Activating PTPN11 mutations play a minor role in pediatric and adult solid tumors

The PTPN11 gene encodes SHP-2, a widely expressed cytoplasmic protein tyrosine phosphatase functioning as a signaling transducer. Germ-line PTPN11 mutations cause Noonan syndrome (NS), a developmental disorder characterized by an increased risk of malignancies. Recently, a novel class of activating mutations in PTPN11 has been documented as a somatic event in a heterogeneous group of leukemias. Because of the relatively higher prevalence of certain solid tumors in children with NS and the positive modulatory function of SHP-2 in RAS signaling, a wider role for activating PTPN11 mutations in cancer has been hypothesized. Here, we screened a number of solid tumors, including those documented in NS or in which deregulated RAS signaling occurs at significant frequency, for PTPN11 mutations. No disease-associated mutation was identified in rhabdomyosarcoma (n = 13), neuroblastoma (n = 32), melanoma (n = 50), thyroid (n = 85), and colon (n = 48) tumors; a novel missense change, promoting an increased basal phosphatase activity of SHP-2, was observed in one glioma specimen. Our data document that deregulated SHP-2 function does not represent a major molecular event in pediatric and adult tumors, further supporting our previous evidence indicating that the oncogenic role of PTPN11 mutations is cell-context specific.     

Genetic heterogeneity in LEOPARD syndrome: two families with no mutations in PTPN11

LEOPARD syndrome (lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness) is an autosomal dominant condition. The main clinical features include multiple lentigines, cardiovascular defects, and facial anomalies, some of which are shared with Noonan syndrome (NS). Recent reports have shown that LEOPARD syndrome can be caused by mutations in PTPN11, the gene in which mutations can produce NS. Here we report the findings of mutation screening and linkage analysis of PTPN11 in three families with LEOPARD syndrome. We identified a novel mutation in one family. The mutation (1529A>C) substitutes proline for glutamine at amino acid 510 (Gln510Pro). No variations in sequence were observed in the other two families, and negative LOD scores excluded linkage to the PTPN11 locus, showing that LEOPARD syndrome is genetically heterogeneous.     

Clinical and molecular characterization of 40 patients with Noonan syndrome

Noonan syndrome (NS, OMIM 163950) is an autosomal dominant disorder, with a prevalence at birth of 1:1000–1:2500 live births, characterized by short stature, facial and skeletal dysmorphisms, cardiovascular defects and haematological anomalies. Missense mutations of PTPN11 gene account for approximately 50% of NS cases, while molecular lesions of other genes of the RAS/MAPK pathway – KRAS, SOS1 and RAF1 – play a minor role in the molecular pathogenesis of the disease. Forty patients were enrolled in the study with a PTPN11 mutation detection rate of 31.5%, including a novel missense mutation, Phe285Ile, in a familial case with high intrafamilial phenotypic variability. All patients negative for PTPN11 mutations were further screened for mutations of the KRAS, SOS1, and RAF1 genes, revealing a Thr266Lys substitution in SOS1 in a single patient, a newborn with a subtle phenotype, characterized by facial dysmorphisms and a mild pulmonic stenosis.     

Genotypic and phenotypic characterization of Noonan syndrome: new data and review of the literature

Noonan syndrome (NS) is an autosomal dominant disorder, characterized by short stature, minor facial anomalies, and congenital heart defects. In approximately 50% of cases the condition is caused by missense mutations in the PTPN11 gene on chromosome 12, resulting in a gain of function of the protein SHP-2. In this study, PTPN11 mutation analysis was performed in 170 NS patients. In 76 (45%) of them a mutation was identified. We report on the distribution of these mutations, as well as on genotype-phenotype relationships. The benefit of the NS scoring system developed by van der Burgt et al. [(1994); Am J Med Genet 53:187-191] is shown, among physicians who consequently based their diagnosis on the NS scoring system the percentage mutation positive subjects was 54%, whereas this percentage was only 39% among physicians who made less use of the scoring system. In two patients with some uncommon manifestations mutations were found in the C-SH2 domain, a region in which defects are not often identified in NS. A trend was observed in patients carrying the 922A --> G change (Asn308Asp) receiving normal education. In one patient with NS and mild juvenile myelomonocytic leukemia (JMML) the mutation 218C --> T (Thr73Ile) was found. This confirms previous findings indicating that individuals with NS with specific mutations in PTPN11 are at risk of developing JMML.     

