ClinVar Genomic variation as it relates to human health

The c.417G>C (p.Glu139Asp) variant in PTPN11 has been reported as a confirmed de novo occurrence in at least 2 patients and at least 5 other independent occurrences in patients with clinical features of a RASopathy (PS2_VeryStrong; PS4; GeneDx, Baylor, BC Children's internal data; SCV000057396.11, SCV000196664.1, SCV000803709.1; GTR Lab ID: 26957, 1006, 249401 PMID: 22315187, 17020470, 11992261). This variant was absent from large population studies (PM2; ExAC, http://exac.broadinstitute.org). In vitro functional studies provide some evidence that the p.Glu139Asp variant may impact protein function (PS3; PMID: 23584145, 15987685, 18372317). The variant is located in the PTPN11 gene, which has been defined by the ClinGen RASopathy Expert Panel as a gene with a low rate of benign missense variants and pathogenic missense variants are common (PP2; PMID: 29493581). Computational prediction tools and conservation analysis suggest that the c.417G>C (p.Glu139Asp) variant may impact the protein (PP3). In summary, this variant meets criteria to be classified as pathogenic for RASopathies in an autosomal dominant manner. Rasopathy-specific ACMG/AMP criteria applied (PMID:29493581): PS2_VeryStrong, PS3, PS4, PM2, PP2, PP3.

Variant summary: The PTPN11 c.417G>C (p.Glu139Asp) variant causes a missense change involving the alteration of a conserved nucleotide located in the SH2 domain (IPR000980) (InterPro). 3/4 in silico tools predict a damaging outcome for this variant (SNPsandGO not captured due to low reliability index). Functional studies demonstrated the SHP2-D139E mutant to exhibit dramatically enhanced catalytic activation (3.5-fold), significant increase in phosphopeptide binding affinity of PTPN11, and approximately 4-fold increase in phosphatase activity after stimulation (Martinelli_2008, Tartaglia_2006). These findings from functional studies further corroborate the established molecular mechanism of disease (Gain of Function) attributed to pathogenic variants in the PTPN11 gene. The variant is absent in the control population datasets of ExAC in 121798 control chromosomes. This variant was reported in multiple patients with Noonan syndrome (Tartaglia_2006, Bertola_2006, Musante_2003). In addition, multiple clinical diagnostic laboratories/reputable databases classified this variant as pathogenic. Taken together, this variant is classified as pathogenic.

The p.Glu139Asp variant in PTPN11 has been identified in many individuals with c linical features of Noonan syndrome, one individual with clinical features of No onan syndrome and acute lymphoblastic leukemia, and one individual with juvenile myelomonocytic leukemia (Ko 2008, Karow 2007, Tartaglia 2002, Chan 2006, Loh 20 04, Bertola 2006, Hung 2007, Lo 2009, LMM unpublished data). This variant has be en reported to have segregated in familial cases and to have occurred de novo as well. In summary, this variant meets our criteria to be classified as pathogeni c. ACMG/AMP criteria applied: PS4, PM2, PM6, PP2, PP1.

This variant has been previously reported as a de novo or heterozygous change in patients with PTPN11-related disorders (PMID: 11992261, 22315187, 19020799, 17339163, 32164556, 28363362). Functional studies have reported that the variant alters the specificity of the C-SH2 domain ultimately leading to functional dysregulation and increased PTPN11 activation (PMID: 18372317, 23584145). It is absent from the gnomAD population database and thus is presumed to be rare. The c.417G>C (p.Glu139Asp) variant affects a highly conserved amino acid and is predicted by multiple in silico tools to have a deleterious effect on protein function. Analysis of the parental samples was negative for the variant, indicating this variant likely occurred as a de novo event. Based on the available evidence, the c.417G>C (p.Glu139Asp) variant is classified as Pathogenic.

Published functional studies demonstrate the p.(E139D) variant alters the specificity of the C-SH2 domain, making it similar to that of the N-SH2 domain, ultimately leading to functional dysregulation (Keilhack et al., 2005; Martinelli et al., 2008); Not observed in large population cohorts (gnomAD); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; Missense variants in this gene are often considered pathogenic (HGMD); This variant is associated with the following publications: (PMID: 11992261, 23584145, 26377682, 26855057, 23321623, 20308328, 24628801, 18372317, 25585602, 12634870, 16358218, 24072241, 15710330, 22315187, 24803665, 28363362, 26918529, 21407260, 19020799, 17339163, 29500832, 21901340, 30293248, 30417923, 30050098, 30410095, 29907801, 31219622, 31560489, 32164556, 32581362, 32978145, 33619735, 34134972, 29493581, 15987685)

The PTPN11 c.417G>C variant is predicted to result in the amino acid substitution p.Glu139Asp. This variant has been reported in multiple individuals with Noonan syndrome (see for example Tartaglia et al. 2002. PubMed ID: 11992261). At PreventionGenetics, we previously detected it in several other patients with Noonan spectrum disorders. Functional studies demonstrate that this variant results in increased release of phosphate compared to wild type, consistent with a gain-of-function mechanism, resulting in hyperactivation of the RAS pathway (Keilhack et al. 2005. PubMed ID: 15987685; Martinelli et al. 2008. PubMed ID: 18372317; Edouard. 2010. PubMed ID: 20308328). This variant has not been reported in a large population database (http://gnomad.broadinstitute.org), indicating this variant is rare. This variant has been interpreted as pathogenic by multiple clinical laboratories in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/variation/40513/). Additionally, a different nucleotide substitution (c.417G>T) resulting in the same amino acid substitution has been reported in multiple individuals with Noonan syndrome (Human Gene Mutation Database; http://www.hgmd.cf.ac.uk/). Based on the available evidence, we consider the c.417G>C (p.Glu139Asp) variant to be pathogenic.

