardiofaciocutaneous syn-16 drome: GeneReviews at GeneTests: Medical Genetics Information Re-source (database online) (University of Washington, Seattle 1997–2007).Tartaglia M, Mehler EL, Goldberg R, 17 Zampino G, Brunner HG, et al: Muta-tions in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 2001;29:465–468.

Musante L, Kehl HG, Majewski F, Mei-18 necke P, Schweiger S, et al: Spectrum of mutations in PTPN11 and geno-type-phenotype correlation in 96 pa-tients with Noonan syndrome and five patients with cardio-facio-cutaneous syndrome. Eur J Hum Genet 2003;11:201–206.Legius E, Schrander-Stumpel C, Schol-19 len E, Pulles-Heintzberger C, Gewillig M, Fryns JP: PTPN11 mutations in LEOPARD syndrome. J Med Genet 2002;39:571–574.Digilio MC, Conti E, Sarkozy A, Min-20 garelli R, Dottorini T, et al: Grouping of multiple-lentigines/LEOPARD and Noo-nan syndromes on the PTPN11 gene. Am J Hum Genet 2002;71:389–394.Razzaque MA, Nishizawa T, Komoike 21 Y, Yagi H, Furutani M, et al: Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet 2007;39:1013–1017.Pandit B, Sarkozy A, Pennacchio LA, 22 Carta C, Oishi K, et al: Gain-of-func-tion RAF1 mutations cause Noonan and LEOPARD syndromes with hyper-trophic cardiomyopathy. Nat Genet 2007;39:1007–1012.Roberts AE, Araki T, Swanson KD, 23 Montgomery KT, Schiripo TA, et al: Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet 2007;39:70–74.Tartaglia M, Pennacchio LA, Zhao C, 24 Yadav KK, Fodale V, et al: Gain-of-function SOS1 mutations cause a dis-tinctive form of Noonan syndrome. Nat Genet 2007;39:75–79.Carta C, Pantaleoni F, Bocchinfuso G, 25 Stella L, Vasta I, et al: Germline mis-sense mutations affecting KRAS iso-form B are associated with a severe Noonan syndrome phenotype. Am J Hum Genet 2006;79:129–135.Schubbert S, Zenker M, Rowe SL, Boll 26 S, Klein C, et al: Germline KRAS mu-tations cause Noonan syndrome. Nat Genet 2006;38:331–336.Niihori T, Aoki Y, Narumi Y, Neri G, 27 Cave H, et al: Germline KRAS and BRAF mutations in cardio-facio-cuta-neous syndrome. Nat Genet 2006;38:294–296.Rodriguez-Viciana P, Tetsu O, Tidy-28 man WE, Estep AL, Conger BA, et al: Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome. Science 2006;311:1287–1290.

Aoki Y, Niihori T, Kawame H, Kuro-29 sawa K, Ohashi H, et al: Germline mu-tations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 2005;37:1038–1040.Davies H, Bignell GR, Cox C, Stephens 30 P, Edkins S, et al: Mutations of the BRAF gene in human cancer. Nature 2002;417:949–954.Forbes S, Clements J, Dawson E, Bam-31 ford S, Webb T, et al: Cosmic 2005. Br J Cancer 2006;94:318–322.Hof P, Pluskey S, Dhe-Paganon S, Eck 32 MJ, Shoelson SE: Crystal structure of the tyrosine phosphatase SHP-2. Cell 1998;92:441–450.Kontaridis MI, Swanson KD, David FS, 33 Barford D, Neel BG: PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activat-ing, effects. J Biol Chem 2006;281: 6785–6792.Wan PT, Garnett MJ, Roe SM, Lee S, 34 Niculescu-Duvaz D, et al: Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004;116:855–867.Roberts PJ, Der CJ: Targeting the Raf-35 MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene 2007;26:3291–3310.Wilhelm SM, Carter C, Tang L, Wilkie 36 D, McNabola A, Rong H, Chen C: BAY 43–9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 2004;64:7099–7109.Strumberg D, Richly H, Hilger RA, 37 Schleucher N, Korfee S, et al: Phase I clinical and pharmacokinetic study of the Novel Raf kinase and vascular en-dothelial growth factor receptor inhib-itor BAY 43–9006 in patients with ad-vanced refractory solid tumors. J Clin Oncol 2005;23:965–972.Oka H, Chatani Y, Hoshino R, Ogawa 38 O, Kakehi Y, et al: Constitutive activa-tion of mitogen-activated protein (MAP) kinases in human renal cell carcinoma. Cancer Res 1995;55: 4182–4187.Rinehart J, Adjei AA, Lorusso PM, Wa-39 terhouse D, Hecht JR, et al: Multicenter phase II study of the oral MEK inhibi-tor, CI-1040, in patients with advanced non-small-cell lung, breast, colon, and pancreatic cancer. J Clin Oncol 2004;22:4456–4462.


