ClinVar Genomic variation as it relates to human health

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 variant has been reported to affect MLH1 protein function (PMID: 20020535, 22753075, 30998989). This variant has been observed in individual(s) with Lynch syndrome (PMID: 18415027, 12547705, 27435373, 30998989, Invitae). ClinVar contains an entry for this variant (Variation ID: 89870). This variant is not present in population databases (ExAC no frequency). This sequence change replaces leucine with phenylalanine at codon 582 of the MLH1 protein (p.Leu582Phe). The leucine residue is moderately conserved and there is a small physicochemical difference between leucine and phenylalanine.

The p.L582F pathogenic mutation (also known as c.1744C>T), located in coding exon 16 of the MLH1 gene, results from a C to T substitution at nucleotide position 1744. The leucine at codon 582 is replaced by phenylalanine, an amino acid with highly similar properties. This alteration showed moderate segregation with disease in a French family that met Amsterdam criteria where the colorectal tumor of the proband displayed loss of MLH1 protein expression on immunohistochemistry (personal communication with Universal Mutation Database depositor). This variant was identified as somatic in conjunction with a second somatic MLH1 pathogenic mutation in an MSI-H colorectal tumor with loss of MLH1 and PMS2 by IHC (Rigter LS et al. Gut, 2018 03;67:447-455). In a cell-free system designed to measure mismatch repair function, the MLH1 protein with the p.L582F variant was determined to have 26.5% relative repair activity compared to wild type (100%) (Drost M et al. Hum Mutat, 2010 Mar;31:247-53). In another in vitro assay, the binding capacity of the MLH1 p.L582F protein variant with PMS2 was measured in a GST pull-down assay and no binding was detected (Andersen SD et al. Hum. Mutat., 2012 Dec;33:1647-55). The p.L582F variant was also classified as potentially damaging in a methylation tolerance-based functional assay (Bouvet D et al. Gastroenterology, 2019 08;157:421-431). Based on internal structural analysis, L582F is more disruptive than an internally pathogenic variant in the same domain (Gueneau E et al. Nat Struct Mol Biol., 2013 Apr;20(4):461-8). This variant is considered to be rare based on population 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 variant is interpreted as a disease-causing mutation.

This variant is denoted MLH1 c.1744C>T at the cDNA level, p.Leu582Phe (L582F) at the protein level, and results in the change of a Leucine to a Phenylalanine (CTC>TTC). This variant has been reported in individuals with early onset colorectal cancer, whose tumor testing demonstrated microsatellite instability and loss of MLH1, and also loss of PMS2 when performed, on mismatch repair immunohistochemistry (IHC) (Hendriks 2003, van Puijenbroek 2008, van der Klift 2016). Functional assays demonstrate MLH1 Leu582Phe to have reduced mismatch repair activity compared to wild-type, while other assays show defective MLH1-PMS2 dimerization (Drost 2010, Andersen 2012). MLH1 Leu582Phe 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 Leucine and Phenylalanine share similar properties, this is considered a conservative amino acid substitution. MLH1 Leu582Phe occurs at a position that is conserved across species and is located in the PMS2/MLH3/PMS1 interaction domain and region of interaction with EXO1 (Pang 1997, Raevaara 2005, Hardt 2011, UniProt). In silico analyses are inconsistent regarding the effect this variant may have on protein structure and function. Based on currently available evidence, it is unclear whether MLH1 Leu582Phe is a pathogenic or benign variant. We consider it to be a variant of uncertain significance.

Variant summary: The MLH1 c.1744C>T (p.L582F) variant involves the alteration of a highly conserved nucleotide and 3/4 in silico tools predict a damaging outcome for this variant. The variant is located within the PMS2/MLH3/PMS1 interaction domain and the abrogated interactions between MLH1 and PMS2 partners in addition to the variant being deficient in an MMR complementation assay were confirmed by functional studies (Drost_2009; Andersen_2012). Tumors for carriers of this variant showed MSH-H and loss of MLH1-PMS2 staining. The variant is absent from the large control datasets of ExAC and gnomAD (121282 and 246040, chrs tested respectively). The variant was identified in several affected individuals (Hendriks_2003; van Puijenbroek_2008; van der Klift_2016). Although several clinical diagnostic laboratories/reputable databases cite the variant with a classification of VUS, the collective evidence points towards a deleterious effect. Taken together, the variant was classified as VUS-Possibly Pathogenic, until more data becomes available.

Not found in the total gnomAD dataset, and the data is high quality. Located in potentially critical domain of the protein. Assessment of experimental evidence suggests this variant results in abnormal protein function.