Noonan syndrome-associated SHP2/PTPN11 mutants cause EGF-dependent prolonged GAB1 binding and sustained ERK2/MAPK1 activation

Noonan syndrome is a developmental disorder with dysmorphic facies, short stature, cardiac defects, and skeletal anomalies, which can be caused by missense PTPN11 mutations. PTPN11 encodes Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2 or SHP-2), a protein tyrosine phosphatase that acts in signal transduction downstream to growth factor, hormone, and cytokine receptors. We compared the functional effects of three Noonan syndrome-causative PTPN11 mutations on SHP2's phosphatase activity, interaction with a binding partner, and signal transduction. All SHP2 mutants had significantly increased basal phosphatase activity compared to wild type, but that activity varied significantly between mutants and was further increased after epidermal growth factor stimulation. Cells expressing SHP2 mutants had prolonged extracellular signal-regulated kinase 2 activation, which was ligand-dependent. Binding of SHP2 mutants to Grb2-associated binder-1 was increased and sustained, and tyrosine phosphorylation of both proteins was prolonged. Coexpression of Grb2-associated binder-1-FF, which lacks SHP2 binding motifs, blocked the epidermal growth factor-mediated increase in SHP2's phosphatase activity and resulted in a dramatic reduction of extracellular signal-regulated kinase 2 activation. Taken together, these results document that Noonan syndrome-associated PTPN11 mutations increase SHP2's basal phosphatase activity, with greater activation when residues directly involved in binding at the interface between the N-terminal Src homology 2 and protein tyrosine phosphatase domains are altered. The SHP2 mutants prolonged signal flux through the RAS/mitogen-activated protein kinase (ERK2/MAPK1) pathway in a ligand-dependent manner that required docking through Grb2-associated binder-1 (GAB1), leading to increased cell proliferation.     

Mild variable Noonan syndrome in a family with a novel PTPN11 mutation

Noonan syndrome (OMIM 163950) is a common genetic condition with variable clinical expression and genetic heterogeneity. About half of the cases can be accounted to activating mutations in the PTPN11 gene encoding SHP-2. We report on a family with mild, variable expression of Noonan syndrome in five individuals. Clinical manifestations included short stature, craniofacial anomalies and thorax deformity, but none of the affected family members had a heart defect. Sequencing of the entire coding region of PTPN11 revealed a novel mutation c.1226G-->C in exon 11 predicting the amino acid exchange G409A. This mutation is not located in the previously known mutation clusters. Our observation and the recent report of a mutation affecting a neighbouring residue (T411M) in a family with a variable phenotype suggest that mutations in this particular region of SHP-2 may have effects on the protein that differ from those of the classical mutations.     

Pathogenic PTPN11 variants involving the poly‐glutamine Gln255‐Gln256‐Gln257 stretch highlight the relevance of helix B in SHP2's functional regulation

Germline PTPN11 mutations cause Noonan syndrome (NS), the most common disorder among RASopathies. PTPN11 encodes SHP2, a protein tyrosine‐phosphatase controlling signaling through the RAS‐MAPK and PI3K‐AKT pathways. Generally, NS‐causing PTPN11 mutations are missense changes destabilizing the inactive conformation of the protein or enhancing its binding to signaling partners. Here, we report on two PTPN11 variants resulting in the deletion or duplication of one of three adjacent glutamine residues (Gln²⁵⁵‐to‐Gln²⁵⁷). While p.(Gln257dup) caused a typical NS phenotype in carriers of a first family, p.(Gln257del) had incomplete penetrance in a second family. Missense mutations involving Gln²⁵⁶ had previously been reported in NS. This poly‐glutamine stretch is located on helix B of the PTP domain, a region involved in stabilizing SHP2 in its autoinhibited state. Molecular dynamics simulations predicted that changes affecting this motif perturb the SHP2's catalytically inactive conformation and/or substrate recognition. Biochemical data showed that duplication and deletion of Gln²⁵⁷ variably enhance SHP2's catalytic activity, while missense changes involving Gln²⁵⁶ affect substrate specificity. Expression of mutants in HEK293T cells documented their activating role on MAPK signaling, uncoupling catalytic activity and modulation of intracellular signaling. These findings further document the relevance of helix B in the regulation of SHP2's function.     

