PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects

Multiple lentigines/LEOPARD syndrome (LS) is a rare, autosomal dominant disorder characterized by Lentigines, Electrocardiogram abnormalities, Ocular hypertelorism, Pulmonic valvular stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness. Like the more common Noonan syndrome (NS), LS is caused by germ line missense mutations in PTPN11, encoding the protein-tyrosine phosphatase Shp2. Enzymologic, structural, cell biological, and mouse genetic studies indicate that NS is caused by gain-of-function PTPN11 mutations. Because NS and LS share several features, LS has been viewed as an NS variant. We examined a panel of LS mutants, including the two most common alleles. Surprisingly, we found that in marked contrast to NS, LS mutants are catalytically defective and act as dominant negative mutations that interfere with growth factor/Erk-mitogen-activated protein kinase-mediated signaling. Molecular modeling and biochemical studies suggest that LS mutations contort the Shp2 catalytic domain and result in open, inactive forms of Shp2. Our results establish that the pathogenesis of LS and NS is distinct and suggest that these disorders should be distinguished by mutational analysis rather than clinical presentation.     

Noonan syndrome-associated SHP-2/Ptpn11 mutants enhance SIRPalpha and PZR tyrosyl phosphorylation and promote adhesion-mediated ERK activation

Noonan syndrome (NS) is an autosomal dominant disorder that is associated with multiple developmental abnormalities. Activated mutations of the protein-tyrosine phosphatase, SHP-2/PTPN11, have been reported in approximately 50% of NS cases. Despite being activated, NS-associated SHP-2 mutants require plasma membrane proximity to evoke disease-associated signaling. Here we show that NS-associated SHP-2 mutants induce hypertyrosyl phosphorylation of the transmembrane glycoproteins, SIRPalpha (signal-regulatory protein alpha) and PZR (protein zero-related), resulting in their increased association with NS-associated SHP-2 mutants. NS-associated SHP-2 mutants enhanced SIRPalpha and PZR tyrosyl phosphorylation either by impairing SIRPalpha dephosphorylation or by promoting PZR tyrosyl phosphorylation. Importantly, during embryogenesis in a mouse model of NS, SIRPalpha and PZR were hypertyrosyl-phosphorylated and bound increased levels of the NS-associated SHP-2 mutant. SIRPalpha and PZR have been implicated in extracellular matrix-dependent signaling. Mouse embryonic fibroblasts derived from a mouse model of NS displayed enhanced ERK activation in response to fibronectin plating. Knockdown of SIRPalpha and PZR in these cells attenuated the enhanced activation of ERK following fibronectin plating. Thus, SIRPalpha and PZR serve as scaffolds that facilitate plasma membrane recruitment and signaling of NS-associated SHP-2 mutants.     

Phosphoproteomics-Mediated Identification of Fer Kinase as a Target of Mutant Shp2 in Noonan and LEOPARD Syndrome

Noonan syndrome (NS) and LEOPARD syndrome (LS) cause congenital afflictions such as short stature, hypertelorism and heart defects. More than 50% of NS and almost all of LS cases are caused by activating and inactivating mutations of the phosphatase Shp2, respectively. How these biochemically opposing mutations lead to similar clinical outcomes is not clear. Using zebrafish models of NS and LS and mass spectrometry-based phosphotyrosine proteomics, we identified a down-regulated peptide of Fer kinase in both NS and LS. Further investigation showed a role for Fer during development, where morpholino-based knockdown caused craniofacial defects, heart edema and short stature. During gastrulation, loss of Fer caused convergence and extension defects without affecting cell fate. Moreover, Fer knockdown cooperated with NS and LS, but not wild type Shp2 to induce developmental defects, suggesting a role for Fer in the pathogenesis of both NS and LS.     

