3-37014476-A-G

Variant summary

Our verdict is Uncertain significance. Variant got 3 ACMG points: 4P and 1B. PM1 PM2 BP4

The NM_000249.4(MLH1):c.722A>G(p.Lys241Arg) variant causes a missense change involving the alteration of a non-conserved nucleotide. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a benign outcome for this variant. 12/21 in silico tools predict a benign outcome for this variant. Variant has been reported in ClinVar as Uncertain significance (★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. K241E) has been classified as Uncertain significance.

• Frequency: Genomes: not found (cov: 33)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Uncertain significance criteria provided, multiple submitters, no conflicts U:5
• Conservation: PhyloP100: 3.31

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 Uncertain_significance. Variant got 3 ACMG points.

• PM1: In a hotspot region, there are 3 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 2 benign, 17 uncertain in NM_000249.4
• PM2: Very rare variant in population databases, with high coverage
• BP4: Computational evidence support a benign effect (MetaRNN=0.3189584).

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.722A>G	p.Lys241Arg	missense_variant	9/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.722A>G	p.Lys241Arg	missense_variant	9/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD3 exomes AF: 0.00000398 AC: 1 AN: 251282 Hom.: 0 AF XY: 0.00000736 AC XY: 1 AN XY: 135810

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.00

Gnomad SAS exome AF: 0.0000327

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.00

Gnomad OTH exome AF: 0.00

GnomAD4 exome Cov.: 28

GnomAD4 genome Cov.: 33

ExAC AF: 0.00000824 AC: 1

ClinVar

Significance: Uncertain significance

Submissions summary: Uncertain:5

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Hereditary cancer-predisposing syndrome Uncertain:3

Uncertain significance, criteria provided, single submitter	clinical testing	Ambry Genetics	Jul 17, 2017	The p.K241R variant (also known as c.722A>G), located in coding exon 9 of the MLH1 gene, results from an A to G substitution at nucleotide position 722. The lysine at codon 241 is replaced by arginine, an amino acid with highly similar properties. This amino acid position is well conserved in available vertebrate species; however, arginine is the reference amino acid in other vertebrate species. In addition, the in silico prediction for this alteration is inconclusive. In addition, this alteration is predicted to be benign by MAPP-MMR in silico analyses (Chao EC et al. Hum. Mutat. 2008 Jun;29:852-60). Since supporting evidence is limited at this time, the clinical significance of this alteration remains unclear. -
Uncertain significance, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Sep 18, 2023	This missense variant replaces lysine with arginine at codon 241 of the MLH1 protein. Computational prediction is inconclusive regarding the impact of this variant on protein structure and function (internally defined REVEL score threshold 0.5 < inconclusive < 0.7, PMID: 27666373). A functional study has shown that this variant does not affect MLH1 protein binding to MSH2 and MSH6 (PMID: 30770470). This variant has not been reported in individuals affected with hereditary cancer in the literature. This variant has been identified in 1/251282 chromosomes in the general population by the Genome Aggregation Database (gnomAD). The available evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance. -
Uncertain significance, criteria provided, single submitter	curation	Sema4, Sema4	Nov 23, 2021	- -

not provided Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

GeneDx

May 03, 2021

Not observed at a significant frequency in large population cohorts (Lek 2016); In silico analysis supports that this missense variant does not alter protein structure/function; Published functional studies demonstrate MSH2 and MSH6 binding affinity similar to wildtype (Zhang 2019); Has not been previously published as pathogenic or benign to our knowledge; This variant is associated with the following publications: (PMID: 30770470) -

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Invitae

Oct 12, 2022

This sequence change replaces lysine, which is basic and polar, with arginine, which is basic and polar, at codon 241 of the MLH1 protein (p.Lys241Arg). This variant is present in population databases (rs778528329, gnomAD 0.003%). This variant has not been reported in the literature in individuals affected with MLH1-related conditions. ClinVar contains an entry for this variant (Variation ID: 405379). 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 not expected to disrupt MLH1 protein function. In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Benign	0.069
BayesDel_addAF	Uncertain	0.10	D
BayesDel_noAF	Uncertain	0.010
Cadd	Benign	18
Dann	Uncertain	1.0
DEOGEN2	Uncertain	0.55	D;.
Eigen	Benign	-0.50
Eigen_PC	Benign	-0.36
FATHMM_MKL	Uncertain	0.96	D
LIST_S2	Benign	0.81	T;T
M_CAP	Benign	0.080	D
MetaRNN	Benign	0.32	T;T
MetaSVM	Benign	-0.32	T
MutationAssessor	Benign	0.68	N;.
MutationTaster	Benign	1.0	D;D;D;D;D;D
PrimateAI	Benign	0.44	T
PROVEAN	Benign	-1.7	N;N
REVEL	Uncertain	0.55
Sift	Benign	0.082	T;T
Sift4G	Benign	0.10	T;T
Polyphen		0.0060	B;.
Vest4		0.44
MutPred		0.46		Loss of ubiquitination at K241 (P = 0.0244);.;
MVP		0.97
MPC		0.072
ClinPred		0.17	T
GERP RS		2.0
Varity_R		0.22
gMVP		0.66

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: rs778528329; hg19: chr3-37055967;