Anna Sarkozy

Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy

Noonan and LEOPARD syndromes are developmental disorders with overlapping features, including cardiac abnormalities, short stature and facial dysmorphia. Increased RAS signaling owing to PTPN11, SOS1 and KRAS mutations causes ∼60% of Noonan syndrome cases, and PTPN11 mutations cause 90% of LEOPARD syndrome cases. Here, we report that 18 of 231 individuals with Noonan syndrome without known mutations (corresponding to 3% of all affected individuals) and two of six individuals with LEOPARD syndrome without PTPN11 mutations have missense mutations in RAF1, which encodes a serine-threonine kinase that activates MEK1 and MEK2. Most mutations altered a motif flanking Ser259, a residue critical for autoinhibition of RAF1 through 14-3-3 binding. Of 19 subjects with a RAF1 mutation in two hotspots, 18 (or 95%) showed hypertrophic cardiomyopathy (HCM), compared with the 18% prevalence of HCM among individuals with Noonan syndrome in general. Ectopically expressed RAF1 mutants from the two HCM hotspots had increased kinase activity and enhanced ERK activation, whereas non–HCM-associated mutants were kinase impaired. Our findings further implicate increased RAS signaling in pathological cardiomyocyte hypertrophy.

Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome

Noonan syndrome is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart defects and skeletal anomalies. Increased RAS-mitogen-activated protein kinase (MAPK) signaling due to PTPN11 and KRAS mutations causes 50% of cases of Noonan syndrome. Here, we report that 22 of 129 individuals with Noonan syndrome without PTPN11 or KRAS mutation have missense mutations in SOS1, which encodes a RAS-specific guanine nucleotide exchange factor. SOS1 mutations cluster at codons encoding residues implicated in the maintenance of SOS1 in its autoinhibited form. In addition, ectopic expression of two Noonan syndrome–associated mutants induces enhanced RAS and ERK activation. The phenotype associated with SOS1 defects lies within the Noonan syndrome spectrum but is distinctive, with a high prevalence of ectodermal abnormalities but generally normal development and linear growth. Our findings implicate gain-of-function mutations in a RAS guanine nucleotide exchange factor in disease for the first time and define a new mechanism by which upregulation of the RAS pathway can profoundly change human development.

Grouping of Multiple-Lentigines/LEOPARD and Noonan Syndromes on the PTPN11 Gene

Multiple-lentigines (ML)/LEOPARD (multiple lentigines, electrocardiographic-conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness) syndrome is an autosomal dominant condition--characterized by lentigines and café au lait spots, facial anomalies, cardiac defects--that shares several clinical features with Noonan syndrome (NS). We screened nine patients with ML/LEOPARD syndrome (including a mother-daughter pair) and two children with NS who had multiple café au lait spots, for mutations in the NS gene, PTPN11, and found, in 10 of 11 patients, one of two new missense mutations, in exon 7 or exon 12. Both mutations affect the PTPN11 phosphotyrosine phosphatase domain, which is involved in <30% of the NS PTPN11 mutations. The study demonstrates that ML/LEOPARD syndrome and NS are allelic disorders. The detected mutations suggest that distinct molecular and pathogenetic mechanisms cause the peculiar cutaneous manifestations of the ML/LEOPARD-syndrome subtype of NS.

Germline missense mutations affecting KRAS isoform B are associated with a severe noonan syndrome phenotype

Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart disease, and multiple skeletal and hematologic defects. NS is an autosomal dominant trait and is genetically heterogeneous. Gain of function of SHP-2, a protein tyrosine phosphatase that positively modulates RAS signaling, is observed in nearly 50% of affected individuals. Here, we report the identification of heterozygous KRAS gene mutations in two subjects exhibiting a severe NS phenotype with features overlapping those of cardiofaciocutaneous and Costello syndromes. Both mutations were de novo and affected exon 6, which encodes the C-terminal portion of KRAS isoform B but does not contribute to KRAS isoform A. Structural analysis indicated that both substitutions (Val152Gly and Asp153Val) perturb the conformation of the guanine ring-binding pocket of the protein, predicting an increase in the guanine diphosphate/guanine triphosphate (GTP) dissociation rate that would favor GTP binding to the KRASB isoform and bypass the requirement for a guanine nucleotide exchange factor.

