3-37014485-G-T

Variant names:

• chr3-37014485-G-T
• rs63750303
• NM_000249.4:c.731G>T

Variant summary

Our verdict is Likely pathogenic. The variant received 8 ACMG points: 8P and 0B. PM1 PM2 PM5 PP3 PP5

The NM_000249.4(MLH1):​c.731G>T​(p.Gly244Val) 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. Variant has been reported in ClinVar as Conflicting classifications of pathogenicity (no stars). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. G244D) has been classified as Pathogenic.

• Frequency: Genomes: not found (cov: 33)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:3 U:2
• Conservation: PhyloP100: 9.52
• Publications: 32 publications found

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]

MLH1 Gene-Disease associations (from GenCC):

• Lynch syndrome

  Inheritance: AD Classification: DEFINITIVE, SUPPORTIVE Submitted by: G2P, ClinGen, Orphanet
• Lynch syndrome 2

  Inheritance: AD Classification: DEFINITIVE, STRONG Submitted by: Ambry Genetics, Genomics England PanelApp
• Muir-Torre syndrome

  Inheritance: AD Classification: DEFINITIVE, STRONG, MODERATE, SUPPORTIVE Submitted by: Genomics England PanelApp, Ambry Genetics, G2P, Orphanet
• mismatch repair cancer syndrome 1

  Inheritance: AR Classification: DEFINITIVE, STRONG, SUPPORTIVE Submitted by: Ambry Genetics, G2P, Labcorp Genetics (formerly Invitae), Orphanet, ClinGen
• Lynch syndrome 1

  Inheritance: AD Classification: STRONG Submitted by: Labcorp Genetics (formerly Invitae)
• ovarian cancer

  Inheritance: AD Classification: STRONG Submitted by: Genomics England PanelApp
• malignant pancreatic neoplasm

  Inheritance: AD Classification: MODERATE Submitted by: Genomics England PanelApp
• rhabdomyosarcoma

  Inheritance: AR Classification: MODERATE Submitted by: Genomics England PanelApp
• prostate cancer

  Inheritance: AD Classification: LIMITED Submitted by: Ambry Genetics
• breast cancer

  Inheritance: AD Classification: NO_KNOWN Submitted by: Ambry Genetics
• hereditary breast carcinoma

  Inheritance: AD Classification: NO_KNOWN Submitted by: ClinGen

ACMG classification

Our verdict: Likely_pathogenic. The variant received 8 ACMG points.

• PM1: In a hotspot region, there are 2 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 2 benign, 38 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 chr3-37014485-G-A is described in ClinVar as Pathogenic. ClinVar VariationId is 90339.Status of the report is reviewed_by_expert_panel, 3 stars.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.809
• PP5: Variant 3-37014485-G-T is Pathogenic according to our data. Variant chr3-37014485-G-T is described in ClinVar as Conflicting_classifications_of_pathogenicity. ClinVar VariationId is 90340.

Variant Effect in Transcripts

ACMG analysis was done for transcript: NM_000249.4. You can select a different transcript below to see updated ACMG assignments.

RefSeq Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	MLH1	NM_000249.4	MANE Select	c.731G>T	p.Gly244Val	missense	Exon 9 of 19	NP_000240.1
	MLH1	NM_001354628.2		c.731G>T	p.Gly244Val	missense	Exon 9 of 18	NP_001341557.1
	MLH1	NM_001354629.2		c.632G>T	p.Gly211Val	missense	Exon 8 of 18	NP_001341558.1

Ensembl Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	MLH1	ENST00000231790.8	TSL:1 MANE Select	c.731G>T	p.Gly244Val	missense	Exon 9 of 19	ENSP00000231790.3
	MLH1	ENST00000456676.7	TSL:1	c.731G>T	p.Gly244Val	missense	Exon 9 of 17	ENSP00000416687.3
	MLH1	ENST00000413740.2	TSL:1	c.731G>T	p.Gly244Val	missense	Exon 9 of 15	ENSP00000416476.2

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD4 exome Cov.: 28

GnomAD4 genome Cov.: 33

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:3 Uncertain:2

Revision: criteria provided, conflicting classifications

Submissions by phenotype

Lynch syndrome Uncertain:2

May 01, 2018

University of Washington Department of Laboratory Medicine, University of Washington

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

MLH1 NM_000249.3:c.731G>T has a 74.5% probability of pathogenicity based on combining prior probability from public data with a likelihood ratio of 26.5 to 1, generated from evidence of seeing this as a somatic mutation in a tumor with loss of heterozygosity at the MLH1 locus. See Shirts et al 2018, PMID 29887214.