Solit DB, Garraway LA, Pratilas CA, 40 Sawai A, Getz G, et al: BRAF mutation predicts sensitivity to MEK inhibition. Nature 2006;439:358–362.Lorusso P, Krishnamurthi S, Rinehart 41 JR, Nabell L, Croghan G, Varterasian M, Sadis SS: A phase 1–2 clinical study of a second generation oral MEK in-hibitor, PD 0325901 in patients with advanced cancer. ASCO Annual Meet-ing Proceedings. J Clin Oncol 2005.Pfizer Inc (Director, Clinical Trials 42 Disclosure Group), MEK Inhibitor PD-325901 To Treat Advanced Breast Cancer, Colon Cancer, And Melanoma. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000- [cited 2008 Feb 20]. Available from: http://clinicaltri-als.gov/ct2/show/NCT00147550?term = pd325901&rank = 2 Identifier: NCT00147550.Yeh TC, Marsh V, Bernat BA, Ballard J, 43 Colwell H, et al: Biological character-ization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-acti-vated protein kinase kinase 1/2 inhibi-tor. Clin Cancer Res 2007;13:1576–1583.Appels NM, Beijnen JH, Schellens JH: 44 Development of farnesyl transferase inhibitors: a review. Oncologist 2005;10:565–578.Lancet JE, Gojo I, Gotlib J, Feldman EJ, 45 Greer J, et al: A phase 2 study of the farnesyltransferase inhibitor tipifarnib in poor-risk and elderly patients with previously untreated acute myelog-enous leukemia. Blood 2007;109: 1387–1394.Fenaux P, Raza A, Mufti GJ, Aul C, 46 Germing U, et al: A multicenter phase 2 study of the farnesyltransferase in-hibitor tipifarnib in intermediate- to high-risk myelodysplastic syndrome. Blood 2007;109:4158–4163.Johnston SR, Hickish T, Ellis P, Hous-47 ton S, Kelland L, et al: Phase II study of the efficacy and tolerability of two dos-ing regimens of the farnesyl trans-ferase inhibitor, R115777, in advanced breast cancer. J Clin Oncol 2003;21:2492–2499.Khuri FR, Glisson BS, Kim ES, Stat-48 kevich P, Thall PF, et al: Phase I study of the farnesyltransferase inhibitor lonafarnib with paclitaxel in solid tu-mors. Clin Cancer Res 2004;10:2968–2976.

Kim ES, Kies MS, Fossella FV, Glisson 49 BS, Zaknoen S, et al: Phase II study of the farnesyltransferase inhibitor lona-farnib with paclitaxel in patients with taxane-refractory/resistant nonsmall cell lung carcinoma. Cancer 2005;104:561–569.Cortes J, Jabbour E, Daley GQ, O’Brien 50 S, Verstovsek S, et al: Phase 1 study of lonafarnib (SCH 66336) and imatinib mesylate in patients with chronic my-eloid leukemia who have failed prior single-agent therapy with imatinib. Cancer 2007;110:1295–1302.Borthakur G, Kantarjian H, Daley G, 51 Talpaz M, O’Brien S, et al: Pilot study of lonafarnib, a farnesyl transferase inhibitor, in patients with chronic my-eloid leukemia in the chronic or accel-erated phase that is resistant or refrac-tory to imatinib therapy. Cancer 2006;106:346–352.Widemann BC, Salzer WL, Arceci RJ, 52 Blaney SM, Fox E, et al: Phase I trial and pharmacokinetic study of the farnesyltransferase inhibitor tipifarnib in children with refractory solid tu-mors or neurofibromatosis type I and plexiform neurofibromas. J Clin Oncol 2006;24:507–516.Widemann BC: National Cancer Insti-53 tute, Tipifarnib in Preventing Cancer in Children With Neurofibromatosis Type 1 and Progressive Plexiform Neurofi-bromas. In: ClinicalTrials.gov [Inter-net]. Bethesda (MD): National Library of Medicine (US). 2000- [cited 2008 Feb 20]. Available from: http://clinicaltrials.gov/ct2/show/NCT00029354?term = tipifarnib+nf1&rank = 1 Identifier: NCT00029354.Castleberry RP, Loh M, Jayaprakash N, 54 Peterson A, Casey V, Chang M, et al: Phase II Window Study of the Farne-syltransferase Inhibitor R115777 (Zarnestra®) in Untreated Juvenile My-elomonocytic Leukemia (JMML): A Children’s Oncology Group Study (ASH Annual Meeting Abstract). Blood 2005;106(suppl 1):727a–728a.Kieran MW, Packer RJ, Onar A, Blaney 55 SM, Phillips P, et al: Phase I and phar-macokinetic study of the oral farnesyl-transferase inhibitor lonafarnib ad-ministered twice daily to pediatric patients with advanced central ner-vous system tumors using a modified continuous reassessment method: a Pediatric Brain Tumor Consortium Study. J Clin Oncol 2007;25:3137–3143.

