Submissions for variant NM_000249.4(MLH1):c.1976G>A (p.Arg659Gln)

gnomAD frequency: 0.00003  dbSNP: rs63749900

Total submissions: 10

Submitter	RCV	SCV	Clinical significance	Condition	Last evaluated	Review status	Method	Comment
Invitae	RCV001081501	SCV000253137	uncertain significance	Hereditary nonpolyposis colorectal neoplasms	2023-12-01	criteria provided, single submitter	clinical testing	This sequence change replaces arginine, which is basic and polar, with glutamine, which is neutral and polar, at codon 659 of the MLH1 protein (p.Arg659Gln). This variant is present in population databases (rs63749900, gnomAD 0.01%). This missense change has been observed in individual(s) with clinical features of Lynch syndrome (PMID: 16083711, 16885385, 28135145). This variant can co-occur with the variant c.1852_1853delinsGC (p.Lys618Ala) in cis (on the same chromosome), as the haplotype c.[1976G>A;1852_1853delinsGC] (p.[Arg659Gln;Lys618Ala]). This haplotype has been observed in individuals with Lynch syndrome (external communication). ClinVar contains an entry for this variant (Variation ID: 89964). 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) has been performed at Invitae for this missense variant, however the output from this modeling did not meet the statistical confidence thresholds required to predict the impact of this variant on MLH1 protein function. Experimental studies are conflicting or provide insufficient evidence to determine the effect of this variant on MLH1 function (PMID: 16083711, 17210669, 17510385, 19863800, 20533529, 23403630, 31697235). This variant disrupts the p.Arg659 amino acid residue in MLH1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 8776590, 9697702, 10037723, 11555625, 11601928, 11793442, 16083711). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance.
Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	RCV000121365	SCV000539645	uncertain significance	not specified	2016-03-31	criteria provided, single submitter	clinical testing	Variant identified in a genome or exome case(s) and assessed due to predicted null impact of the variant or pathogenic assertions in the literature or databases. Disclaimer: This variant has not undergone full assessment. The following are preliminary notes: Several papers describe as non-pathogenic; ExAC: 2/10400 African; ClinVar: VUS by expert panel
GeneDx	RCV000656865	SCV000565165	uncertain significance	not provided	2021-02-01	criteria provided, single submitter	clinical testing	Observed in families meeting Amsterdam or Bethesda criteria, with corresponding colon and endometrial tumors showing microsatellite instability (MSI-H) and loss of MLH1 staining by immunohistochemistry (Raevaara 2005, Hampel 2006, Yurgelun 2017); Published functional studies are inconclusive: some demonstrate protein expression, mismatch repair activity, nuclear localization, and PMS2 interaction similar to wild type, while others show reduced protein expression, increased mutation rate, and inconsistent dominant mutator effect, and some suggest this variant may cause a splice defect (Raevaara 2005, Takahashi 2007, Wanat 2007, Vogelsang 2009, Kosinski 2010, Hinrichsen 2013, Xiong 2015); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 17210669, 16885385, 17510385, 20533529, 23403630, 19863800, 25525159, 22949387, 21120944, 25871441, 17192056, 17370310, 18383312, 17594722, 27629256, 23741719, 24728327, 28135145, 16083711, 31697235, 30212499, 32980694, 33309985)
Quest Diagnostics Nichols Institute San Juan Capistrano	RCV000656865	SCV000601375	uncertain significance	not provided	2022-10-22	criteria provided, single submitter	clinical testing	The frequency of this variant in the general population, 0.00012 (3/24942 chromosomes, http://gnomad.broadinstitute.org), is uninformative in assessment of its pathogenicity. In the published literature, the variant has been reported in a hereditary non-polyposis colorectal cancer family with 10 affected members and fulfilled Amsterdam criteria (PMID: 16083711 (2005)). Published functional studies have reported conflicting results on the effect of this variant on MLH1 protein activity (PMID: 16083711 (2005), 17510385 (2009), 20533529 (2010), 19863800 (2009)). Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded conflicting predictions that this variant is benign or damaging. Based on the available information, we are unable to determine the clinical significance of this variant.