A 3-bp deletion mutation of PTPN11 in an infant with severe Noonan syndrome including hydrops fetalis and juvenile myelomonocytic leukemia

A de novo 3-bp deletion (179-181delGTG) was identified at exon 3 of the PTPN11 gene in a female infant with severe Noonan phenotype including hydrops fetalis and juvenile myelomonocytic leukemia. Since the 3-bp deletion is predicted to result in loss of the 60th glycine in the N-SH2 domain that is directly involved in the intramolecular interaction between the N-SH2 and the PTP domains of the PTPN11 protein, this mutation would disrupt the N-SH2/PTP binding in the absence of a phosphopeptide, leading to an excessive phosphatase activity. The results expand the spectrum of PTPN11 mutations in Noonan syndrome (NS), and suggest that a PTPN11 mutation leads to a wide range of clinical features of Noonan syndrome.     

PTPN11 mutation in a large family with Noonan syndrome and dizygous twinning

Noonan syndrome (NS, MIM 163950) is an autosomal dominant condition characterised by facial dysmorphy, congenital cardiac defects and short stature. Recently missense mutations in PTPN11, the gene encoding the nonreceptor protein tyrosine phosphatase SHP-2 on 12q24, were identified in 50% of analysed Noonan cases. A large four-generation Belgian family with NS and some features suggestive of cardio-facio-cutaneous syndrome (CFC) was previously used to fine map the Noonan syndrome candidate region to a 5 cM region in 12q24. We now report the identification of a mutation (Gln79Arg) in the PTPN11 gene in this large family. In D. melanogaster and C. elegans the PTPN11 gene has been implicated in oogenesis. In this family two affected females had dizygous twins. This suggests that PTPN11 might also be involved in oogenesis and twinning in humans.     

Phosphatase-defective LEOPARD syndrome mutations in PTPN11 gene have gain-of-function effects during Drosophila development

Missense mutations in the PTPN11 gene, which encodes the proteintyrosine phosphatase SHP-2, cause clinically similar but distinctivedisorders, LEOPARD (LS) and Noonan (NS) syndromes. The LS isan autosomal dominant disorder with pleomorphic developmentalabnormalities including lentigines, cardiac defects, short statureand deafness. Biochemical analyses indicated that LS allelesengender loss-of-function (LOF) effects, while NS mutationsresult in gain-of-function (GOF). These biochemical findingslead to an enigma that how PTPN11 mutations with opposite effectson function result in disorders that are so similar. To studythe developmental effects of the commonest LS PTPN11 alleles(Y279C and T468M), we generated LS transgenic fruitflies usingcorkscrew (csw), the Drosophila orthologue of PTPN11. Ubiquitousexpression of the LS csw mutant alleles resulted in ectopicwing veins and, for the Y279C allele, rough eyes with increasedR7 photoreceptor numbers. These were GOF phenotypes mediatedby increased RAS/MAPK signaling and requiring the LS mutant’sresidual phosphatase activity. Our findings provide the firstevidence that LS mutant alleles have GOF developmental effectsdespite reduced phosphatase activity, providing a rationalefor how PTPN11 mutations with GOF and LOF produce similar butdistinctive syndromes.     

