ClinVar Genomic variation as it relates to human health

This missense variant replaces phenylalanine with serine at codon 432 of the MSH6 protein. Computational prediction is inconclusive regarding the impact of this variant on protein structure and function (internally defined REVEL score threshold 0.5 < inconclusive < 0.7, PMID: 27666373). This substitution disrupts a conserved Phe-X-Glu motif that contacts mis-paired DNA bases (PMID: 17531815). A functional study has shown that this variant impacts MMR function in vitro and the equivalent variant in the mouse Msh6 homolog also disrupts DNA mismatch binding and Msh6 function in a genetic screen for tolerance to 6-thioguanine (PMID: 31965077). This variant has been observed in individuals affected with Lynch syndrome-associated cancer (PMID: 24933100, 28765196, 35884469). 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 Likely Pathogenic.

This variant is denoted MSH6 c.1295T>C at the cDNA level, p.Phe432Ser (F432S) at the protein level, and results in the change of a Phenylalanine to a Serine (TTT>TCT). This variant has been identified in at least one woman with endometrioid endometrial cancer, and a family history of colorectal and/or endometrial cancer, whose tumor displayed presence of MLH1, MSH2, MSH6, and PMS2 by immunohistochemistry and low levels of microsatellite instability (MSI-L) (Batte 2014). In addition, Dufner et al. (2000) performed in vitro functional studies on an alternate variant at the same residue, Phe432Ala, which exhibited that this variant was unable to form stable complexes, abolished DNA-binding, induced ATP hydrolysis and abolished mismatch repair activity. MSH6 Phe432Ser was not observed in approximately 6,500 individuals of European and African American ancestry in the NHLBI Exome Sequencing Project, suggesting it is not a common benign variant in these populations. Since Phenylalanine and Serine differ in polarity, charge, size or other properties, this is considered a non-conservative amino acid substitution. MSH6 Phe432Ser occurs at a position that is conserved across species and is located in domain I of the MutS domain and in the MSH2 binding sites (Kariola 2002, Terui 2013). In silico analyses predict that this variant is probably damaging to protein structure and function. Based on the currently available evidence, we consider MSH6 Phe432Ser to be a likely pathogenic variant.

This variant is considered likely pathogenic. Functional studies indicate this variant impacts protein function [PMID: 31965077]. This variant is expected to disrupt protein structure [Myriad internal data].

In the published literature, the variant has been reported in individuals with colorectal cancer and/or endometrial cancer (PMID: 28765196 (2017), 24933100 (2014)). A functional study indicated the variant caused impaired DNA mismatch binding and severely deficient MMR activity in vitro (PMID 31965077 (2020)). Based on the available information, this variant is classified as pathogenic.

This sequence change replaces phenylalanine, which is neutral and non-polar, with serine, which is neutral and polar, at codon 432 of the MSH6 protein (p.Phe432Ser). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with MSH6-related conditions (PMID: 24933100, 28765196). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 183760). 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 MSH6 protein function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects MSH6 function (PMID: 31965077). In summary, the currently available evidence indicates that the variant is pathogenic, but additional data are needed to prove that conclusively. Therefore, this variant has been classified as Likely Pathogenic.

This missense variant replaces phenylalanine with serine at codon 432 of the MSH6 protein. Computational prediction is inconclusive regarding the impact of this variant on protein structure and function (internally defined REVEL score threshold 0.5 < inconclusive < 0.7, PMID: 27666373). This substitution disrupts a conserved Phe-X-Glu motif that contacts mis-paired DNA bases (PMID: 17531815). A functional study has shown that this variant impacts MMR function in vitro and the equivalent variant in the mouse Msh6 homolog also disrupts DNA mismatch binding and Msh6 function in a genetic screen for tolerance to 6-thioguanine (PMID: 31965077). This variant has been observed in individuals affected with Lynch syndrome-associated cancer (PMID: 24933100, 28765196, 35884469). 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 Likely Pathogenic.

