ClinVar Genomic variation as it relates to human health

Satisfies class 5 criteria

Variant summary: MSH2 c.998G>A (p.Cys333Tyr) results in a non-conservative amino acid change located in the DNA mismatch repair protein MutS, core domain (IPR007696) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function and several publications report experimental evidence of the variant to be MMR-deficient (examples: Ollivia_2006; Gammie_2007; Houlleberghs_2016). The variant was absent in 251466 control chromosomes (gnomAD). The c.998G>A has been reported in the literature in multiple individuals affected with Lynch Syndrome. Tumors from these patients were indicated to have MSI-I and lack of MSH2 staining on IHC, consistent with a deficiency of DNA mismatch repair (examples: Ward_2005; Fazekas-Lavu_2017; Chubb_2015; Shia_2005). Eight submitters including an expert panel (InSiGHT) have submitted clinical-significance assessments for this variant to ClinVar after 2014 and all classified the variant as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

This sequence change replaces cysteine, which is neutral and slightly polar, with tyrosine, which is neutral and polar, at codon 333 of the MSH2 protein (p.Cys333Tyr). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with Lynch syndrome-related cancers (PMID: 12624141, 15849733, 16175654, 21642682, 25559809, 26681312, 28769567). ClinVar contains an entry for this variant (Variation ID: 91273). Advanced modeling performed at Invitae incorporating data from internal and/or published experimental studies (PMID: 33357406) indicates that this missense variant is expected to disrupt MSH2 function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects MSH2 function (PMID: 17101317, 17720936, 18951462, 26951660). This variant disrupts the p.Cys333 amino acid residue in MSH2. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 11379475, 17720936, 20176959). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic.

The p.C333Y pathogenic mutation (also known as c.998G>A), located in coding exon 6 of the MSH2 gene, results from a G to A substitution at nucleotide position 998. The cysteine at codon 333 is replaced by tyrosine, an amino acid with highly dissimilar properties. This alteration is in the core domain of the MSH2 protein and in vitro functional studies showed normal MSH6 interaction but reduced mismatch repair efficiency compared to the wild-type (7.0% versus 44.7%) and decreased protein stability (Ollila S et al. Gastroenterology. 2006 Nov;131(5):1408-17; Nielsen S et al. PLoS Genet. 2017 04;13(4):e1006739). Additional functional studies have shown p.C333Y to result in loss of mismatch repair activity, decreased MSH2 levels (1% of wild type) and loss of subunit interaction (Gammie AE et al. Genetics. 2007 Oct;177(2):707-21). This alteration was also found to be pathogenic by a functional screening method utilizing oligo targeting mutagenesis and analysis of cell colonies resistant to methylation (Houlleberghs H et al. Proc. Natl. Acad. Sci. U.S.A. 2016 Apr;113(15):4128-33). In addition, the p.C333Y alteration has been detected in multiple probands with family histories meeting the Amsterdam criteria whose tumors demonstrated absent MSH2/MSH6 staining on IHC and tested negative for germline mutations in the EPCAM and MSH6 genes (Fazekas-Lavu M et al. Ther Clin Risk Manag 2017 Jul;13:915-918; Ambry internal data). This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

Not found in the gnomAD exomes dataset, and the data is high quality (0/251466 chr). Statistically enriched in patients compared to ethnically matched controls. Found in at least one symptomatic patient. Predicted to have a damaging effect on the protein. One other pathogenic or likely pathogenic variant affects the same amino acid. Assessment of experimental evidence suggests this variant results in abnormal protein function.

