Submissions for variant NM_000249.4(MLH1):c.229T>C (p.Cys77Arg)

dbSNP: rs63749859

Total submissions: 5

Submitter	RCV	SCV	Clinical significance	Condition	Last evaluated	Review status	Method	Comment
International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	RCV000075596	SCV000106593	pathogenic	Lynch syndrome	2013-09-05	reviewed by expert panel	research	Multifactorial likelihood analysis posterior probability >0.99
Ambry Genetics	RCV000220766	SCV000274513	pathogenic	Hereditary cancer-predisposing syndrome	2021-12-15	criteria provided, single submitter	clinical testing	The p.C77R pathogenic mutation (also known as c.229T>C) is located in coding exon 3 of the MLH1 gene. This alteration results from a T to C substitution at nucleotide position 229. The cysteine at codon 77 is replaced by arginine, an amino acid with highly dissimilar properties. This alteration was detected in multiple individuals meeting Amsterdam criteria (Pensabene M et al. Hered Cancer Clin Pract, 2016 Sep;14:18; Ambry internal data) and shows good segregation with disease in at least three families (Nystrom-Lahti M et al. Genes Chromosomes Cancer. 2002 Feb;33(2):160-7; Vietri MT et al. Med Oncol 2021 Jan;38(2):13). Functional analyses showed that this alteration does not affect dimerization with PMS2; however, an in vitro mismatch repair (MMR) assay indicated that it produces a nonfunctional MLH1 protein (Nystrom-Lahti M et al. Genes Chromosomes Cancer. 2002 Feb;33(2):160-7). In another study involving the same family, additional functional assays showed decreased protein expression, severely deficient MMR activity, and decreased protein subcellular localization (Raevaara TE et al. Gastroenterology. 2005 Aug;129(2):537-49). The C77R variant also demonstrated a loss of MMR function and reduced MMR efficiency in yeast and cell free in vitro assays (Ellison AR et al. Nucleic Acids Res. 2004 Oct 8;32(18):5321-38; Ou J et al. Hum Mutat. 2007 Nov;28(11):1047-54). 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 alteration is interpreted as a disease-causing mutation.
Women's Health and Genetics/Laboratory Corporation of America, LabCorp	RCV001192575	SCV001360803	pathogenic	Hereditary nonpolyposis colon cancer	2019-01-22	criteria provided, single submitter	clinical testing	Variant summary: MLH1 c.229T>C (p.Cys77Arg) results in a non-conservative amino acid change located in the Histidine kinase/HSP90-like ATPase domain (IPR003594) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 246190 control chromosomes (gnomAD). c.229T>C has been reported in the literature in multiple individuals affected with Lynch Syndrome, where the variant segregated with the disease (Panariello 1998, Nystrom-Lahti 2002). These data indicate that the variant is very likely to be associated with disease. Publications also reported experimental evidence evaluating an impact on protein function, demonstrating preserved interaction with PMS2, but deficient MMR function (Nystrom-Lahti 2002, Raevaara 2005, Borras 2012). In addition, based on a multifactorial likelihood analysis, the variant of interest was classified as pathogenic (Thompson 2014). A ClinVar submission from a clinical diagnostic laboratory (evaluation after 2014) cite the variant as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.
Invitae	RCV001854300	SCV002149596	pathogenic	Hereditary nonpolyposis colorectal neoplasms	2021-09-17	criteria provided, single submitter	clinical testing	For these reasons, this variant has been classified as Pathogenic. This variant disrupts the p.Cys77 amino acid residue in MLH1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 12810663, 17135187, 17210669, 17510385, 22949379, 22949387). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. Experimental studies have shown that this missense change affects MLH1 function (PMID: 11793442, 15475387, 16083711, 17210669, 22736432). 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. ClinVar contains an entry for this variant (Variation ID: 90110). This missense change has been observed in individual(s) with clinical features of Lynch syndrome (PMID: 10660333, 11793442, 27602174). This variant is not present in population databases (ExAC no frequency). This sequence change replaces cysteine with arginine at codon 77 of the MLH1 protein (p.Cys77Arg). The cysteine residue is highly conserved and there is a large physicochemical difference between cysteine and arginine.
Department of Pathology and Laboratory Medicine, Sinai Health System	RCV001358424	SCV001554150	pathogenic	not provided		no assertion criteria provided	clinical testing	The MLH1 p.Cys77Arg was shown to decrease MMR activity in multiple functional assays using yeast, increasing the likelihood that it has clinical significance (Ellison 2004, Wanat 2007). Raevaara et al (2005) used site directed mutagenesis to assess non-truncating MLH1 variants for protein expression/stability, subcellular localization, protein-protein interaction and repair efficiency, with the finding that the variant was pathogenic based on deficient MMR activity, decreased protein expression and amino acid conservation. Nystrom-Lahti et al (2002) tested the functionality of this variant with similar findings of MMR deficiency with no effect on hetero-dimerization with PMS2, implying pathogenicity based on nonfunctional protein production. An in silico model using multivariate analysis also indicated that the variant is pathogenic, with a MAPP-MMR score of >4.55 (Chao 2008). The variant was identified in dbSNP (ID: rs63749859) as “With Pathogenic allele”, Clinvitae database (classification pathogenic), Mismatch Repair Genes Variant Database, MMR Gene Unclassified Variants Database, InSiGHT Colon Cancer Gene Variant Database (26x, as Pathogenic), ClinVar database (classification pathogenic, submitters InSIGHT and Ambry Genetics), and UMD (2x with an “unclassified variant” classification). This variant was not identified in the 1000 Genomes Project, NHLBI GO Exome Sequencing Project, the Exome Aggregation Consortium database (August 8, 2016), Zhejiang Colon Cancer Database (LOVD), COSMIC, and GeneInsight-COGR database. The p.Cys77 residue is conserved across mammals and other organisms, and computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) suggest that the variant may impact the protein; however, this information is not predictive enough to assume pathogenicity. The variant occurs outside of the splicing consensus sequence and two of five in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) predict a greater than 10% difference in splicing, however, this information is not predictive enough to assume pathogenicity. In summary, based on the above information, this variant is classified as pathogenic.