PTPN11: PS1, PS2, PM2, PS4:Moderate, PP3, PS3:Supporting

PS3, PS4, PM2, PP1, PP5

The variant has been previously reported as de novoo in at least four similarly affected unrelated individuals (PMID: 22315187, 17020470, 11992261, 3billion dataset, PS2, PS4). Functional studies provide strong evidence of the variant having a damaging effect on the gene or gene product (PMID: 15987685, 18372317, 23584145, PS3). The variant was observed as assumed (i.e. paternity and maternity not confirmed) de novoo (3billion dataset, PM6). It is not observed in the gnomAD v2.1.1 dataset (PM2). In silico tool predictions suggest damaging effect of the variant on gene or gene product (REVEL: 0.769, 3Cnet: 0.960, PP3). Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change (ClinVar ID: 40512 - c.417G>T;p.(Glu139Asp)) PS1.Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product (PMID: 23584145; 15987685) - The c.417G>C;p.(Glu139Asp) missense variant has been observed in affected individual(s) and ClinVar contains an entry for this variant (ClinVar ID: 40513; PMID: 22315187; PMID: 17020470; PMID: 11992261) - PS4. The variant is located in a mutational hot spot and/or critical and well-established functional domain (SH2) - PM1. This variant is not present in population databases (rs397507520- gnomAD; ABraOM no frequency - http://abraom.ib.usp.br/) - PM2. Missense variant in PTPN11 that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease - PP2. Multiple lines of computational evidence support a deleterious effect on the gene or gene product - PP3. In summary, the currently available evidence indicates that the variant is pathogenic.

This variant occurs de novo in an individual tested at Athena Diagnostics and in published literature (PMID: 21706501). Furthermore, it has been reported in multiple unrelated symptomatic individuals (PMID: 30417923, 31560489, 28363362, 29907801, 19020799). This variant has not been reported in large, multi-ethnic general populations (http://gnomad.broadinstitute.org). Assessment of experimental evidence suggests this variant results in abnormal protein function (PMID: 18372317).This observation is not an independent occurrence and has been identified in the same individual by RCIGM, the other laboratory participating in the GEMINI study.

ACMG classification criteria: PS2 strong, PS3 strong, PS4 strong, PM2 moderated, PP2 supporting, PP3 supporting

The PTPN11 c.417G>C; p.Glu139Asp variant (rs397507520) is frequently found in patients diagnosed with Noonan syndrome (Houweling 2010, Hung 2007, Jongmans 2004, Ko 2008, Musante 2002, Nair 2015, Pauli 2012, Tartaglia 2002, Tartaglia 2006). This variant is also reported in ClinVar (Variation ID: 40513). It is absent from the Genome Aggregation Database, indicating it is not a common polymorphism. The glutamate residue is located in the C-SH2 domain of PTPN11 (Hof 1998, Muller 2013, Qiu 2014), and the p.Glu139Asp variant is shown to alter the binding specificity of PTPN11 to phospho-tyrosine peptides (Martinelli 2008, Qiu 2014). Functional characterization of the p.Glu139Asp protein indicates increased catalytic activity upon stimulation (Eduoard 2010, Keilhack 2005, Martinelli 2008, Tartaglia 2006), consistent with the established disease mechanisms of Noonan syndrome. Based on available information, this variant is considered to be pathogenic. References: Eduoard T et al. Functional effects of PTPN11 (SHP2) mutations causing LEOPARD syndrome on epidermal growth factor-induced phosphoinositide 3-kinase/AKT/glycogen synthase kinase 3beta signaling. Mol Cell Biol. 2010; 30(10):2498-507. Hof P et al. Crystal structure of the tyrosine phosphatase SHP-2. Cell. 1998; 92(4): 441-450. Houweling A et al. Prenatal detection of Noonan syndrome by mutation analysis of the PTPN11 and the KRAS genes. Prenat Diagn. 2010; 30(3):284-6. Hung C et al. Mutational analysis of PTPN11 gene in Taiwanese children with Noonan syndrome. J Formos Med Assoc. 2007; 106(2):169-72. Jongmans M et al. Genetics and variation in phenotype in Noonan syndrome. Horm Res. 2004; 62 Suppl 3:56-9. Keilhack H et al. Diverse biochemical properties of Shp2 mutants. Implications for disease phenotypes. J Biol Chem. 2005; 280(35):30984-93. Ko J et al. PTPN11, SOS1, KRAS, and RAF1 gene analysis, and genotype-phenotype correlation in Korean patients with Noonan syndrome. J Hum Genet. 2008; 53(11-12):999-1006. Martinelli S et al. Diverse driving forces underlie the invariant occurrence of the T42A, E139D, I282V and T468M SHP2 amino acid substitutions causing Noonan and LEOPARD syndromes. Hum Mol Genet. 2008; 17(13):2018-29. Muller PJ et al. Protein tyrosine phosphatase SHP2/PTPN11 mistargeting as a consequence of SH2-domain point mutations associated with Noonan Syndrome and leukemia. J Proteomics. 2013 Jun 12;84:132-47. Musante L et al. Spectrum of mutations in PTPN11 and genotype-phenotype correlation in 96 patients with Noonan syndrome and five patients with cardio-facio-cutaneous syndrome. Eur J Hum Genet. 2003; 11(2):201-6. Nair S et al. Optic nerve pilomyxoid astrocytoma in a patient with Noonan syndrome. Pediatr Blood Cancer. 2015; 62(6):1084-6. Pauli S et al. Occurrence of acute lymphoblastic leukemia and juvenile myelomonocytic leukemia in a patient with Noonan syndrome carrying the germline PTPN11 mutation p.E139D. Am J Med Genet A 2012; 158A(3):652-8. Qiu W et al. Structural insights into Noonan/LEOPARD syndrome-related mutants of protein-tyrosine phosphatase SHP2 (PTPN11). BMC Struct Biol. 2014; 14:10. Tartaglia M et al. PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity. Am J Hum Genet. 2002; 70(6): 1555-1563. Tartaglia M et al. Diversity and functional consequences of germline and somatic PTPN11 mutations in human disease. Am J Hum Genet. 2006; 78(2): 279-290.