Mutations of the PTPN11 and RAS genes in rhabdomyosarcoma and pediatric hematological malignancies

PTPN11 has been identified as a causative gene in Noonan syndrome (NS), responsible for about 50% of cases of NS. Given the association between NS and an increased risk of some malignancies, notably leukemia and probably some solid tumors including neuroblastoma (NB) and rhabdomyosarcoma (RMS), recent studies have reported that gain-of-function somatic mutations in PTPN11 occur in some hematological malignancies, especially de novo juvenile myelomonocytic leukemia (JMML) and in some solid tumors such as NB, although at a low frequency. In a screen for mutations of PTPN11 in 7 cell lines and 30 fresh tumors of RMS and in 25 cell lines and 40 fresh tumors of NB, we identified a missense mutation (A72T) in an embryonal RMS patient. In the RMS samples, we also detected mutations of NRAS in 1 cell line and 1 patient; both mutations were in embryonal RMSs and had no PTPN11 mutations. No mutations of PTPN11 were detected in NB. In 95 leukemia cell lines and 261 fresh leukemia samples including 22 JMMLs, 9 kinds of missense mutations were detected in 17 leukemia samples, which included 11 (50.0%) mutations in JMML samples and lower frequencies in other hematological malignancies. Furthermore, we identified 4 (18.2%) NRAS mutations and 1 (4.5%) KRAS mutation in 5 JMML samples, 1 of which had a concomitant PTPN11 mutation. Our data suggest that mutations of PTPN11 as well as RAS play a role in the pathogenesis of not only myeloid hematological malignancies but also a subset of RMS malignancies.     

Cancer risk in patients with Noonan syndrome carrying a PTPN11 mutation

Noonan syndrome (NS) is characterized by short stature, facial dysmorphisms and congenital heart defects. PTPN11 mutations are the most common cause of NS. Patients with NS have a predisposition for leukemia and certain solid tumors. Data on the incidence of malignancies in NS are lacking. Our objective was to estimate the cancer risk and spectrum in patients with NS carrying a PTPN11 mutation. In addition, we have investigated whether specific PTPN11 mutations result in an increased malignancy risk. We have performed a cohort study among 297 Dutch NS patients with a PTPN11 mutation (mean age 18 years). The cancer histories were collected from the referral forms for DNA diagnostics, and by consulting the Dutch national registry of pathology and the Netherlands Cancer Registry. The reported frequencies of cancer among NS patients were compared with the expected frequencies using population-based incidence rates. In total, 12 patients with NS developed a malignancy, providing a cumulative risk for developing cancer of 23% (95% confidence interval (CI), 8-38%) up to age 55 years, which represents a 3.5-fold (95% CI, 2.0-5.9) increased risk compared with that in the general population. Hematological malignancies occurred most frequently. Two malignancies, not previously observed in NS, were found: a malignant mastocytosis and malignant epithelioid angiosarcoma. No correlation was found between specific PTPN11 mutations and cancer occurrence. In conclusion, this study provides first evidence of an increased risk of cancer in patients with NS and a PTPN11 mutation, compared with that in the general population. Our data do not warrant specific cancer surveillance.     

Exclusion of PTPN11 mutations in Costello syndrome: further evidence for distinct genetic etiologies for Noonan,cardio-facio-cutaneous and Costello syndromes

Costello syndrome (CS) is a rare, multiple congenital anomaly syndrome with characteristic dysmorphic features, cardiac anomalies and a tendency to develop certain cancers. Phenotypically there is some overlap with other genetic disorders, notably cardio-facio-cutaneous (CFC) syndrome and Noonan syndrome (NS), suggesting that these syndromes may be allelic. We recently identified PTPN11, which encodes the non-receptor protein tyrosine phosphatase, SHP-2, as a major NS disease gene. In this report, we screened a cohort of 27 patients, with the clinical diagnosis of CS, for PTPN11 mutations using denaturing high performance liquid chromatography analysis. No mutations of the PTPN11 gene were found in the CS patients. Common polymorphisms in introns 6 and 7 and exon 8 were identified in four individuals. With our previous exclusion of PTPN11 mutations in CFC syndrome, these data suggest distinct genetic etiologies for Noonan, CFC and Costello syndromes.     