Yang SH, Meta M, Qiao X, Frost D, 56 Bauch J, et al: A farnesyltransferase inhibitor improves disease phenotypes in mice with a Hutchinson-Gilford progeria syndrome mutation. J Clin Invest 2006;116:2115–2121.Fong LG, Frost D, Meta M, Qiao X, 57 Yang SH, Coffinier C, Young SG: A protein farnesyltransferase inhibitor ameliorates disease in a mouse model of progeria. Science 2006;311: 1621–1623.Kieran MW: Phase II Trial of Lona-58 farnib (a Farnesyltransferase Inhibi-tor) for Progeria. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000- [cited 2008 Feb 20]. Available from: http://clinicaltrials.gov/ct2/show/NCT00425607?term = progeria&rank = 2 Identifier: NCT00425607.Araki T, Mohi MG, Ismat FA, Bronson 59 RT, Williams IR, et al: Mouse model of Noonan syndrome reveals cell type- and gene dosage-dependent effects of Ptpn11 mutation. Nat Med 2004;10: 849–857.Nakamura T, Colbert M, Krenz M, 60 Molkentin JD, Hahn HS, Dorn GW, 2nd, Robbins J: Mediating ERK 1/2 signaling rescues congenital heart de-fects in a mouse model of Noonan syn-drome. J Clin Invest 2007;117:2123–2132.Pao W, Wang TY, Riely GJ, Miller VA, 61 Pan Q, et al: KRAS mutations and pri-mary resistance of lung adenocarcino-mas to gefitinib or erlotinib. PLoS Med 2005;2:e17.Senawong T, Phuchareon J, Ohara O, 62 McCormick F, Rauen KA, Tetsu O: Germline mutations of MEK in car-dio-facio-cutaneous (CFC) syndrome are sensitive to MEK and RAF inhibi-tion: Implications for therapeutic op-tions. Hum Mol Genet 2007;17: 419–430.Choong K, Freedman MH, Chitayat D, 63 Kelly EN, Taylor G, Zipursky A: Juve-nile myelomonocytic leukemia and Noonan syndrome. J Pediatr Hematol Oncol 1999;21:523–537.Hasle H: Myelodysplastic and myelo-64 proliferative disorders in children. Curr Opin Pediatr 2007;19:1–8.Driscoll K, Isakoff M, Ferrer F: Update 65 on pediatric genitourinary oncology. Curr Opin Urol 2007;17:281–286.


Kim S, Chung DH: Pediatric solid ma-66 lignancies: neuroblastoma and Wilms’ tumor. Surg Clin North Am 2006;86:469–487, xi.Al-Rahawan MM, Chute DJ, Sol-67 Church K, Gripp KW, Stabley DL, et al: Hepatoblastoma and heart transplan-tation in a patient with cardio-facio-cutaneous syndrome. Am J Med Genet A 2007;143:1481–1488.Makita Y, Narumi Y, Yoshida M, Nii-68 hori T, Kure S, et al: Leukemia in Car-dio-facio-cutaneous (CFC) syndrome: a patient with a germline mutation in BRAF proto-oncogene. J Pediatr He-matol Oncol 2007;29:287–290.

van Den Berg H, Hennekam RC: Acute 69 lymphoblastic leukaemia in a patient with cardiofaciocutaneous syndrome. J Med Genet 1999;36:799–800.Silva AJ, Frankland PW, Marowitz Z, 70 Friedman E, Laszlo GS, et al: A mouse model for the learning and memory deficits associated with neurofibroma-tosis type I. Nat Genet 1997;15: 281–284.Costa RM, Federov NB, Kogan JH, 71 Murphy GG, Stern J, et al: Mechanism for the learning deficits in a mouse model of neurofibromatosis type 1. Nature 2002;415:526–530.

Li W, Cui Y, Kushner SA, Brown RA, 72 Jentsch JD, et al: The HMG-CoA re-ductase inhibitor lovastatin reverses the learning and attention deficits in a mouse model of neurofibromatosis type 1. Curr Biol 2005;15:1961–1967.Sebti SM, Tkalcevic GT, Jani JP: Lovas-73 tatin, a cholesterol biosynthesis inhibi-tor, inhibits the growth of human H-ras oncogene transformed cells in nude mice. Cancer Commun 1991;3:141–147.Cannon T: Trial to Evaluate the Safety 74 of Lovastatin in Adults With Neurofi-bromatosis Type I (NF1). 2008.Nexavar (sorafenib) Prescribing Infor-75 mation (Bayer Pharmaceuticals 2006).Force T, Krause DS, Van Etten RA: Mo-76 lecular mechanisms of cardiotoxicity of tyrosine kinase inhibition. Nat Rev Cancer 2007;7:332–344.