Ambry Genetics	RCV000568527	SCV000669521	uncertain significance	Hereditary cancer-predisposing syndrome	2023-01-11	criteria provided, single submitter	clinical testing	The p.R659Q variant (also known as c.1976G>A) is located in coding exon 17 of the MLH1 gene. This alteration results from a G to A substitution at nucleotide position 1976. The arginine at codon 659 is replaced by glutamine, an amino acid with highly similar properties. This alteration has been identified in three probands families with tumor testing demonstrating high microsatellite instability and/or absent MLH1 staining on immunohistochemistry (IHC) and two of the three had family histories that met Amsterdam I/II criteria for Lynch syndrome (Raevaara TE et al. Gastroenterology. 2005 Aug;129(2):537-49; Hampel H et al. Cancer Res., 2006 Aug;66:7810-7; Yurgelun MB et al. J Clin Oncol. 2017 Apr;35(10):1086-1095; Ambry internal data). However, multiple in vitro functional studies including protein expression/stability, subcellular localization, protein-protein interaction, and MMR repair efficiency indicated that this alteration was not likely to be pathogenic (Raevaara TE et al. Gastroenterology. 2005 Aug;129(2):537-49; Kansikas M et al. Hum Mutat. 2011 Jan;32(1):107-15; Abildgaard AB et al. Elife, 2019 11;8). Several other functional studies also indicated that this variant was unlikely to be pathogenic based on in vitro mismatch repair and expression assays that were comparable to wild type (Takahashi M et al. Cancer Res. 2007 May 15;67(10):4595-604; Ou J et al. Hum Mutat. 2007 Nov;28(11):1047-54; Hinrichsen I et al. Clin Cancer Res. 2013 May 1;19(9):2432-41). In contrast, this variant was reported as a pathogenic alteration in an in vivo yeast assay (Vogelsang M et al. BMC Cancer. 2009 Oct 28;9:382). This alteration was also reported in healthy control groups or cohorts that underwent sequencing (Bodian DL et al. PLoS One. 2014 Apr 11;9(4):e94554; Fujita M et al. Clin Gastroenterol Hepatol, 2020 Dec). This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Since supporting evidence is conflicting at this time, the clinical significance of this alteration remains unclear.
Color Diagnostics, LLC DBA Color Health	RCV000568527	SCV000911479	likely benign	Hereditary cancer-predisposing syndrome	2016-12-16	criteria provided, single submitter	clinical testing
Women's Health and Genetics/Laboratory Corporation of America, LabCorp	RCV000121365	SCV001370693	uncertain significance	not specified	2023-05-01	criteria provided, single submitter	clinical testing	Variant summary: MLH1 c.1976G>A (p.Arg659Gln) results in a conservative amino acid change located in the C-terminal domain (IPR032189) of the encoded protein sequence. Four of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 1.6e-05 in 251070 control chromosomes (gnomAD). The available data on variant occurrences in the general population are insufficient to allow any conclusion about variant significance. c.1976G>A has been reported in the literature in individuals/families fulfilling Amsterdam and/or Bethesda criteria for HNPCC, with corresponding tumors showing high microsatellite instability and absent MLH1 following immunohistochemistry (e.g. Hampel_2006 (no PMID), Raevaara_2005, Yurgelun_2017). The variant has also been reported in healthy controls (e.g. Mizukami_2020, Fujita_2022). These reports do not provide unequivocal conclusions about association of the variant with Hereditary Nonpolyposis Colorectal Cancer. In one of the reported CRC patients a KRAS variant (G13D, classified as pathogenic somatic variant) was detected following tumor testing (Yurgelun_2017). Co-occurrences with pathogenic variants have been reported (MLH1 c.2T>C, p.Met1?; BRIP1 c.2010dupT, p.Glu671X; UMD and internal testing), providing supporting evidence for a benign role. Multiple functional studies demonstrated that even though the expression of the variant protein is reduced, there is mild to no deficiency in MMR assays and the protein is stable exhibiting normal cellular localization and normal protein interaction (e.g. Abildgaard_2019, Hinrichsen_2013, Ou_2017, Raevaara_2005, Takahashi_2007). Yeast assays examining the dominant mutator effect and reversion rate were conflicting in their outcomes with some showing a negative effect of the variant while others did not or showed a low mutator phenotype (e.g. Takahashi_2007, Wanat_2007). p.Arg659Gln affects a codon that has been linked to aberrant splicing and subsequent skipping of exon 17, and some publications suggest it could have potential impact on splicing (Nystrom-Lahti_1999 and Raevaara_2005). However, 4/4 computational tools predict no significant impact of the variant on normal splicing and RNA studies did not reveal any aberrant splicing (Hampel_2019). The following publications have been ascertained in the context of this evaluation (PMID: 31697235, 33309985, 16885385, 23403630, 21120944, 32980694, 33558524, 17594722, 16083711, 17510385, 22949387, 27629256, 19863800, 17210669, 28135145, 10534773). Seven submitters have provided clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. Based on the evidence outlined above, the variant was classified as uncertain significance.
Sema4, Sema4	RCV000568527	SCV002528700	uncertain significance	Hereditary cancer-predisposing syndrome	2021-10-07	criteria provided, single submitter	curation
All of Us Research Program, National Institutes of Health	RCV003997120	SCV004843245	likely benign	Lynch syndrome	2023-08-15	criteria provided, single submitter	clinical testing
ITMI	RCV000121365	SCV000085546	not provided	not specified	2013-09-19	no assertion provided	reference population