Germinal mosaicism in Noonan syndrome: A family with two affected siblings of normal parents

Noonan syndrome (NS; OMIM 163950) is an autosomal dominant disorder with variable clinical expression and genetic heterogeneity. Clinical manifestations include characteristic facial features, short stature, and cardiac anomalies. Mutations in protein-tyrosine phosphatase, non-receptor-type 11 (PTPN11), encoding SHP-2, account for about half of NS patients. We report on a Moroccan family with two children with NS and apparently unaffected parents. The molecular studies showed the heterozygous mutation c.922A>G of PTPN11 gene in the two affected sibs. Neither the parents, nor the oldest brother carries this mutation in hematologic cells. The mutation was also absent in buccal epithelial cells and fingernails of both parents. We believe this is the first report of germ cell mosaicism in NS and suggest an empirical risk for recurrence of that is less than 1%.     

PTPN11 mutations in Noonan syndrome type I: detection of recurrent mutations in exons 3 and 13

We surveyed 16 subjects with the clinical diagnosis of Noonan Syndrome (NS1) from 12 families and their relevant family members for mutations in PTPN11/SHP2 using direct DNA sequencing. We found three different mutations among five families. Two unrelated subjects shared the same de novo missense substitution in exon 13 (S502T); an additional two unrelated families had a mutation in exon 3 (Y63C); and one subject had the amino acid substitution Y62D, also in exon 3. None of the three mutations were present in ethnically matched controls. In the mature protein model, the exon 3 mutants and the exon 13 mutant amino acids cluster at the interface between the N' SH2 domain and the phosphatase catalytic domain. Six of eight subjects with PTPN11/SHP2 mutations had pulmonary valve stenosis while no mutations were identified in those subjects (N = 4) with hypertrophic cardiomyopathy. An additional four subjects with possible Noonan syndrome were evaluated, but no mutations in PTPN11/SHP2 were identified. These results confirm that mutations in PTPN11/SHP2 underlie a common form of Noonan syndrome, and that the disease exhibits both allelic and locus heterogeneity. The observation of recurrent mutations supports the hypothesis that a special class of gain-of-function mutations in SHP2 give rise to Noonan syndrome.     

Structural,Functional, and Clinical Characterization of a Novel PTPN11 Mutation Cluster Underlying Noonan Syndrome

Germline mutations in PTPN11, the gene encoding the Src‐homology 2 (SH2) domain‐containing protein tyrosine phosphatase (SHP2), cause Noonan syndrome (NS), a relatively common, clinically variable, multisystem disorder. Here, we report on the identification of five different PTPN11 missense changes affecting residues Leu²⁶¹, Leu²⁶², and Arg²⁶⁵ in 16 unrelated individuals with clinical diagnosis of NS or with features suggestive for this disorder, specifying a novel disease‐causing mutation cluster. Expression of the mutant proteins in HEK293T cells documented their activating role on MAPK signaling. Structural data predicted a gain‐of‐function role of substitutions at residues Leu²⁶² and Arg²⁶⁵ exerted by disruption of the N‐SH2/PTP autoinhibitory interaction. Molecular dynamics simulations suggested a more complex behavior for changes affecting Leu²⁶¹, with possible impact on SHP2's catalytic activity/selectivity and proper interaction of the PTP domain with the regulatory SH2 domains. Consistent with that, biochemical data indicated that substitutions at codons 262 and 265 increased the catalytic activity of the phosphatase, while those affecting codon 261 were only moderately activating but impacted substrate specificity. Remarkably, these mutations underlie a relatively mild form of NS characterized by low prevalence of cardiac defects, short stature, and cognitive and behavioral issues, as well as less evident typical facial features.     

A novel PTPN11 mutation in LEOPARD syndrome

PTPN11 gene mutations are common to both patients with Noonan (NS) and LEOPARD syndrome (LS). So far only two recurrent mutations have been identified in LS patients by different research groups, i.e., Tyr279Cys and Thr468Met. In this work we describe the third PTPN11 mutation that has been found in a single LS patient. The mutation (c.1517A>C) substitutes a proline for a glutamine at amino acid 506 (Gln506Pro) in the phosphatase domain (PTP) of the PTPN11 peptide SHP2. This region is a mutation hotspot. Changes at amino acids 501 to 504 cause NS. Gln506Pro is predicted, by modeling analysis, to seriously disrupt the normal contacts between the regulating N-SH2 and the active PTP domains, leading to hyperactivity of the phosphatase. This report demonstrates that rarer mutations other than Tyr279Cys and Thr468Met can be found in LS patients and the need of screening the whole gene in those negative for the commonest mutations.     