Apr 19, 2019

Service de Génétique Médicale, Institut Central des Hôpitaux

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1

Jul 17, 2023

Myriad Genetics, Inc.

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant is considered likely pathogenic. Functional studies indicate this variant impacts protein function [PMID: 30998989, 17510385]. This variant is expected to disrupt protein structure [Myriad internal data].

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Aug 04, 2020

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

For these reasons, this variant has been classified as Pathogenic. A different missense substitution at this codon (p.Gly244Asp) has been determined to be likely pathogenic (PMID: 16982745, 17594722). This suggests that the glycine residue is critical for MLH1 protein function and that other missense substitutions at this position may also be pathogenic. Experimental studies have shown that this missense change results in impaired mismatch repair function in vitro (PMID: 17510385, 30998989, 31697235). This variant has been reported in individuals affected with clinical features of Lynch syndrome (PMID: 21642682, 30998989, Invitae). ClinVar contains an entry for this variant (Variation ID: 90340). This variant is not present in population databases (ExAC no frequency). This sequence change replaces glycine with valine at codon 244 of the MLH1 protein (p.Gly244Val). The glycine residue is highly conserved and there is a moderate physicochemical difference between glycine and valine.

Hereditary cancer-predisposing syndrome Pathogenic:1

Oct 31, 2023

Ambry Genetics

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The p.G244V variant (also known as c.731G>T), located in coding exon 9 of the MLH1 gene, results from a G to T substitution at nucleotide position 731. The glycine at codon 244 is replaced by valine, an amino acid with dissimilar properties. This alteration has been detected in a family meeting Amsterdam II criteria for Lynch syndrome and the tumor of the proband demonstrated high microsatellite instability, but had normal mismatch repair (MMR) protein expression by immunohistochemistry (Ambry internal data). In one study, protein expression levels in cells expressing this variant were determined to be greater than 75% when compared to the wild type level and MMR activity was reduced compared wild type MLH1 in a complementation assay (Takahashi M et al. Cancer Res. 2007 May;67(10):4595604). This variant also demonstrated deficient activity in a methylation tolerance-based functional assay (Bouvet D et al. Gastroenterology, 2019 08;157:421-431). In another study, steady state levels for this variant were reported to be reduced compared to wild type MLH1 and the p.G244V substitution was predicted to result in destabilization relative to wild type MLH1 (Abildgaard AB et al. Elife, 2019 11;8). The equivalent allele in Bacillus subtilis MutL demonstrated reduced MMR activity and expression, but did not confer a dominant negative effect (Bolz NJ et al. J Bacteriol, 2012 Oct;194:5361-7). Based on internal structural analysis, this variant is anticipated to result in a significant decrease in structural stability (Ambry internal data; Wu H et al. Acta Crystallogr F Struct Biol Commun, 2015 Aug;71:981-5). 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 majority of available evidence to date, this variant is likely to be pathogenic.

Computational scores

Source: dbNSFP v4.9

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.86
BayesDel_addAF	Pathogenic	0.49	D
BayesDel_noAF	Pathogenic	0.47
CADD	Pathogenic	28
DANN	Uncertain	1.0
DEOGEN2	Pathogenic	0.86	D
Eigen	Uncertain	0.68
Eigen_PC	Pathogenic	0.67
FATHMM_MKL	Pathogenic	1.0	D
LIST_S2	Uncertain	0.95	D
M_CAP	Pathogenic	0.41	D
MetaRNN	Pathogenic	0.81	D
MetaSVM	Uncertain	0.45	D
MutationAssessor	Pathogenic	3.7	H
PhyloP100		9.5
PrimateAI	Uncertain	0.61	T
PROVEAN	Pathogenic	-6.7	D
REVEL	Pathogenic	0.78
Sift	Uncertain	0.013	D
Sift4G	Uncertain	0.016	D
Polyphen		0.80	P
Vest4		0.96
MutPred		0.43		Loss of sheet (P = 0.0457)
MVP		0.99
MPC		0.43
ClinPred		0.99	D
GERP RS		5.7
Varity_R		0.96
gMVP		0.92
Mutation Taster		=0/100	disease causing (ClinVar)

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

Other links and lift over

dbSNP: rs63750303; hg19: chr3-37055976; COSMIC: COSV51615490;