Distinct and Overlapping Functions of ptpn11 Genes in Zebrafish Development

The PTPN11 (protein-tyrosine phosphatase, non-receptor type 11) gene encodes SHP2, a cytoplasmic PTP that is essential for vertebrate development. Mutations in PTPN11 are associated with Noonan and LEOPARD syndrome. Human patients with these autosomal dominant disorders display various symptoms, including short stature, craniofacial defects and heart abnormalities. We have used the zebrafish as a model to investigate the role of Shp2 in embryonic development. The zebrafish genome encodes two ptpn11 genes, ptpn11a and ptpn11b. Here, we report that ptpn11a is expressed constitutively and ptpn11b expression is strongly upregulated during development. In addition, the products of both ptpn11 genes, Shp2a and Shp2b, are functional. Target-selected inactivation of ptpn11a and ptpn11b revealed that double homozygous mutants are embryonic lethal at 5–6 days post fertilization (dpf). Ptpn11a-/-ptpn11b-/- embryos showed pleiotropic defects from 4 dpf onwards, including reduced body axis extension and craniofacial defects, which was accompanied by low levels of phosphorylated Erk at 5 dpf. Interestingly, defects in homozygous ptpn11a-/- mutants overlapped with defects in the double mutants albeit they were milder, whereas ptpn11b-/- single mutants did not show detectable developmental defects and were viable and fertile. Ptpn11a-/-ptpn11b-/- mutants were rescued by expression of exogenous ptpn11a and ptpn11b alike, indicating functional redundance of Shp2a and Shp2b. The ptpn11 mutants provide a good basis for further unravelling of the function of Shp2 in vertebrate development.     

Diversity and functional consequences of germline and somatic PTPN11 mutations in human disease

Germline mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome (NS) and the clinically related LEOPARD syndrome (LS), whereas somatic mutations in the same gene contribute to leukemogenesis. On the basis of our previously gathered genetic and biochemical data, we proposed a model that splits NS- and leukemia-associated PTPN11 mutations into two major classes of activating lesions with differential perturbing effects on development and hematopoiesis. To test this model, we investigated further the diversity of germline and somatic PTPN11 mutations, delineated the association of those mutations with disease, characterized biochemically a panel of mutant SHP-2 proteins recurring in NS, LS, and leukemia, and performed molecular dynamics simulations to determine the structural effects of selected mutations. Our results document a strict correlation between the identity of the lesion and disease and demonstrate that NS-causative mutations have less potency for promoting SHP-2 gain of function than do leukemia-associated ones. Furthermore, we show that the recurrent LS-causing Y279C and T468M amino acid substitutions engender loss of SHP-2 catalytic activity, identifying a previously unrecognized behavior for this class of missense PTPN11 mutations.     

Targeted/exome sequencing identified mutations in ten Chinese patients diagnosed with Noonan syndrome and related disorders

Background

Noonan syndrome (NS) and Noonan syndrome with multiple lentigines (NSML) are autosomal dominant developmental disorders. NS and NSML are caused by abnormalities in genes that encode proteins related to the RAS-MAPK pathway, including PTPN11, RAF1, BRAF, and MAP2K. In this study, we diagnosed ten NS or NSML patients via targeted sequencing or whole exome sequencing (TS/WES).

Methods

TS/WES was performed to identify mutations in ten Chinese patients who exhibited the following manifestations: potential facial dysmorphisms, short stature, congenital heart defects, and developmental delay. Sanger sequencing was used to confirm the suspected pathological variants in the patients and their family members.

Results

TS/WES revealed three mutations in the PTPN11 gene, three mutations in RAF1 gene, and four mutations in BRAF gene in the NS and NSML patients who were previously diagnosed based on the abovementioned clinical features. All the identified mutations were determined to be de novo mutations. However, two patients who carried the same mutation in the RAF1 gene presented different clinical features. One patient with multiple lentigines was diagnosed with NSML, while the other patient without lentigines was diagnosed with NS. In addition, a patient who carried a hotspot mutation in the BRAF gene was diagnosed with NS instead of cardiofaciocutaneous syndrome (CFCS).

Conclusions

TS/WES has emerged as a useful tool for definitive diagnosis and accurate genetic counseling of atypical cases. In this study, we analyzed ten Chinese patients diagnosed with NS and related disorders and identified their correspondingPTPN11, RAF1, and BRAF mutations. Among the target genes, BRAF showed the same degree of correlation with NS incidence as that of PTPN11 or RAF1.

