Paper by Van der Klift et al. 2015 shows aberrant splicing (exon 10 exclusion) in patient sample and minigene assay, confirmed by minigene assay in Soukarieh et al., 2016 (Plos Genet).

This variant is considered likely pathogenic. Functional studies indicate this variant impacts protein function [PMID: 17210669, 20020535]. This variant is expected to disrupt protein structure [Myriad internal data].

Variant summary: MLH1 c.793C>A (p.Arg265Ser) results in a non-conservative amino acid change in the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. Consensus agreement among computation tools predict no significant impact on normal splicing. At least one publication reports experimental evidence that this variant affects mRNA splicing by causing skipping of exon 10 and causing a premature stop codon subject to nonsense mediated decay (vanderKlift_2015). The variant allele was found at a frequency of 4e-06 in 251460 control chromosomes. c.793C>A has been reported in the literature in individuals affected with Lynch Syndrome (Zavodna_2006, vanderKlift_2015, Hardt_2011, Ferguson_2014). These data indicate that the variant is likely to be associated with disease. The following publications have been ascertained in the context of this evaluation (PMID: 25081409, 21404117, 16830052, 26247049). ClinVar contains an entry for this variant (Variation ID: 90380). Based on the evidence outlined above, the variant was classified as pathogenic.

MLH1 NM_000249.3:c.793C>A has a 97.7% probability of pathogenicity based on combining prior probability from public data with a likelihood ratio of 1.56 to 1, generated from evidence of seeing this as a somatic mutation in a tumor without loss of heterozygosity at the MLH1 locus. See Shirts et al 2018, PMID 29887214.

The c.793C>A pathogenic mutation (also known as p.R265S), located in coding exon 10 of the MLH1 gene, results from a C to A substitution at nucleotide position 793. The arginine at codon 265 is replaced by serine, an amino acid with dissimilar properties. This mutation has been identified as germline in multiple individuals with Lynch syndrome, several whose tumors demonstrated high microsatellite instability and/or loss of MLH1/PMS2 proteins on immunohistochemistry (Zavodna K et al. Neoplasma. 2006;53:269-76; Niessen RC et al. Gut, 2006 Dec;55:1781-8; Alemayehu A et al. Genes Chromosomes Cancer. 2008 Oct;47:906-14; Hardt K et al. Fam. Cancer. 2011 Jun;10:273-84; Ferguson SE et al. Cancer. 2014 Dec;120:3932-9; van der Klift HM et al. Hum Mutat, 2016 11;37:1162-1179; Latham A et al. J Clin Oncol, 2019 02;37:286-295; Ambry internal data). This alteration is located in the ATP-binding domain of the MLH1 protein and has been demonstrated to reduce mismatch repair proficiency in two different yeast strains when compared to wildtype (Wanat JJ et al. Hum. Mol. Genet. 2007 Feb;16:445-52). In an in vitro complementation assay, this variant was determined to be functionally deficient (Drost M et al. Genet Med, 2019 07;21:1486-1496). Additionally, minigene and RNA assays have shown that this alteration results in a transcript with near-complete skipping of coding exon 10 (van der Klift HM et al. Mol Genet Genomic Med. 2015 Jul;3:327-45; Soukarieh O et al. PLoS Genet. 2016 Jan;12:e1005756). This nucleotide position is highly conserved in available vertebrate species. In silico splice site analysis for this alteration is inconclusive. RNA studies have demonstrated that this alteration results in abnormal splicing in the set of samples tested (Ambry internal data). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

This sequence change replaces arginine, which is basic and polar, with serine, which is neutral and polar, at codon 265 of the MLH1 protein (p.Arg265Ser). RNA analysis indicates that this missense change induces altered splicing and may result in an absent or disrupted protein product. This variant is present in population databases (rs63751194, gnomAD 0.0009%). This missense change has been observed in individuals with clinical features of Lynch syndrome (PMID: 21404117, 25081409, 27435373). ClinVar contains an entry for this variant (Variation ID: 90380). Based on a multifactorial likelihood algorithm using genetic, in silico, and/or statistical data, this variant has been determined to have a high probability of being pathogenic (PMID: 24362816). Studies have shown that this missense change results in skipping of exon 10 and introduces a premature termination codon (PMID: 26247049; Invitae). The resulting mRNA is expected to undergo nonsense-mediated decay. For these reasons, this variant has been classified as Pathogenic.

The p.Arg265Ser variant has been previously reported in the literature (Zavodna_2006_16830052, Alemayehu_2008_18618713, Niessen_2006_16636019). It was identified in 3 out of 572 proband chromosomes (frequency 0.005) in individuals with colorectal cancer, however, no normal population controls were included in these studies. In addition, this variant has been previously reported by our laboratory in two families. In one family the variant was shown to segregate with disease in 4 affected individuals with Lynch syndrome (two were obligate carriers), and did not segregate in at least two unaffected individuals, increasing the likelihood this variant is pathogenic. The p.Arg265 residue is conserved across mammals/species and computational analyses (PolyPhen, SIFT, AlignGVGD, BLOSUM, MAPP-MMR) suggest that the p.Arg265Ser variant may impact the protein. However, this information is not predictive enough to assume pathogenicity. However, functional studies including in vitro MMR complementation assays, protein expression assays, promoter methylation studies, LOH analysis, and a yeast-based reversion rate assay, suggests this variant is likely pathogenic (Drost_2010_20020535, Alemayehu_2008_18618713, Wanat_2007_17210669). Of note, another variant at the same nucleotide position (c.793C>T) causing a different missense substitution (p.Arg265Cys) has been previously identified in Lynch Syndrome families in literature and by our laboratory, has been well-characterized by similar functional studies as the p.Arg265Ser variant, and has been classified as pathogenic, further suggesting that p.Arg265Ser may also have clinical significance. In summary, based on the above information this variant meets our laboratory's criteria to be classified as Pathogenic.