rs63751094

Positions:

• chr3-36996624-A-G
• chr3-36996624-A-T

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.122A>G​(p.Asp41Gly) 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 Pathogenic (★★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. D41H) 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: 8.87

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 helix (size 13) in uniprot entity MLH1_HUMAN there are 19 pathogenic changes around while only 8 benign (70%) 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 chr3-36996623-G-C is described in Lovd as [Pathogenic].
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.992
• PP5: Variant 3-36996624-A-G is Pathogenic according to our data. Variant chr3-36996624-A-G is described in ClinVar as [Pathogenic]. Clinvar id is 89684.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-36996624-A-G is described in Lovd as [Pathogenic]. Variant chr3-36996624-A-G is described in Lovd as [Benign].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
MLH1	NM_000249.4	c.122A>G	p.Asp41Gly	missense_variant	2/19	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.122A>G	p.Asp41Gly	missense_variant	2/19	1	NM_000249.4	ENSP00000231790	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 30

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:5

Revision: reviewed by expert panel

Submissions by phenotype

Lynch syndrome Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Cancer Variant Interpretation Group UK, Institute of Cancer Research, London	Jul 13, 2018	Data used in classification: Single UK family with 11 informative meiosis (1:2048) (PP1_very_strong). Same amino acid residue as Asp41His (classified by INSIGHT as pathogenic) (PM5). The variant was observed in 3 independent UK families undergoing clinical diagnostic testing. There are additional reports of this variant on InSight (11 entries) and MLH1 LOVD (10 entries) (PS4_mod). The variant is absent GNOMAD NFE (63,369 individuals) and also in the remainder of the GNOMAD populations (75,263 individuals) (PM2_mod). 2/2 UK pedigrees for which we have tumour data demonstrated MSI in the tumour of the proband. Kruger S et al. Hum Mutat. 2004;24(4):351-2 report Amsterdam positive family (proband with colon cancer at 35 (ascending); [father: metachronous colon cancers: ascending colon (44), transverse colon (52) paternal grandmother: colon cancer (40)] ) with MSI-high but no loss of expression on IHC (PS3_mod). Same amino acid residue as Asp41His (classified by INSIGHT as pathogenic) (PM5). Predicted deleterious on AlignGVGD, SIFT, Polyphen2 HumVar (and also MAPP) (PP3). Additional Information (not included in classification): mRNA studies using sample from affected patient undertaken at molecular diagnostic laboratory demonstrated frame-shifting alteration in cDNA sequence: Minigene reported in Van der klift HM, et al. Mol Genet Genomic Med. 2015;3(4):327-45, reports mixed sequence products. Prior classification as Class 5 using multifactorial analysis (Insight paper 2012). Comment: Initially it was thought that this variant exerted pathogenicity through effect on splicing. However, in silico splicing predictions and splicing assay results are mixed. It may exert effect through missense impact on protein. Regardless of mechanisms, there are (i) multiple lines of generic functional evidence supporting this variant resulting in MMR deficiency and (ii) extremely strong genetic epidemiological evidence supporting pathogenicity (which is likewise agnostic to mechanism). -
Pathogenic, reviewed by expert panel	curation	International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	Dec 19, 2018	Multifactorial likelihood analysis posterior probability >0.99 (1.000) -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Myriad Genetics, Inc.

Jul 11, 2023

This variant is considered likely pathogenic. Functional studies indicate this variant impacts protein function [PMID: 23403630]. This variant is expected to disrupt protein structure [Myriad internal data]. -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Sep 22, 2022

This variant is not present in population databases (gnomAD no frequency). For these reasons, this variant has been classified as Pathogenic. This variant disrupts the p.Asp41 amino acid residue in MLH1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 12624141, 19419416, 25060679, 25477341; Invitae). 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: 15475387, 23403630). 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: 89684). This missense change has been observed in individuals with Lynch syndrome (PMID: 15300854, 15365996, 15849733, 20233461, 28874130, 29360550). This sequence change replaces aspartic acid, which is acidic and polar, with glycine, which is neutral and non-polar, at codon 41 of the MLH1 protein (p.Asp41Gly). -

Hereditary cancer-predisposing syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Jan 11, 2022

The p.D41G variant (also known as c.122A>G), located in coding exon 2 of the MLH1 gene, results from an A to G substitution at nucleotide position 122. The aspartic acid at codon 41 is replaced by glycine, an amino acid with similar properties. This variant has been detected in multiple patients who meet Amsterdam criteria and/or have colon cancer (Krüger S et al. Hum. Mutat., 2004 Oct;24:351-2; Wardell CP et al. J. Hepatol., 2018 05;68:959-969; Rossi BM et al. BMC Cancer, 2017 Sep;17:623; van der Klift HM et al. Hum. Mutat., 2016 11;37:1162-1179; Pigatto F et al. Hered Cancer Clin Pract, 2004 Nov;2:175-84; Mangold E et al. Int. J. Cancer, 2005 Sep;116:692-702; Sharp A et al. Hum. Mutat., 2004 Sep;24:272; Hardt K et al. Fam. Cancer, 2011 Jun;10:273-84). This variant demonstrated reduced expression and deficient mismatch repair (MMR) activity in an in vitro complementation assay (Hinrichsen I et al. Clin. Cancer Res., 2013 May;19:2432-41). In a yeast-based functional assay, this variant demonstrated partial loss of MMR activity (Ellison AR et al. Nucleic Acids Res., 2004 Oct;32:5321-38). RNA studies for this variant based on minigene assay and patient RNA analysis demonstrated out-of-frame exon 2 skipping, but this could not be confirmed in subsequent studies (Sharp A et al. Hum. Mutat., 2004 Sep;24:272; Tournier I et al. Hum. Mutat., 2008 Dec;29:1412-24; van der Klift HM et al. Mol Genet Genomic Med, 2015 Jul;3:327-45). 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	0.99
BayesDel_addAF	Pathogenic	0.54	D
BayesDel_noAF	Pathogenic	0.54
CADD	Pathogenic	30
DANN	Benign	0.90
DEOGEN2	Pathogenic	0.97	D
Eigen	Pathogenic	0.83
Eigen_PC	Pathogenic	0.79
FATHMM_MKL	Pathogenic	0.98	D
LIST_S2	Pathogenic	1.0	D
M_CAP	Pathogenic	0.52	D
MetaRNN	Pathogenic	0.99	D
MetaSVM	Pathogenic	0.87	D
MutationAssessor	Pathogenic	5.0	H
MutationTaster	Benign	1.0	D;D;D;D;D;D
PrimateAI	Uncertain	0.74	T
PROVEAN	Pathogenic	-6.2	D
REVEL	Pathogenic	0.94
Sift	Pathogenic	0.0	D
Sift4G	Pathogenic	0.0	D
Polyphen		1.0	D
Vest4		0.97
MutPred		0.97		Gain of ubiquitination at K43 (P = 0.1079);
MVP		0.99
MPC		0.46
ClinPred		1.0	D
GERP RS		5.8
Varity_R		0.98
gMVP		0.91

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.010		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: rs63751094; hg19: chr3-37038115; COSMIC: COSV51613778; COSMIC: COSV51613778;