     

A suggested role for mitochondria in Noonan syndrome

Noonan syndrome (NS) is an autosomal dominant disorder, and a main feature is congenital heart malformation. About 50% of cases are caused by gain-of-function mutations in the tyrosine phosphatase SHP2/PTPN11, a downstream regulator of ERK/MAPK. Recently it was reported that SHP2 also localizes to the mitochondrial intercristae/intermembrane space (IMS), but the role of SHP2 in mitochondria is unclear. The mitochondrial oxidative phosphorylation (OxPhos) system provides the vast majority of cellular energy and produces reactive oxygen species (ROS). Changes in ROS may interfere with organ development such as that observed in NS patients. Several phosphorylation sites have been found in OxPhos components including cytochrome c oxidase (CcO) and cytochrome c (Cytc), and we hypothesized that OxPhos complexes may be direct or indirect targets of SHP2. We analyzed mitochondrial function using mouse fibroblasts from wild-types, SHP2 knockdowns, and D61G SHP2 mutants leading to constitutively active SHP2, as found in NS patients. Levels of OxPhos complexes were similar except for CcO and Cytc, which were 37% and 28% reduced in the D61G cells. However, CcO activity was significantly increased, as we also found for two lymphoblast cell lines from NS patients with two independent mutations in PTPN11. D61G cells showed lower mitochondrial membrane potential and 30% lower ATP content compared to controls. ROS were significantly increased; aconitase activity, a marker for ROS-induced damage, was decreased; and catalase activity was increased in D61G cells. We propose that decreased energy levels and/or increased ROS may explain, at least in part, some of the clinical features in NS that overlap with children with mitochondrial disorders.     

Mitochondrial DNA haplogroup ‘R’ is associated with Noonan syndrome of South India

Mutations in PTPN11 gene was responsible for ～50% of the Noonan syndrome (NS), however, we did not find any mutation in PTPN11 in any of seven NS patients analysed. Whereas, the complete mtDNA sequencing revealed 146 mutations, of which five, including one heteroplasmic (A11144R; Thr → Ala) non-synonymous mutation, were novel and exclusively observed in NS patients. Interestingly all the seven probands and their maternal relatives were clustered under a major haplogroup R and its novel sub-haplogroups (R7b1b, R30a1, R30c, T2b7, U9a1) exclusive in NS, therefore we strongly suggest that these haplogroups may influence NS in South Indian populations.     

Defects of CRB2 Cause Steroid-Resistant Nephrotic Syndrome

Nephrotic syndrome (NS), the association of gross proteinuria, hypoalbuminaemia, edema, and hyperlipidemia, can be clinically divided into steroid-sensitive (SSNS) and steroid-resistant (SRNS) forms. SRNS regularly progresses to end-stage renal failure. By homozygosity mapping and whole exome sequencing, we here identify recessive mutations in Crumbs homolog 2 (CRB2) in four different families affected by SRNS. Previously, we established a requirement for zebrafish crb2b, a conserved regulator of epithelial polarity, in podocyte morphogenesis. By characterization of a loss-of-function mutation in zebrafish crb2b, we now show that zebrafish crb2b is required for podocyte foot process arborization, slit diaphragm formation, and proper nephrin trafficking. Furthermore, by complementation experiments in zebrafish, we demonstrate that CRB2 mutations result in loss of function and therefore constitute causative mutations leading to NS in humans. These results implicate defects in podocyte apico-basal polarity in the pathogenesis of NS.     

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.     

In-Silico and In-Vitro Analysis of Human SOS1 Protein Causing Noonan Syndrome - A Novel Approach to Explore the Molecular Pathways