Variant summary: MSH6 c.1295T>C (p.Phe432Ser) results in a non-conservative amino acid change located in the DNA mismatch repair protein MutS-like, N-terminal of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 251280 control chromosomes. c.1295T>C has been reported in the literature in individuals affected with HNPCC-associated cancers (Batte_2014, Borras_2017, Kim_2022). These data indicate that the variant may be associated with disease. The variant was reported as damaging via the complete in vitro MMR activity (CIMRA) assay used to quantify the functional activity of variants in MMR genes (Drost_2020). The following publications have been ascertained in the context of this evaluation (PMID: 24933100, 28765196, 11641390, 10938287, 35884469, 31965077). ClinVar contains an entry for this variant (Variation ID: 183760). Based on the evidence outlined above, the variant was classified as likely pathogenic.

The p.F432S pathogenic mutation (also known as c.1295T>C), located in coding exon 4 of the MSH6 gene, results from a T to C substitution at nucleotide position 1295. The phenylalanine at codon 432 is replaced by serine, an amino acid with highly dissimilar properties. This alteration has been observed in multiple individuals with Lynch syndrome-associated cancers and families that meet Amsterdam criteria including an individual with clinical features consistent with constitutional mismatch repair deficiency syndrome (CMMRD) (Nassar AH et al. Genet Med, 2020 Apr;22:709-718; Kim YN et al. Cancers (Basel), 2022 Jul;14; Ambry internal data). This alteration was reported in an individual diagnosed with endometrial cancer whose tumor demonstrated low microsatellite instability and normal MSH6 expression on immunohistochemistry (IHC) (Batte BA et al. Gynecol. Oncol., 2014 Aug;134:319-25; Borras E et al. Cancer Prev Res (Phila), 2017 Oct;10:580-587). This alteration has been reported as functionally defective based on results from a complementation assay performed in mammalian cells (Drost M et al. Genet. Med., 2020 May;22:847-856). Another alteration at the same codon, p.F432A, was shown to have reduced DNA binding and abolished mismatch repair activity in an in vitro functional assay (Dufner P et al. J. Biol. Chem., 2000 Nov;275:36550-5). Based on internal structural analysis using published crystal structures, this alteration is important to protein function and moderately destabilizing to the local structure (Warren JJ et al. Mol. Cell, 2007 May;26:579-92; Sharma M et al. Biophys. J., 2014 Jun;106:2483-92; Reyes GX et al. Chromosoma, 2015 Dec;124:443-62). This variant was not reported in population-based cohorts in the Genome Aggregation Database (gnomAD). 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.

The MSH6 p.Phe432Ser variant was not identified in the literature, nor was it identified in the dbSNP, NHLBI Exome Sequencing Project (Exome Variant Server), HGMD, COSMIC, MutDB, “Mismatch Repair Genes Variant Database”, “MMR Gene Unclassified Variants Database”, “InSiGHT Colon Cancer Database”, “Zhejiang Colon Cancer Database”, ClinVar database, or UMD. The p.Phe432 residue is conserved across mammals and computational analyses (PolyPhen, SIFT, AlignGVGD, BLOSUM) suggest that the p.Phe432Ser variant may impact the protein. However, this information is not predictive enough to assume pathogenicity. Functional studies using site-directed mutagenesis have shown that a different alteration at this position, a substitution of phenylalanine for alanine at residue 432, abolished not only mismatch recognition but also the binding of the MSH2-MSH6 complex to DNA in general (Drotschmann 2001, Dufner 2000, Dalhus 2009), increasing the likelihood that the mutations at this highly conserved residue are of clinical significance. In summary, based on the above information, the clinical significance of this variant cannot be determined with certainty at this time, although we would lean towards a more pathogenic role for this variant. This variant is classified as predicted pathogenic.

PS3, PS4_mod, PM2_sup, PP4