rs63749859

chr3-37000976-T-Cchr3-37000976-T-G

Variant summary

Our verdict is Pathogenic. Variant got 18 ACMG points: 18P and 0B. PM1 PM2 PM5 PP3_Strong PP5_Very_Strong

The NM_000249.4(MLH1):c.229T>C(p.Cys77Arg) variant causes a missense change involving the alteration of a conserved nucleotide. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a pathogenic outcome for this variant. 12/20 in silico tools predict a damaging outcome for this variant. Variant has been reported in ClinVar as Pathogenic (★★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. C77Y) has been classified as Pathogenic.

• Frequency: Genomes: not found (cov: 32)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Pathogenic reviewed by expert panel P:5
• Conservation: PhyloP100: 7.56

Genes affected

MLH1 (HGNC:7127): (mutL homolog 1) The protein encoded by this gene can heterodimerize with mismatch repair endonuclease PMS2 to form MutL alpha, part of the DNA mismatch repair system. When MutL alpha is bound by MutS beta and some accessory proteins, the PMS2 subunit of MutL alpha introduces a single-strand break near DNA mismatches, providing an entry point for exonuclease degradation. The encoded protein is also involved in DNA damage signaling and can heterodimerize with DNA mismatch repair protein MLH3 to form MutL gamma, which is involved in meiosis. This gene was identified as a locus frequently mutated in hereditary nonpolyposis colon cancer (HNPCC). [provided by RefSeq, Aug 2017]

ACMG classification

Verdict is Pathogenic. Variant got 18 ACMG points.

• PM1: In a hotspot region, there are 5 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 0 benign, 15 uncertain in NM_000249.4
• PM2: Very rare variant in population databases, with high coverage
• PM5: Other missense variant is known to change same aminoacid residue: Variant chr3-37000977-G-A is described in ClinVar as [Pathogenic]. Clinvar id is 90112.Status of the report is reviewed_by_expert_panel, 3 stars.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.991
• PP5: Variant 3-37000976-T-C is Pathogenic according to our data. Variant chr3-37000976-T-C is described in ClinVar as [Pathogenic]. Clinvar id is 90110.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37000976-T-C is described in Lovd as [Pathogenic]. Variant chr3-37000976-T-C is described in Lovd as [Benign].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.229T>C	p.Cys77Arg	missense_variant	3/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.229T>C	p.Cys77Arg	missense_variant	3/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 28

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:5

Revision: reviewed by expert panel

Submissions by phenotype

Hereditary nonpolyposis colon cancer Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Women's Health and Genetics/Laboratory Corporation of America, LabCorp

Jan 22, 2019

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. -

Lynch syndrome Pathogenic:1

Pathogenic, reviewed by expert panel

research

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Sep 05, 2013

Multifactorial likelihood analysis posterior probability >0.99 -

not provided Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

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. -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Invitae

Sep 17, 2021

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. -

Hereditary cancer-predisposing syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Dec 15, 2021

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. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	1.0
BayesDel_addAF	Pathogenic	0.55	D
BayesDel_noAF	Pathogenic	0.56
Cadd	Pathogenic	29
Dann	Uncertain	1.0
DEOGEN2	Pathogenic	0.94	D
Eigen	Pathogenic	0.98
Eigen_PC	Pathogenic	0.91
FATHMM_MKL	Pathogenic	0.97	D
LIST_S2	Pathogenic	0.98	D
M_CAP	Pathogenic	0.31	D
MetaRNN	Pathogenic	0.99	D
MetaSVM	Pathogenic	1.1	D
MutationAssessor	Pathogenic	3.9	H
MutationTaster	Benign	1.0	D;D;D;D;D;D
PrimateAI	Pathogenic	0.88	D
PROVEAN	Pathogenic	-10	D
REVEL	Pathogenic	0.96
Sift	Pathogenic	0.0	D
Sift4G	Pathogenic	0.0	D
Polyphen		1.0	D
Vest4		0.96
MutPred		0.96		Gain of disorder (P = 0.0348);
MVP		0.98
MPC		0.53
ClinPred		1.0	D
GERP RS		5.9
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7
Varity_R		1.0
gMVP		0.96

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.0		Details are displayed if max score is > 0.2

Find out detailed SpliceAI scores and Pangolin per-transcript scores at spliceailookup.broadinstitute.org

Publications

LitVar

Other links and lift over

dbSNP: rs63749859; hg19: chr3-37042467;