3-37000991-A-G

Position:

• chr3-37000991-A-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.244A>G​(p.Thr82Ala) variant causes a missense change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.00000274 in 1,459,020 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a pathogenic outcome for this variant. Variant has been reported in ClinVar as Likely pathogenic (★★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. T82I) has been classified as Pathogenic.

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 0.0000027 (0 hom.)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Likely pathogenic reviewed by expert panel P:13
• Conservation: PhyloP100: 8.79

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 4 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 chr3-37000992-C-T is described in ClinVar as [Pathogenic]. Clinvar id is 90118.Status of the report is reviewed_by_expert_panel, 3 stars.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.978
• PP5: Variant 3-37000991-A-G is Pathogenic according to our data. Variant chr3-37000991-A-G is described in ClinVar as [Likely_pathogenic]. Clinvar id is 90116.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37000991-A-G is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.244A>G	p.Thr82Ala	missense_variant	3/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.244A>G	p.Thr82Ala	missense_variant	3/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD3 exomes AF: 0.00000796 AC: 2 AN: 251394 Hom.: 0 AF XY: 0.00000736 AC XY: 1 AN XY: 135878

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.0000992

Gnomad EAS exome AF: 0.00

Gnomad SAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.00000880

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.00000274 AC: 4 AN: 1459020 Hom.: 0 Cov.: 29 AF XY: 0.00000275 AC XY: 2 AN XY: 726086

Gnomad4 AFR exome AF: 0.00

Gnomad4 AMR exome AF: 0.00

Gnomad4 ASJ exome AF: 0.0000383

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.0000116

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.00000180

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 32

Bravo AF: 0.00000756

ExAC AF: 0.00000824 AC: 1

ClinVar

Significance: Likely pathogenic

Submissions summary: Pathogenic:13

Revision: reviewed by expert panel

Submissions by phenotype

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:3

Likely pathogenic, criteria provided, single submitter	clinical testing	Baylor Genetics	Jul 23, 2023	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	Myriad Genetics, Inc.	Jul 12, 2023	This variant is considered likely pathogenic. Functional studies indicate this variant impacts protein function [PMID: 22736432]. This variant is expected to disrupt protein structure [Myriad internal data]. -
Pathogenic, criteria provided, single submitter	research	Centre for Mendelian Genomics, University Medical Centre Ljubljana	Dec 09, 2022	PS3, PS4_STR, PM2_SUP, PM5, PP1 -

Lynch syndrome Pathogenic:3

Likely pathogenic, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Mar 14, 2016	Variant summary: This c.244A>G variant affects a conserved nucleotide, resulting in amino acid change from Thr to Ala. 4/4 in-silico tools predict this variant to be damaging. This variant was found in 1/122056 control chromosomes from the broad and large populations of ExAC at a frequency of 0.0000082, which does not exceed maximal expected frequency of a pathogenic allele (0.0007105) in this gene. This variant has been reported in multiple patients (at least five independent occurrences) affected with HNPCC or HNPCC-related cancers. There are no reported cosegregation studies for this variant. The variant has been functionally characterized to impair the MMR activity, strongly suggesting for pathogenic outcome. Multiple clinical laboratories as well as reputable databases have classified this variant as likely pathogenic. Multifactorial likelihood analysis for the variant is also in line with likely pathogenic outcome. Taken together this variant has currently been classfied as a Probable Disease Variant/Likely Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Mar 02, 2023	This missense variant replaces threonine with alanine at codon 82 of the MLH1 protein. Computational prediction suggests that this variant may have deleterious impact on protein structure and function (internally defined REVEL score threshold >= 0.7, PMID: 27666373). Functional studies have shown that the mutant protein shows <10% of normal DNA-mismatch repair activity in vitro (PMID: 22736432, 31881334) (PMID: 22736432, 31881334). This variant has been reported in individuals affected with Lynch syndrome (PMID: 21239990, 26300997) and in individuals affected with colorectal cancer with tumors showing microsatellite instability (PMID: 21404117, 22736432). This variant has been identified in 2/251394 chromosomes in the general population by the Genome Aggregation Database (gnomAD). A different missense variant p.Thr82Ile is known to be pathogenic, indicating the importance of threonine residue at this position for MLH1 function. Based on the available evidence, this variant is classified as Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	May 13, 2020	The p.Thr82Ala variant in MLH1 has been reported in 10 European individuals with Lynch syndrome or MLH1-associated cancers (Sjursen 2010, Hardt 2011, Pastrello 2011, Simbolo 2015, Stradella 2019). It has also been identified in 2/123,762 European/Ashkenazi Jewish chromosomes by gnomAD (http://gnomad.broadinstitute.org). Computational prediction tools and conservation analysis suggest that this variant may impact the protein, though this information is not predictive enough to determine pathogenicity. In vitro functional studies provide some evidence that this variant impacts protein function (Ellison 2004, Borras 2012). This variant was classified as Likely Pathogenic on June 13, 2018 by the ClinGen-approved InSiGHT expert panel (Variation ID 90116). Another variant involving this codon (p.Thr82Ile) has been identified in individuals with Lynch syndrome and is classified as pathogenic by the ClinGen-approved InSiGHT expert panel. In summary, this variant meets criteria to be classified as pathogenic for autosomal dominant Lynch syndrome. ACMG/AMP Criteria applied: PM2, PS4_Strong, PP3, PS3_Supporting, PM5. -