AimsPerform in-silico analysis of human SOS1 mutations to elucidate their pathogenic role in Noonan syndrome (NS).BackgroundNS is an autosomal dominant genetic disorder caused by single nucleotide mutation in PTPN11, SOS1, RAF1, and KRAS genes. NS is thought to affect approximately 1 in 1000. NS patients suffer different pathogenic effects depending on the mutations they carry. Analysis of the mutations would be a promising predictor in identifying the pathogenic effect of NS.MethodsWe performed computational analysis of the SOS1 gene to identify the pathogenic nonsynonymous single nucleotide polymorphisms (nsSNPs) th a t cause NS. SOS1 variants were retrieved from the SNP database (dbSNP) and analyzed by in-silico tools I-Mutant, iPTREESTAB, and MutPred to elucidate their structural and functional characteristics.ResultsWe found that 11 nsSNPs of SOS1 that were linked to NS. 3D modeling of the wild-type and the 11 nsSNPs of SOS1 showed that SOS1 interacts with cardiac proteins GATA4, TNNT2, and ACTN2. We also found that GRB2 and HRAS act as intermediate molecules between SOS1 and cardiac proteins. Our in-silico analysis findings were further validated using induced cardiomyocytes (iCMCs) derived from NS patients carrying SOS1 gene variant c.1654A>G (NSiCMCs) and compared to control human skin fibroblast-derived iCMCs (C-iCMCs). Our in vitro data confirmed that the SOS1, GRB2 and HRAS gene expressions as well as the activated ERK protein, were significantly decreased in NS-iCMCs when compared to C-iCMCs.ConclusionThis is the first in-silico and in vitro study demonstrating that 11 nsSNPs of SOS1 play deleterious pathogenic roles in causing NS.     

Paternal germline origin and sex-ratio distortion in transmission of PTPN11 mutations in Noonan syndrome

Germline mutations in PTPN11--the gene encoding the nonreceptor protein tyrosine phosphatase SHP-2--represent a major cause of Noonan syndrome (NS), a developmental disorder characterized by short stature and facial dysmorphism, as well as skeletal, hematologic, and congenital heart defects. Like many autosomal dominant disorders, a significant percentage of NS cases appear to arise from de novo mutations. Here, we investigated the parental origin of de novo PTPN11 lesions and explored the effect of paternal age in NS. By analyzing intronic portions that flank the exonic PTPN11 lesions in 49 sporadic NS cases, we traced the parental origin of mutations in 14 families. Our results showed that all mutations were inherited from the father, despite the fact that no substitution affected a CpG dinucleotide. We also report that advanced paternal age was observed among cohorts of sporadic NS cases with and without PTPN11 mutations and that a significant sex-ratio bias favoring transmission to males was present in subjects with sporadic NS caused by PTPN11 mutations, as well as in families inheriting the disorder.     

Targeted Molecular Sequencing Revealed Allelic Heterogeneity of <Emphasis Type="Italic">BRAF</Emphasis> and <Emphasis Type="Italic">PTPN11</Emphasis> Genes among Arab Noonan Syndrome Patients

Noonan syndrome (NS) is a very rare heterogenous genetic disorder often characterized by short stature, facial dysmorphisms, congenital heart defects and learning disabilities in affected children. In the current study, we sought to discover the disease causal mutations, inherited or de novo, for Noonan Syndrome among Arab patients. We screened the coding regions and splice sites of 10 known RAS/MAP Kinase pathway genes in 17 NS-trios and 100 random healthy volunteers by oilgonucleotide chip testing and Sanger sequencing methods. We found pathogenic heteroallelic de novo mutations in BRAF or PTPN11 gene in 7/17 (41.17%) of NS patients. None of the 200 chromosomes of healthy volunteers had those pathogenic mutations. Genotype-phenotype analysis showed positive correlation between BRAF and PTPN11 gene mutations and classical NS clinical manifestations. Characteristic facies is the major observed clinical manifestation among PTPN11-mutation positive cases (c.236A>G, c.854T>C, c.923A>G), whereas both characteristic facies and ectodermal manifestations are seen as dominant clinical features among BRAF-mutation positive cases (c.730A>C, c.770A>G, c.1406G>A). In addition to genotyping and clinical phenotyping, we performed computational structural analysis to examine the impact of amino acid substitution mutations on the conformation and folding of BRAF and PTPN11 proteins. Our results suggested that BRAF (c.730A>C, c.770A>G, c.1406G>A) and PTPN11 (c.236A>G, c.854T>C, c.923A>G) gene mutations elicits structural and functional alterations at protein level, which would eventually lead to dysregulation of RAS-MAPK signaling cascade, which plays critical a role in cell cycle and senescence. In conclusion, our study suggest that molecular screening of BRAF and PTPN11 genetic mutations in Arab NS patients may assist in deriving competitive outcomes related to clinical phenotyping and disease diagnosis.     