This sequence change replaces glutamic acid, which is acidic and polar, with aspartic acid, which is acidic and polar, at codon 139 of the PTPN11 protein (p.Glu139Asp). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individuals with Noonan syndrome (PMID: 11992261, 17339163, 19020799, 22315187). ClinVar contains an entry for this variant (Variation ID: 40513). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae indicates that this missense variant is expected to disrupt PTPN11 protein function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects PTPN11 function (PMID: 15987685, 18372317, 23584145, 24628801). For these reasons, this variant has been classified as Pathogenic.

The p.E139D pathogenic mutation (also known as c.417G>C) is located in coding exon 4 of the PTPN11 gene. This alteration results from a G to C substitution at nucleotide position 417. The glutamic acid at codon 139 is replaced by aspartic acid. This alteration, and another nucleotide change resulting in the same amino acid substitution (c.417G>T), have been detected in multiple individuals reported to have Noonan syndrome or suspected Noonan syndrome, having been reported as occurring de novo in at least one case (Tartaglia M et al. Am J Hum Genet. 2002;70:1555-63; Jongmans M et al. Horm Res. 2004;62 Suppl 3:56-9; Bakker M. Prenat Diagn. 2011;31:833-40; Nair S et al. Pediatr Blood Cancer. 2015;62:1084-6; Chinton J et al. Arch Argent Pediatr, 2019 10;117:330-337; Athota JP et al. BMC Med Genet, 2020 03;21:50). Some individuals with this alteration were also reported to develop leukemia (JPauli S et al. Am J Med Genet. 2012;158A:652-8; Park HD et al. Ann Hematol. 2012;91:511-7). In addition, this alteration was determined to be the result of de novo mutation or germline mosaicism through whole exome sequencing in 3 families with isolated cases of Noonan syndrome (Ambry internal data). Furthermore, a functional study found this mutation altered the binding specificity of the C-SH2 domain so that they were similar to the N-SH2 domain and increased the phosphopeptide binding affinity (Martinelli S et al. Hum Mol Genet. 2008;17(13):2018-29). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. Following criteria are met: 0103 - Both loss of function and gain of function are known mechanisms of disease for this gene. Metachondromatosis (MIM#156250) and Noonan syndrome with multiple lentigines have been associated with loss of function variants, whereas Noonan syndrome 1 (MIM#163950) is caused by gain of function variants (PMIDs: 11992261, 24935154, 21533187; ClinGen expert panel). (I) 0107 - This gene is associated with autosomal dominant disease. (I) 0115 - Variants in this gene are known to have variable expressivity. Noonan syndrome is known to have variable expressivity (GeneReviews). (I) 0200 - Variant is predicted to result in a missense amino acid change from glutamic acid to aspartic acid. (I) 0251 - This variant is heterozygous. (I) 0301 - Variant is absent from gnomAD (both v2 and v3). (SP) 0600 - Variant is located in the annotated SH2 domain (DECIPHER). (I) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. It is one of the most common variants causing Noonan syndrome and has been classified as pathogenic by the ClinGen expert panel (ClinVar). (SP) 1203 - This variant has been shown to be de novo in the proband (parental status confirmed) (by trio analysis). (SP) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

Variant classified using ACMG guidelines