Victoria A. Joshi

65 Landsdowne St.

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165

Author Index

Allanson, J.E. 9

Araki, T. 138

Binder, G. 104

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

Kerr, B. 83

Kratz, C. 119

Kucherlapati, R. 151

Legius, E. 128

Marino, B. 40, 109

Neel, B.G. 138

Noonan, J.A. 1

Rauen, K.A. 73

Roberts, A.E. 66, 151

Sarkozy, A. 40, 55, 109

Schmid, M. VII

Sol-Church, K. 94

Tartaglia, M. 20, 55

Tidyman, W.E. 73

Versacci, P. 109

Zampino, G. 55

Zenker, M. IX

166

Subject Index

Animal models 138AZD6244 156

BRAF 33, 76, 114

Café-au-lait spots 128Cancer predisposition 94Cardiac valve defects 138Cardiofaciocutaneous syndrome (CFC, CFCS) 66, 73,

114, 119clinical diagnosis 66, 73clinical features 67

cardiovascular features 68, 114development 67ectodermal features 69facial features 67gastrointestinal features 70growth 67malignancy 70neurological features 68ophthalmologic features 69renal features 70skeletal feature 70

history 3molecular causes 73natural history 71treatment 80

CFC, see Cardiofaciocutaneous syndromeCFCS, see Cardiofaciocutaneous syndromeCongenital heart defect (CHD) 10, 109 Costello syndrome (CS) 83, 94, 114, 122

development 87diagnosis 89

endocrine abnormalities 87history 4, 83HRAS mutations 94, 101hypertrophic cardiomyopathy (HCM) 86, 114natural history 85phenotype 88skeletal abnormalities 88tumor risk 85

Cryptorchidism 14CS, see Costello syndrome

Drosophila models 142

Gain of function 94Genotype-phenotype correlation 40, 61, 106Germline mutation 94, 98, 123, 138Growth hormone (GH) 104

therapy 105

HRAS 94, 119G12 variants 96Germline mutation 98Rare variants 97Somatic mosaicism 99

Hypertrophic cardiomyopathy (HCM) 86, 111, 114

IGF-I 105Inhibitor 151

Juvenile myelomonocytic leukemia (JMML) 15, 120,138

KRAS 13, 27, 45, 78

Subject Index 167

Learning difficulties 128LEOPARD syndrome (LS) 3, 20, 55, 109, 114

clinical aspects 55disease genes 59history 3, 55molecular pathogenesis 55

Leukemia 138Lonafarnib 156LS, see LEOPARD syndrome

MEK1/2 36, 52, 77, 114Mental retardation 138Mouse models 144Myeloproliferative disorder (MPD) 15, 119, 138

Neuroblastoma 119Neuro-Cardio-Facial-Cutaneous (NCFC) Syndromes

134, 138Neurofibromatosis-Noonan syndrome (NFNS) 5, 128

history 5Neurofibromatosis type 1 (NF1) 5, 128NF1, see neurofibromatosis type 1NFNS, see Neurofibromatosis-Noonan syndromeNoonan syndrome (NS) 1, 9, 20, 40, 59, 109, 119,

128, 138animal models 138cardiovascular anomalies 10central nervous system 13craniofacial features 9development 12gastrointestinal system 14genes 21genitourinary system 14genotype-phenotype correlation 40, 106growth 11, 104hearing 12hematology 14history 1immunological findings 15lymphatics 14

molecular genetics 20musculoskeletal findings 13ocular anomalies 12short stature 104skin 14

NS (see Noonan syndrome)

Oncogene 94

Parental origin 98PD325901 156Pectus 13Protein-tyrosine phosphatase (PTP) 138, 140PTPN11 11, 23, 41, 59, 104, 109, 113, 119, Puberty 104Pulmonary stenosis 10, 110, 115

RAF1 33, 49, 61, 109, 114Ras-MAPK pathway 75, 94, 110, 128, 151RAS

function 100signaling 21, 62structure 100

Rhabdomyosarcoma 85, 119

SH2 domain 140short stature 104SHP2 105, 139Signal transduction 76Somatic mosaicism 99Sorafenib 156SOS1 14, 31, 46

Tipifarnib 156Treatment 80, 115, 151

Watson syndrome 128

Zebrafish model 143

martin zenker-noonan syndrome and related disorders - a matter of deregulated ras signaling...

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