rs63750850

Positions:

• chr3-36996689-G-A
• chr3-36996689-G-C
• chr3-36996689-G-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.187G>A​(p.Asp63Asn) 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. 13/21 in silico tools predict a damaging 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. D63E) has been classified as Pathogenic.

• Frequency: Genomes: not found (cov: 32)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Pathogenic/Likely pathogenic criteria provided, multiple submitters, no conflicts P:6
• Conservation: PhyloP100: 9.51

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 (HGNC:):

ACMG classification

Verdict is Pathogenic. Variant got 18 ACMG points.

• PM1: In a binding_site (size 0) in uniprot entity MLH1_HUMAN there are 4 pathogenic changes around while only 1 benign (80%) 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 chr3-36996691-C-A is described in Lovd as [Pathogenic].
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.994
• PP5: Variant 3-36996689-G-A is Pathogenic according to our data. Variant chr3-36996689-G-A is described in ClinVar as [Likely_pathogenic]. Clinvar id is 89920.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars. Variant chr3-36996689-G-A is described in Lovd as [Pathogenic]. Variant chr3-36996689-G-A is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
MLH1	NM_000249.4	c.187G>A	p.Asp63Asn	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.187G>A	p.Asp63Asn	missense_variant	2/19	1	NM_000249.4	ENSP00000231790.3

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 30

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic/Likely pathogenic

Submissions summary: Pathogenic:6

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Hereditary cancer-predisposing syndrome Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	May 23, 2024	The p.D63N pathogenic mutation (also known as c.187G>A), located in coding exon 2 of the MLH1 gene, results from a G to A substitution at nucleotide position 187. The aspartic acid at codon 63 is replaced by asparagine, an amino acid with highly similar properties. This alteration was reported in a Hungarian family satisfying Amsterdam I criteria for HNPCC/Lynch syndrome (Papp J et al. World J. Gastroenterol. 2007 May; 13(19):2727-32). In this family, p.D63N segregated with disease, being detected in the affected proband (colorectal cancer at 25y) and his affected father (colorectal cancer at 40y) and was absent from 7 cancer-free relatives. This variant has also been identified in multiple individuals with tumors demonstrating loss of MLH1 and PMS2 by immunohistochemistry (Ambry internal data). An in vitro functional study reported that the p.D63N variant abolished ATP binding in the hMLH1 subunit, deleteriously affecting expression of the heterodimer (Räschle M et al. J. Biol. Chem. 2002 Jun;277:21810-20). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). 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. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Feb 05, 2020	This missense variant replaces aspartic acid with asparagine at codon 63 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). Splice site prediction tools suggest that this variant may not impact RNA splicing. An experimental study has shown that this variant affects protein stability (PMID: 11948175). This variant has been reported in two related individuals affected with colorectal cancer and absent in multiple asymptomatic family members (PMID: 17569143). Different missense variants affecting the same codon, p.Asp63Glu and p.Asp63Gly are associated with disease (Clinvar variation ID: 89934, 422297), indicating that aspartic acid residue at this position is important for MLH1 function. This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Likely Pathogenic. -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Baylor Genetics

Jun 30, 2022

- -

Lynch syndrome Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

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

Feb 26, 2019

Variant summary: MLH1 c.187G>A (p.Asp63Asn) results in a conservative amino acid change located in the Histidine kinase/HSP90-like ATPase domain of the encoded protein sequence. Four of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 246206 control chromosomes. c.187G>A has been reported in the literature in individuals affected with Lynch Syndrome (Papp_2007, Espenschied_2017). These data indicate that the variant may be associated with disease. At least one publication reports experimental evidence evaluating an impact on protein function. The most pronounced variant effect results in dramatically reduced expression of both MLH1 and PMS2 proteins and reduced ATP'ase activity (Raschle_2002). Three clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories classified the variant as pathogenic/likely pathogenic. Based on the evidence outlined above, the variant was classified as likely pathogenic. -

not provided Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

GeneDx

Sep 29, 2016

This variant is denoted MLH1 c.187G>A at the cDNA level, p.Asp63Asn (D63N) at the protein level, and results in the change of an Aspartic Acid to an Asparagine (GAC>AAC). MLH1 Asp63Asn was observed in a Hungarian family meeting Amsterdam I criteria and reported to co-segregate with disease in two family members with a history of early-onset colon cancer but was not present in seven unaffected family members (Papp 2007). Raschle et. al. (2002) showed that MLH1 Asp63Asn results in a dramatic reduction in MLH1 and PMS2 protein expression. In addition, another variant at the same residue, MLH1 Asp63Glu, is considered pathogenic by the International Society for Gastrointestinal Hereditary Tumours Incorporated (InSiGHT). MLH1 Asp63Glu has been reported to segregate with Lynch syndrome-related cancers in at least one family and has been shown to result in decreased MLH1 protein expression, deficient mismatch repair activity, and decreased nuclear localization of MLH1 and PMS2 (Raevaara 2005, Hardt 2011). MLH1 Asp63Asn was not observed in approximately 6,500 individuals of European and African American ancestry in the NHLBI Exome Sequencing Project, indicating it is not a common benign variant in these populations. Since Aspartic Acid and Asparagine differ in some properties, this is considered a semi-conservative amino acid substitution. MLH1 Asp63Asn occurs at a position that is conserved across species and is located in ATP-binding domain (Raevaara 2005). In addition, in silico analyses predict that this variant is probably damaging to protein structure and function. Based on currently available information, we consider MLH1 Asp63Asn to be a likely pathogenic variant. -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Aug 17, 2023

This sequence change replaces aspartic acid, which is acidic and polar, with asparagine, which is neutral and polar, at codon 63 of the MLH1 protein (p.Asp63Asn). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with Lynch syndrome (PMID: 17569143). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 89920). 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: 11948175). This variant disrupts the p.Asp63 amino acid residue in MLH1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 16083711, 17594722, 21120944). 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	0.33	D
BayesDel_noAF	Pathogenic	0.23
CADD	Pathogenic	32
DANN	Pathogenic	1.0
DEOGEN2	Pathogenic	0.97	D
Eigen	Pathogenic	1.2
Eigen_PC	Pathogenic	1.1
FATHMM_MKL	Pathogenic	0.98	D
LIST_S2	Pathogenic	1.0	D
M_CAP	Pathogenic	0.63	D
MetaRNN	Pathogenic	0.99	D
MetaSVM	Pathogenic	0.94	D
MutationAssessor	Pathogenic	5.0	H
PrimateAI	Pathogenic	0.79	T
PROVEAN	Pathogenic	-4.6	D
REVEL	Pathogenic	0.95
Sift	Pathogenic	0.0	D
Sift4G	Pathogenic	0.0	D
Polyphen		1.0	D
Vest4		0.98
MutPred		0.98		Gain of MoRF binding (P = 0.2407);
MVP		0.99
MPC		0.40
ClinPred		1.0	D
GERP RS		5.6
Varity_R		0.99
gMVP		0.94

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: rs63750850; hg19: chr3-37038180;