ClinVar Genomic variation as it relates to human health

Variant reclassification due to new tumour Likelihood Ratios: Multifactorial likelihood analysis posterior probability > 0.95 (0.965)

PS3, PS4_STR, PM2_SUP, PM5, PP1

This variant is considered likely pathogenic. Functional studies indicate this variant impacts protein function [PMID: 22736432]. This variant is expected to disrupt protein structure [Myriad internal data].

This sequence change replaces threonine, which is neutral and polar, with alanine, which is neutral and non-polar, at codon 82 of the MLH1 protein (p.Thr82Ala). This variant is present in population databases (rs587778998, gnomAD 0.01%). This missense change has been observed in individuals with Lynch syndrome (PMID: 21239990, 21404117, 22736432, 26300997, 28514183). ClinVar contains an entry for this variant (Variation ID: 90116). 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 MLH1 protein function. Experimental studies have shown that this missense change affects MLH1 function (PMID: 22736432). This variant disrupts the p.Thr82 amino acid residue in MLH1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 10422993, 17510385, 19690142, 20587412, 23403630). 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.

This missense variant replaces threonine with alanine at codon 82 of the MLH1 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 the mutant protein shows <10% of normal DNA-mismatch repair activity in vitro (PMID: 22736432, 31881334) (PMID: 22736432, 31881334). This variant has been reported in individuals affected with Lynch syndrome (PMID: 21239990, 26300997) and in individuals affected with colorectal cancer with tumors showing microsatellite instability (PMID: 21404117, 22736432). This variant has been identified in 2/251394 chromosomes in the general population by the Genome Aggregation Database (gnomAD). A different missense variant p.Thr82Ile is known to be pathogenic, indicating the importance of threonine residue at this position for MLH1 function. Based on the available evidence, this variant is classified as Pathogenic.

The p.T82A variant (also known as c.244A>G), located in coding exon 3 of the MLH1 gene, results from an A to G substitution at nucleotide position 244. The threonine at codon 82 is replaced by alanine, an amino acid with similar properties. This variant has been identified in probands with HNPCC or HNPCC-related cancers and concordant tumors when available (Pastrello C et al. Genet Med. 2011 Feb;13(2):115-24,Hardt K et al. Fam Cancer. 2011 Jun;10(2):273-84, Borras E et al. Hum Mutat. 2012 Nov;33(11):1576-88, Simbolo M et al. Hered. Cancer Clin Pract. 2015 Aug 21;13(1), Ambry internal data). This variant is located in the ATPase functional domain and impaired MMR activity compared to wild-type (Borras E et al. Hum Mutat. 2012 Nov;33(11)). This paper also cites literature demonstrating alterations at the same codon, including p.T82K, p.T82M, and p.T82I, to be defective in MMR activity in yeast and/or human cells. This alteration has been classified as pathogenic by multifactorial analysis, which integrates the following lines of evidence to produce a quantitative likelihood of pathogenicity: in silico prediction models, segregation with disease, tumor characteristics, mutation co-occurrence, and functional assay results (Thompson B et al. Nat Genet. 2014 Feb;46(2):107-15; available at [www.insight-group.org/variants/classifications/]). 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 majority of available evidence to date, this variant is likely to be pathogenic.

Variant summary: MLH1 c.244A>G (p.Thr82Ala) 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. The variant allele was found at a frequency of 7.9e-06 in 252112 control chromosomes. c.244A>G has been reported in the literature in multiple individuals affected with Hereditary Nonpolyposis Colorectal Cancer (Hardg_2011, Hiljadnikova-Bajro_2011, Pasrello_2011, Borras_2012, Simbolo_2015, Espenscheid_2017). These data indicate that the variant is very likely to be associated with disease. A different variant affecting the same codon has been classified as pathogenic by our lab (c.245C>T, p.Thr82Ile), supporting the critical relevance of codon 82 to MLH1 protein function. At least one publication reports experimental evidence evaluating an impact on protein function. The most pronounced variant effect results in 10%-<30% of normal MMR activity (Borras_2012). The following publications have been ascertained in the context of this evaluation (PMID: 21239990, 22736432, 21404117, 24362816, 26300997, 28514183). ClinVar contains an entry for this variant (Variation ID: 90116). Based on the evidence outlined above, the variant was classified as pathogenic.

This missense variant replaces threonine with alanine at codon 82 of the MLH1 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 the mutant protein shows <10% of normal DNA-mismatch repair activity in vitro (PMID: 22736432, 31881334). This variant has been reported in individuals affected with Lynch syndrome (PMID: 21239990, 26300997) and in individuals affected with colorectal cancer with tumors showing microsatellite instability (PMID: 21404117, 22736432). This variant has been identified in 2/251394 chromosomes in the general population by the Genome Aggregation Database (gnomAD). A different missense variant occurring at the same codon, p.Thr82Ile, is known to be disease-causing, indicating the importance of threonine residue at this position for MLH1 function. Based on the available evidence, this variant is classified as Pathogenic.

The p.Thr82Ala variant in MLH1 has been reported in 10 European individuals with Lynch syndrome or MLH1-associated cancers (Sjursen 2010, Hardt 2011, Pastrello 2011, Simbolo 2015, Stradella 2019). It has also been identified in 2/123,762 European/Ashkenazi Jewish chromosomes by gnomAD (http://gnomad.broadinstitute.org). Computational prediction tools and conservation analysis suggest that this variant may impact the protein, though this information is not predictive enough to determine pathogenicity. In vitro functional studies provide some evidence that this variant impacts protein function (Ellison 2004, Borras 2012). This variant was classified as Likely Pathogenic on June 13, 2018 by the ClinGen-approved InSiGHT expert panel (Variation ID 90116). Another variant involving this codon (p.Thr82Ile) has been identified in individuals with Lynch syndrome and is classified as pathogenic by the ClinGen-approved InSiGHT expert panel. In summary, this variant meets criteria to be classified as pathogenic for autosomal dominant Lynch syndrome. ACMG/AMP Criteria applied: PM2, PS4_Strong, PP3, PS3_Supporting, PM5.

The p.Thr82Ala variant has been previously reported in the literature and by our laboratory. It was identified in one individual who was reported to have high microsatellite instability and was MLH1 deficient. In addition, it was shown to segregate in one affected family member (Pastrello_2011_21239990). Our laboratory has identified this variant in one family and the variant segregated with disease in 5 affected individuals. Although the pattern of MSI and IHC deficient tumours was inconsistent across individuals from this family, the observation of this variant segregating with Lynch syndrome related cancers increases the likelihood this variant is pathogenic. In addition, functional studies in yeast demonstrated that different variants at this same amino acid residue (T82S, T82K, T82M) were associated with a loss of mismatch repair function between 8% and 67%, increasing the likelihood that this variant may affect normal protein function (Ellison_2004_15475387). In summary, based on the above information, this variant is classified as pathogenic.