Spectrum of mutations in PTPN11 and genotype-phenotype correlation in 96 patients with Noonan syndrome and five patients with cardio-facio-cutaneous syndrome

Noonan syndrome (NS) is a relatively common, but genetically heterogeneous autosomal dominant malformation syndrome. Characteristic features are proportionate short stature, dysmorphic face, and congenital heart defects. Only recently, a gene involved in NS could be identified. It encodes the non-receptor protein tyrosine phosphatase SHP-2, which is an important molecule in several intracellular signal transduction pathways that control diverse developmental processes, most importantly cardiac semilunar valvulogenesis. We have screened this gene for mutations in 96 familial and sporadic, well-characterised NS patients and identified 15 different missense mutations in a total of 32 patients (33%), including 23 index patients. Most changes clustered in one exon which encodes parts of the N-SH2 domain. Five of the mutations were recurrent. Interestingly, no mutations in the PTPN11 gene were detected in five additional patients with cardio-facio-cutaneous (CFC) syndrome, which shows clinical similarities to NS.     

Transgenic Drosophila models of Noonan syndrome causing PTPN11 gain-of-function mutations

Mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase SHP-2, causes Noonan syndrome (NS), an autosomal dominant disorder with pleomorphic developmental abnormalities. Certain germline and somatic PTPN11 mutations cause leukemias. Mutations have gain-of-function (GOF) effects with the commonest NS allele, N308D, being weaker than the leukemia-causing mutations. To study the effects of disease-associated PTPN11 alleles, we generated transgenic fruitflies with GAL4-inducible expression of wild-type or mutant csw, the Drosophila orthologue of PTPN11. All three transgenic mutant CSWs rescued a hypomorphic csw allele's eye phenotype, documenting activity. Ubiquitous expression of two strong csw mutant alleles were lethal, but did not perturb development from some CSW-dependent receptor tyrosine kinase pathways. Ubiquitous expression of the weaker N308D allele caused ectopic wing veins, identical to the EGFR GOF phenotype. Epistatic analyses established that csw(N308D)'s ectopic wing vein phenotype required intact EGF ligand and receptor, and that this transgene interacted genetically with Notch, DPP and JAK/STAT signaling. Expression of the mutant csw transgenes increased RAS-MAP kinase activation, which was necessary but not sufficient for transducing their phenotypes. The findings from these fly models provided hypotheses testable in mammalian models, in which these signaling cassettes are largely conserved. In addition, these fly models can be used for sensitized screens to identify novel interacting genes as well as for high-throughput screening of therapeutic compounds for NS and PTPN11-related cancers.     

Mutation screening of the PTPN11 gene in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiac disease and a major cause of sudden death. It is an autosomal dominant disorder predominantly caused by mutations in genes encoding for sarcomeric proteins. Only 50-60% of HCM probands have mutations in known genes suggesting the presence of additional disease genes. Noonan and LEOPARD syndromes are characterised by multiple dysmorphia and cardiac defects with HCM present in approximately 20% of cases. Both syndromes are caused by mutations in the PTPN11 gene which codes for the protein tyrosine phosphatase SHP-2. It is suspected but unproven that the cardiac phenotype may predominate or even be present in isolation. In order to determine possible involvement of this gene in the pathogenesis of HCM, we performed mutation screening of the PTPN11 coding region in 250 selected HCM probands (200 patients without mutations in sarcomeric genes and 50 with identified mutations). No mutations in PTPN11 were identified. Our data suggests that mutations in the PTPN11 gene are not a cause of HCM in the absence of Noonan/LEOPARD syndromes.