Diverse biochemical properties of Shp2 mutants. Implications for disease phenotypes

Mutations in the Src homology 2 (SH2)-containing protein-tyrosine phosphatase Shp2 (PTPN11) underlie half of the cases of the autosomal dominant genetic disorder Noonan syndrome, and somatic Shp2 mutations are found in several hematologic and solid malignancies. Earlier studies of small numbers of mutants suggested that disease-associated mutations cause constitutive (SH2 binding-independent) activation and that cancer-associated mutants are more active than those associated with Noonan syndrome. We have characterized a larger panel of Shp2 mutants and find that this "activity-centric" model cannot explain the behaviors of all pathogenic Shp2 mutations. Instead, enzymatic, structural, and mathematical modeling analyses show that these mutants can affect basal activation, SH2 domain-phosphopeptide affinity, and/or substrate specificity to varying degrees. Furthermore, there is no absolute correlation between the mutants' extents of basal activation and the diseases they induce. We propose that activated mutants of Shp2 modulate signaling from specific stimuli to a subset of effectors and provide a theoretical framework for understanding the complex relationship between Shp2 activation, intracellular signaling, and pathology.     

Shp2 Knockdown and Noonan/LEOPARD Mutant Shp2–Induced Gastrulation Defects

Shp2 is a cytoplasmic protein-tyrosine phosphatase that is essential for normal development. Activating and inactivating mutations have been identified in humans to cause the related Noonan and LEOPARD syndromes, respectively. The cell biological cause of these syndromes remains to be determined. We have used the zebrafish to assess the role of Shp2 in early development. Here, we report that morpholino-mediated knockdown of Shp2 in zebrafish resulted in defects during gastrulation. Cell tracing experiments demonstrated that Shp2 knockdown induced defects in convergence and extension cell movements. In situ hybridization using a panel of markers indicated that cell fate was not affected by Shp2 knock down. The Shp2 knockdown–induced defects were rescued by active Fyn and Yes and by active RhoA. We generated mutants of Shp2 with mutations that were identified in human patients with Noonan or LEOPARD Syndrome and established that Noonan Shp2 was activated and LEOPARD Shp2 lacked catalytic protein-tyrosine phosphatase activity. Expression of Noonan or LEOPARD mutant Shp2 in zebrafish embryos induced convergence and extension cell movement defects without affecting cell fate. Moreover, these embryos displayed craniofacial and cardiac defects, reminiscent of human symptoms. Noonan and LEOPARD mutant Shp2s were not additive nor synergistic, consistent with the mutant Shp2s having activating and inactivating roles in the same signaling pathway. Our results demonstrate that Shp2 is required for normal convergence and extension cell movements during gastrulation and that Src family kinases and RhoA were downstream of Shp2. Expression of Noonan or LEOPARD Shp2 phenocopied the craniofacial and cardiac defects of human patients. The finding that defective Shp2 signaling induced cell movement defects as early as gastrulation may have implications for the monitoring and diagnosis of Noonan and LEOPARD syndrome.     

Protein-tyrosine Phosphatase Shp2 Positively Regulates Macrophage Oxidative Burst