Hereditary cancer-predisposing syndrome Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	May 16, 2023	This missense variant replaces threonine with alanine at codon 82 of the MLH1 protein. Computational prediction suggests that this variant may have deleterious impact on protein structure and function (internally defined REVEL score threshold >= 0.7, PMID: 27666373). Functional studies have shown that the mutant protein shows <10% of normal DNA-mismatch repair activity in vitro (PMID: 22736432, 31881334). This variant has been reported in individuals affected with Lynch syndrome (PMID: 21239990, 26300997) and in individuals affected with colorectal cancer with tumors showing microsatellite instability (PMID: 21404117, 22736432). This variant has been identified in 2/251394 chromosomes in the general population by the Genome Aggregation Database (gnomAD). A different missense variant occurring at the same codon, p.Thr82Ile, is known to be disease-causing, indicating the importance of threonine residue at this position for MLH1 function. Based on the available evidence, this variant is classified as Pathogenic. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Mar 28, 2020	The p.T82A variant (also known as c.244A>G), located in coding exon 3 of the MLH1 gene, results from an A to G substitution at nucleotide position 244. The threonine at codon 82 is replaced by alanine, an amino acid with similar properties. This variant has been identified in probands with HNPCC or HNPCC-related cancers and concordant tumors when available (Pastrello C et al. Genet Med. 2011 Feb;13(2):115-24,Hardt K et al. Fam Cancer. 2011 Jun;10(2):273-84, Borras E et al. Hum Mutat. 2012 Nov;33(11):1576-88, Simbolo M et al. Hered. Cancer Clin Pract. 2015 Aug 21;13(1), Ambry internal data). This variant is located in the ATPase functional domain and impaired MMR activity compared to wild-type (Borras E et al. Hum Mutat. 2012 Nov;33(11)). This paper also cites literature demonstrating alterations at the same codon, including p.T82K, p.T82M, and p.T82I, to be defective in MMR activity in yeast and/or human cells. This alteration has been classified as pathogenic by multifactorial analysis, which integrates the following lines of evidence to produce a quantitative likelihood of pathogenicity: in silico prediction models, segregation with disease, tumor characteristics, mutation co-occurrence, and functional assay results (Thompson B et al. Nat Genet. 2014 Feb;46(2):107-15; available at [www.insight-group.org/variants/classifications/]). 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. -

not specified Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

ARUP Laboratories, Molecular Genetics and Genomics, ARUP Laboratories

Dec 27, 2016

- -

Carcinoma of colon Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

The p.Thr82Ala variant has been previously reported in the literature and by our laboratory. It was identified in one individual who was reported to have high microsatellite instability and was MLH1 deficient. In addition, it was shown to segregate in one affected family member (Pastrello_2011_21239990). Our laboratory has identified this variant in one family and the variant segregated with disease in 5 affected individuals. Although the pattern of MSI and IHC deficient tumours was inconsistent across individuals from this family, the observation of this variant segregating with Lynch syndrome related cancers increases the likelihood this variant is pathogenic. In addition, functional studies in yeast demonstrated that different variants at this same amino acid residue (T82S, T82K, T82M) were associated with a loss of mismatch repair function between 8% and 67%, increasing the likelihood that this variant may affect normal protein function (Ellison_2004_15475387). In summary, based on the above information, this variant is classified as pathogenic. -

Lynch syndrome 1 Pathogenic:1

Likely pathogenic, reviewed by expert panel

curation

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Jun 13, 2018

Variant reclassification due to new tumour Likelihood Ratios: Multifactorial likelihood analysis posterior probability > 0.95 (0.965) -

not provided Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital

Jul 31, 2024

- -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Feb 08, 2023

This sequence change replaces threonine, which is neutral and polar, with alanine, which is neutral and non-polar, at codon 82 of the MLH1 protein (p.Thr82Ala). This variant is present in population databases (rs587778998, gnomAD 0.01%). This missense change has been observed in individuals with Lynch syndrome (PMID: 21239990, 21404117, 22736432, 26300997, 28514183). ClinVar contains an entry for this variant (Variation ID: 90116). 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. Experimental studies have shown that this missense change affects MLH1 function (PMID: 22736432). This variant disrupts the p.Thr82 amino acid residue in MLH1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 10422993, 17510385, 19690142, 20587412, 23403630). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.98
BayesDel_addAF	Pathogenic	0.52	D
BayesDel_noAF	Pathogenic	0.50
CADD	Pathogenic	28
DANN	Uncertain	1.0
DEOGEN2	Pathogenic	0.96	D
Eigen	Pathogenic	1.1
Eigen_PC	Pathogenic	1.0
FATHMM_MKL	Pathogenic	0.98	D
LIST_S2	Pathogenic	1.0	D
M_CAP	Uncertain	0.26	D
MetaRNN	Pathogenic	0.98	D
MetaSVM	Pathogenic	1.1	D
MutationAssessor	Pathogenic	5.0	H
MutationTaster	Benign	1.0	D;D;D;D;D;D
PrimateAI	Uncertain	0.62	T
PROVEAN	Pathogenic	-4.6	D
REVEL	Pathogenic	0.92
Sift	Pathogenic	0.0	D
Sift4G	Pathogenic	0.0	D
Polyphen		1.0	D
Vest4		0.87
MutPred		0.89		Loss of ubiquitination at K84 (P = 0.0982);
MVP		0.99
MPC		0.40
ClinPred		1.0	D
GERP RS		5.9
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7
Varity_R		0.95
gMVP		0.89

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: rs587778998; hg19: chr3-37042482; COSMIC: COSV51620354;