Observed in individuals with MSH2-related cancers, including colon tumors exhibiting microsatellite instability and/or loss of MSH2 staining by immunohistochemistry (Parc et al., 2003; Mangold et al., 2005; Ward et al., 2005; Chubb et al., 2015; Fazekas-Lavu et al., 2017; Published functional studies demonstrate a damaging effect: increased protein turnover with reduced steady-state protein levels, loss of interaction with the MSH3 protein, and decreased MMR efficiency (Ollila et al., 2006; Gammie et al., 2007; Arlow et al., 2013; Houlleberghs et al., 2016); Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 17594722, 17101317, 15849733, 17720936, 18951462, 19669161, 23248292, 22949387, 21120944, 21642682, 16175654, 18383312, 25559809, 26333163, 12624141, 26951660, 26681312, 28422960, 28769567, 30322717, 33357406, 30787465, 18822302, 32660107)

Criteria applied: PS3,PS4,PM5_STR,PM2_SUP

ACMG criteria used to clasify this variant:PP3_STR, PS3_MOD, PS4_MOD, PM1_SUP, PM2_SUP

This missense variant replaces cysteine with tyrosine at codon 333 of the MSH2 protein. Computational prediction suggests that this variant may have deleterious impact on protein structure and function (internally defined REVEL score threshold >= 0.7, PMID: 27666373). Functional studies have shown that this variant disrupts MSH2 protein function, decreasing protein stability and expression and resulting in defective mismatch repair (PMID: 17101317, 17720936, 18951462, 23248292, 26951660, 28422960, 33357406). This variant has been reported in numerous individuals affected with Lynch syndrome-related cancers, including colorectal, ovarian and endometrial cancers (PMID: 12624141, 15849733, 16175654, 17101317, 19669161, 21642682, 25173403, 25559809, 26681312, 28769567). Different variants affecting the same codon, c.997T>C (p.Cys333Arg), c.998G>T (p.Cys333Phe), (MSH2):c.999T>G (p.Cys333Trp), are also described as disease-causing (ClinVar variation ID: 91272, 638846, 450154), supporting that this position is important for the protein function. This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Pathogenic.

The MSH2 p.Cys333Tyr variant was identified in 2 of 5014 proband chromosomes (frequency: 0.0004) from individuals or families with Lynch Syndrome and CRC (Mangold 2005, Ward 2005). The variant was also identified in dbSNP (ID: rs63750828) as “With Pathogenic allele”, ClinVar (as uncertain significance by InSight, as pathogenic by GeneDx and Ambry Genetics), UMD-LSDB (6 x as UV), Insight Colon Cancer Gene Variant Database (reported 35x, as class 5, pathogenic), Mismatch Repair Genes Variant Database (2 x), MMR Gene Unclassified Variants Database (2 x ), Insight Hereditary Tumors Database (13 x, class 5, pathogenic) databases. The variant was not identified in Genesight-COGR, Cosmic, MutDB, Zhejiang Colon Cancer Database, databases. The variant was (also) identified by our laboratory in 1 individual with HNPCC. The variant was not identified in the 1000 Genomes Project, the NHLBI GO Exome Sequencing Project or the Exome Aggregation Consortium (August 8th 2016) control databases. Functional studies have included co-immunoprecipitation and Western blot analysis which demonstrated normal interaction of this MSH2 variant protein with MSH6 protein. However, an in-vitro mismatch-repair assay demonstrated complete deficiency of this variant and the authors predicted p.Cys333Tyr as pathogenic (Ollila 2006). In addition verification of the three-step model in assessing the pathogenicity of mismatch repair gene variants, based on MMR assay indicates pathogenicity (Kansikkas 2010). Lack of adequate levels of variant Msh2 protein is the most common reason for failure of DNA mismatch repair among the defective human missense variants. Functional characterization of MSH2 missense variant p.Cys333Tyr showed loss of interaction with all partners (Gammie 2007). The p.Cys333 residue is conserved across mammals and other organisms, and computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) suggest that the variant may impact the protein; however, this information is not predictive enough to assume pathogenicity. The variant occurs outside of the splicing consensus sequence and in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) do not predict a difference in splicing. The variant is located with the DNA mismatch repair protein MutS, core DNA mismatch repair protein, MSH2 functional domain(s) increasing the liklihood that it may have clinical significance. In summary, based on the above information, this variant meets our laboratory’s criteria to be classified as pathogenic.

Variant interpreted as Pathogenic and reported on 11-11-2020 by Invitae. GenomeConnect-Invitae Patient Insights Network assertions are reported exactly as they appear on the patient-provided report from the testing laboratory. Registry team members make no attempt to reinterpret the clinical significance of the variant. Phenotypic details are available under supporting information.