Submissions for variant NM_000249.4(MLH1):c.1976G>T (p.Arg659Leu)

dbSNP: rs63749900

Total submissions: 3

Submitter	RCV	SCV	Clinical significance	Condition	Last evaluated	Review status	Method	Comment
International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	RCV000075448	SCV000106446	pathogenic	Lynch syndrome	2013-09-05	reviewed by expert panel	research	Multifactorial likelihood analysis posterior probability >0.99
Ambry Genetics	RCV000572238	SCV000676046	pathogenic	Hereditary cancer-predisposing syndrome	2016-05-20	criteria provided, single submitter	clinical testing	The p.R659L variant (also known as c.1976G>T), located in coding exon 17 of the MLH1 gene, results from a G to T substitution at nucleotide position 1976. The arginine at codon 659 is replaced by leucine, an amino acid with dissimilar properties. This alteration has been reported in multiple individuals either meeting classic ‘Amsterdam’ criteria for HNPCC, or a modified set of criteria (Cravo M et al. Gut 2002 Mar; 50(3):405-12; Fidalgo P et al. Eur. J. Hum. Genet. 2000 Jan; 8(1):49-53; Lage PA et al. Cancer 2004 Jul; 101(1):172-7; Nyström-Lahti M et al. Genes Chromosomes Cancer 1999 Dec; 26(4):372-5). Further, this alteration has been shown to segregate with disease in one family, as two affected members were shown to carry this alteration while one unaffected member did not (Cravo M et al. Gut 2002 Mar; 50(3):405-12). This alteration is located in the c-terminal ATP binding and hydrolysis domain of the MLH1 protein, which is responsible for constitutive dimerization with PMS2. In a study examining pathogenic effects due to interference with dimerization with PMS2, this alteration was reported to results in statistically significant reduction of MLH1 expression by multiple independent Western blot transfection experiments (Kosinski J et al. Hum. Mutat. 2010 Aug; 31(8):975-82). This alteration has also 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, clinical phenotype including 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/]). Using the BDGP and ESEfinder splice site prediction tools, this alteration is predicted to create a new alternate splice acceptor site. Functional analysis of this alteration using patient RNA sequencing via RT-PCR revealed a deletion of 93 base pairs due to skipping of exon 17; authors note that no sequence alterations of the intron-exon boundaries were identified suggesting this alteration will thereby lead to a non-functional protein (Nyström-Lahti M et al. Genes Chromosomes Cancer 1999 Dec; 26(4):372-5). Based on the available evidence, p.R659L is classified as a pathogenic mutation.
Color Diagnostics, LLC DBA Color Health	RCV000572238	SCV001346828	likely pathogenic	Hereditary cancer-predisposing syndrome	2020-04-23	criteria provided, single submitter	clinical testing	This missense variant replaces arginine with leucine at codon 659 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 this variant reduces MLH1 protein expression, disrupts nuclear localization and interaction with PMS2, and results in reduced DNA mismatch repair activity (PMID: 11839723, 20533529, 23403630, 30504929). An RNA study using cells from a heterozygous carrier has detected a low level of an alternate splicing transcript due to out-of-frame exon 17 skipping (PMID: 10534773). This variant has been reported in two related individuals affected with Lynch syndrome (PMID: 10534773, 10713887, 11839723, 15222003) and in an unrelated individual affected with Lynch syndrome (PMID: 22776989). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Loss of MLH1 function is a known mechanism of disease (clinicalgenome.org). Based on the available evidence, this variant is classified as Likely Pathogenic.