Macrophages are vital to innate immunity and express pattern recognition receptors and integrins for the rapid detection of invading pathogens. Stimulation of Dectin-1 and complement receptor 3 (CR3) activates Erk- and Akt-dependent production of reactive oxygen species (ROS). Shp2, a protein-tyrosine phosphatase encoded by Ptpn11, promotes activation of Ras-Erk and PI3K-Akt and is crucial for hematopoietic cell function; however, no studies have examined Shp2 function in particulate-stimulated ROS production. Maximal Dectin-1-stimulated ROS production corresponded kinetically to maximal Shp2 and Erk phosphorylation. Bone marrow-derived macrophages (BMMs) from mice with a conditionally deleted allele of Ptpn11 (Shp2^flox/flox;Mx1Cre+) produced significantly lower ROS levels compared with control BMMs. Although YFP-tagged phosphatase dead Shp2-C463A was strongly recruited to the early phagosome, its expression inhibited Dectin-1- and CR3-stimulated phospho-Erk and ROS levels, placing Shp2 phosphatase function and Erk activation upstream of ROS production. Further, BMMs expressing gain of function Shp2-D61Y or Shp2-E76K and peritoneal exudate macrophages from Shp2D61Y/+;Mx1Cre+ mice produced significantly elevated levels of Dectin-1- and CR3-stimulated ROS, which was reduced by pharmacologic inhibition of Erk. SIRPα (signal regulatory protein α) is a myeloid inhibitory immunoreceptor that requires tyrosine phosphorylation to exert its inhibitory effect. YFP-Shp2C463A-expressing cells have elevated phospho-SIRPα levels and an increased Shp2-SIRPα interaction compared with YFP-WT Shp2-expressing cells. Collectively, these findings indicate that Shp2 phosphatase function positively regulates Dectin-1- and CR3-stimulated ROS production in macrophages by dephosphorylating and thus mitigating the inhibitory function of SIRPα and by promoting Erk activation.     

Mutations in EMP2 Cause Childhood-Onset Nephrotic Syndrome

Nephrotic syndrome (NS) is a genetically heterogeneous group of diseases that are divided into steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS). SRNS inevitably leads to end-stage kidney disease, and no curative treatment is available. To date, mutations in more than 24 genes have been described in Mendelian forms of SRNS; however, no Mendelian form of SSNS has been described. To identify a genetic form of SSNS, we performed homozygosity mapping, whole-exome sequencing, and multiplex PCR followed by next-generation sequencing. We thereby detected biallelic mutations in EMP2 (epithelial membrane protein 2) in four individuals from three unrelated families affected by SRNS or SSNS. We showed that EMP2 exclusively localized to glomeruli in the kidney. Knockdown of emp2 in zebrafish resulted in pericardial effusion, supporting the pathogenic role of mutated EMP2 in human NS. At the cellular level, we showed that knockdown of EMP2 in podocytes and endothelial cells resulted in an increased amount of CAVEOLIN-1 and decreased cell proliferation. Our data therefore identify EMP2 mutations as causing a recessive Mendelian form of SSNS.     

PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity

Noonan syndrome (NS) is a developmental disorder characterized by facial dysmorphia, short stature, cardiac defects, and skeletal malformations. We recently demonstrated that mutations in PTPN11, the gene encoding the non-receptor-type protein tyrosine phosphatase SHP-2 (src homology region 2-domain phosphatase-2), cause NS, accounting for approximately 50% of cases of this genetically heterogeneous disorder in a small cohort. All mutations were missense changes and clustered at the interacting portions of the amino-terminal src-homology 2 (N-SH2) and protein tyrosine phosphatase (PTP) domains. A gain of function was postulated as a mechanism for the disease. Here, we report the spectrum and distribution of PTPN11 mutations in a large, well-characterized cohort with NS. Mutations were found in 54 of 119 (45%) unrelated individuals with sporadic or familial NS. There was a significantly higher prevalence of mutations among familial cases than among sporadic ones. All defects were missense, and several were recurrent. The vast majority of mutations altered amino acid residues located in or around the interacting surfaces of the N-SH2 and PTP domains, but defects also affected residues in the C-SH2 domain, as well as in the peptide linking the N-SH2 and C-SH2 domains. Genotype-phenotype analysis revealed that pulmonic stenosis was more prevalent among the group of subjects with NS who had PTPN11 mutations than it was in the group without them (70.6% vs. 46.2%; P<.01), whereas hypertrophic cardiomyopathy was less prevalent among those with PTPN11 mutations (5.9% vs. 26.2%; P<.005). The prevalence of other congenital heart malformations, short stature, pectus deformity, cryptorchidism, and developmental delay did not differ between the two groups. A PTPN11 mutation was identified in a family inheriting Noonan-like/multiple giant-cell lesion syndrome, extending the phenotypic range of disease associated with this gene.