Germline BRAF mutations in noonan, LEOPARD, and cardiofaciocutaneous Syndromes: Molecular diversity and associated phenotypic spectrum

Noonan, LEOPARD, and cardiofaciocutaneous syndromes (NS, LS, and CFCS) are developmental disorders with overlapping features including distinctive facial dysmorphia, reduced growth, cardiac defects, skeletal and ectodermal anomalies, and variable cognitive deficits. Dysregulated RAS–mitogen‐activated protein kinase (MAPK) signal traffic has been established to represent the molecular pathogenic cause underlying these conditions. To investigate the phenotypic spectrum and molecular diversity of germline mutations affecting BRAF, which encodes a serine/threonine kinase functioning as a RAS effector frequently mutated in CFCS, subjects with a diagnosis of NS (N=270), LS (N=6), and CFCS (N=33), and no mutation in PTPN11, SOS1, KRAS, RAF1, MEK1, or MEK2, were screened for the entire coding sequence of the gene. Besides the expected high prevalence of mutations observed among CFCS patients (52%), a de novo heterozygous missense change was identified in one subject with LS (17%) and five individuals with NS (1.9%). Mutations mapped to multiple protein domains and largely did not overlap with cancer‐associated defects. NS‐causing mutations had not been documented in CFCS, suggesting that the phenotypes arising from germline BRAF defects might be allele specific. Selected mutant BRAF proteins promoted variable gain of function of the kinase, but appeared less activating compared to the recurrent cancer‐associated p.Val600Glu mutant. Our findings provide evidence for a wide phenotypic diversity associated with mutations affecting BRAF, and occurrence of a clinical continuum associated with these molecular lesions. Hum Mutat 0:1–8, 2009.

Correlation between PTPN11 gene mutations and congenital heart defects in Noonan and LEOPARD syndromes

Noonan syndrome (MIM 163950), an autosomal dominant disorder with an estimated prevalence of 1/1000–2500 at birth, is characterised by short stature, facial anomalies, pterygium colli, and congenital heart disease.1,2 Although pulmonary valve stenosis with dysplastic leaflets, hypertrophic cardiomyopathy, and atrial septal defects (ASD) are the most common congenital heart defects in Noonan syndrome,3 a broad spectrum of cardiac phenotypes has been recognised.2–6 About half the affected individuals have PTPN11 gene mutations.7–9 This gene, which maps to chromosome 12q22-qter,10 encodes for the human SH2 domain containing protein tyrosine phosphatase (SHP2).11 PTPN11 gene mutations have also been detected in multiple lentigines/LEOPARD syndrome (multiple lentigines, ECG conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth and sensorineural deafness; ML/LS, MIM 151100),12,13 in Noonan-like/multiple giant cell lesion syndrome (MIM 163955),8 in two families presumably affected by cardio-facio-cutaneous syndrome (CFCS, MIM 115150),14 but not in sporadic CFCS.15 The clinical and genetic heterogeneity of these disorders suggests a possible relation between different PTPN11 gene mutations and distinct clinical features. A genotype–phenotype correlation study in Noonan syndrome found an association between pulmonary stenosis and PTPN11 mutations.8 Our aim in this study was to screen a large cohort of patients with Noonan syndrome and ML/LS in order to expand the genotype–phenotype correlation analysis, with particular emphasis on cardiac diseases. ### Key points

Spectrum of atrial septal defects associated with mutations of NKX2.5 and GATA4 transcription factors

Atrial septal defect (ASD) is a common cardiovascular malformation, affecting over 1 in 1000 live births, accounting for 10% of congenital heart defects (CHD).1 ASD refers to a communication between the right and left atria, anatomically classified into the deficient atrial septum structure. ASD ostium secundum (ASDos) is the prevalent defect, representing 85% of all ASDs.2 ASD may be isolated or associated with other CHDs, such as pulmonary valve stenosis (PVS), ventricular septal defect (VSD), or conduction defects. In addition, persistent left to right blood shunt may result in atrial and ventricular dysfunctions and atrial arrhythmias, in the absence of surgical or catheter based repair. The atrial septum is one of the cardiac structures most sensitive to environmental or genetic factors. Several lines of evidence have highlighted a role for different proteins and transcription factors in the septogenesis process;3 however, only two genes, encoding for the transcription factors NKX2.5 and GATA4, have been implicated so far in non-syndromic ASDs.4,5 Clinical and molecular analyses have shown that mutations in these two genes are responsible for ASDs in association with distinct cardiac features.4–15 Mutations in NKX2.5 , a member of the NK-2 class of homeobox genes, have been described in autosomal dominant ASDos with progressive atrioventricular (AV) block, and in 1–4% of sporadic ASD patients.4,6–12 Most of these mutations occur within the homeodomain, a critical protein domain that interacts specifically with DNA, and are associated with conduction anomalies. Low penetrance NKX2.5 gene mutations, mainly outside the homeodomain, have been found in 5% of patients with tetralogy of Fallot, and in a number of individuals with other CHDs and normal conduction.4,6,9–13 Recently, heterozygous mutations in the GATA4 zinc finger transcription factor gene have been identified in three families with …

NF1 Gene Mutations Represent the Major Molecular Event Underlying Neurofibromatosis-Noonan Syndrome

Neurofibromatosis type 1 (NF1) demonstrates phenotypic overlap with Noonan syndrome (NS) in some patients, which results in the so-called neurofibromatosis-Noonan syndrome (NFNS). From a genetic point of view, NFNS is a poorly understood condition, and controversy remains as to whether it represents a variable manifestation of either NF1 or NS or is a distinct clinical entity. To answer this question, we screened a cohort with clinically well-characterized NFNS for mutations in the entire coding sequence of the NF1 and PTPN11 genes. Heterozygous NF1 defects were identified in 16 of the 17 unrelated subjects included in the study, which provides evidence that mutations in NF1 represent the major molecular event underlying this condition. Lesions included nonsense mutations, out-of-frame deletions, missense changes, small inframe deletions, and one large multiexon deletion. Remarkably, a high prevalence of inframe defects affecting exons 24 and 25, which encode a portion of the GAP-related domain of the protein, was observed. On the other hand, no defect in PTPN11 was observed, and no lesion affecting exons 11-27 of the NF1 gene was identified in 100 PTPN11 mutation-negative subjects with NS, which provides further evidence that NFNS and NS are genetically distinct disorders. These results support the view that NFNS represents a variant of NF1 and is caused by mutations of the NF1 gene, some of which have been demonstrated to cause classic NF1 in other individuals.

Clinical and molecular analysis of 30 patients with multiple lentigines LEOPARD syndrome

Multiple lentigines LEOPARD syndrome (MIM 151100) is an autosomal dominant multiple congenital anomaly syndrome, with high penetrance and markedly variable expression.1 The acronym LEOPARD was coined by Gorlin et al. in 1971 as a mnemonic of the major features of this disorder: multiple l entigines, E CG conduction abnormalities, o cular hypertelorism, p ulmonic stenosis, a bnormal genitalia, r etardation of growth, and sensorineural d eafness.2 It is also known as cardiocutaneous syndrome, Moynahan syndrome, lentiginosis profuse, and progressive cardiomyopathic lentiginosis.3,4 Voron et al. proposed some diagnostic criteria for multiple lentigines LEOPARD syndrome.5 More than 100 cases have been described, and one review has been published.5,6 Multiple lentigines LEOPARD syndrome shares many features with Noonan syndrome (MIM 163950),7–9 in which lentigines and deafness usually are not present. About 40% of patients with Noonan syndrome have missense mutations in the PTPN11 gene, which encodes for the protein tyrosine phosphatase SHP2.10–14 Multiple lentigines LEOPARD syndrome has proved to be allelic to Noonan syndrome,15,16 with two recurrent PTPN11 mutations in exons 7 (Tyr279Cys) and 12 (Thr468Met). Recently, we reported a novel PTPN11 mutation (Gln506Pro) in a unique patient with multiple lentigines LEOPARD syndrome, which suggests that mutations other than Tyr279Cys and Thr468Met could be found in these patients.17 As PTPN11 mutations in multiple lentigines LEOPARD syndrome and Noonan syndrome are exclusive to these conditions, the distinctive manifestations of these disorders likely result from different molecular mechanisms. For instance, as commented in a recent report, the cardiac phenotypes in patients with Noonan syndrome and those with multiple lentigines LEOPARD syndrome with PTPN11 mutations are rather dissimilar, with pulmonary valve stenosis prevailing in the former and hypertrophic cardiomyopathy in the latter.18 We examined the PTPN11 gene in a consecutive series …

Leopard syndrome : Clinical diagnosis in the first year of life

LEOPARD syndrome (LS) is an autosomal dominant syndrome characterized by multiple lentigines and café‐au‐lait spots, electrocardiographic‐conduction abnormalities, ocular hypertelorism/obstructive cardiomyopathy, pulmonary stenosis, abnormalities of the genitalia in males, retardation of growth, and deafness. LS shares many features with Noonan syndrome (NS), in which lentigines and deafness are usually not present. Molecular studies have shown that LS and NS are allelic disorders, caused by different missense mutations in PTPN11, a gene encoding the protein tyrosine phosphatase SHP‐2 located at chromosome 12q22‐qter. The clinical diagnosis of LS is generally difficult in the first months of life because the distinctive lentigines are generally not present at birth and develop during childhood. From January 2002 to December 2004, we suspected LS clinically in 10 patients admitted to our genetic counseling services in the first 12 months of life. A PTPN11 gene mutation was detected in 8/10 (80%) patients. In one patient without a PTPN11 mutation a subsequent clinical diagnosis of neurofibromatosis type 1 (NF1) was made, following the evaluation of the mother, who had previously undiagnosed classic NF1. The age of LS patients with PTPN11 mutation ranged between 1 and 11 months (mean age ± SD 7.5 ± 3.96 months). Review of the clinical characteristics of patients with LS confirmed by molecular study during the first year of life demonstrates that the diagnosis of LS in the first months of age can be clinically suspected in patients presenting with three main features, that is, characteristic facial features (100%), hypertrophic cardiomyopathy (HCM) (87%), and cafe‐au‐lait spots (75%). Characteristic facial features can be mild or severe, and consist of hypertelorism, downslanting palpebral fissures, ptosis, and dysmorphic ears. The clinical suspicion of LS may be confirmed by molecular screening for PTPN11 mutations. An early diagnosis of the disease is useful for the prospective care of associated medical problems and for precise genetic counseling.