rs63751194

chr3-37017508-C-Achr3-37017508-C-Gchr3-37017508-C-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.793C>A(p.Arg265Ser) variant causes a missense, splice region 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. 1/1 splice prediction tools predict no significant impact on normal splicing. Variant has been reported in ClinVar as Pathogenic (★★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. R265C) has been classified as Pathogenic.

• Frequency: Genomes: not found (cov: 32)
• Consequence: MLH1 NM_000249.4 missense, splice_region
• Clinical Significance: Pathogenic reviewed by expert panel P:9
• Conservation: PhyloP100: 7.28

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 2 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
• PM5: Other missense variant is known to change same aminoacid residue: Variant chr3-37017508-C-T is described in ClinVar as [Pathogenic]. Clinvar id is 29654.Status of the report is reviewed_by_expert_panel, 3 stars.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.991
• PP5: Variant 3-37017508-C-A is Pathogenic according to our data. Variant chr3-37017508-C-A is described in ClinVar as [Pathogenic]. Clinvar id is 90380.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37017508-C-A is described in Lovd as [Likely_pathogenic]. Variant chr3-37017508-C-A is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.793C>A	p.Arg265Ser	missense_variant, splice_region_variant	10/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.793C>A	p.Arg265Ser	missense_variant, splice_region_variant	10/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD3 exomes AF: 0.00000398 AC: 1 AN: 251460 Hom.: 0 AF XY: 0.00 AC XY: 0 AN XY: 135902

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: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.00000879

Gnomad OTH exome AF: 0.00

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:9

Revision: reviewed by expert panel

Submissions by phenotype

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Baylor Genetics	Dec 06, 2020	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	Myriad Genetics, Inc.	Jul 18, 2023	This variant is considered likely pathogenic. Functional studies indicate this variant impacts protein function [PMID: 17210669, 20020535]. This variant is expected to disrupt protein structure [Myriad internal data]. -

Lynch syndrome Pathogenic:2

Pathogenic, reviewed by expert panel	curation	International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	Oct 18, 2018	Paper by Van der Klift et al. 2015 shows aberrant splicing (exon 10 exclusion) in patient sample and minigene assay, confirmed by minigene assay in Soukarieh et al., 2016 (Plos Genet). -
Likely pathogenic, criteria provided, single submitter	clinical testing	University of Washington Department of Laboratory Medicine, University of Washington	May 01, 2018	MLH1 NM_000249.3:c.793C>A has a 97.7% probability of pathogenicity based on combining prior probability from public data with a likelihood ratio of 1.56 to 1, generated from evidence of seeing this as a somatic mutation in a tumor without loss of heterozygosity at the MLH1 locus. See Shirts et al 2018, PMID 29887214. -

Carcinoma of colon Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

The p.Arg265Ser variant has been previously reported in the literature (Zavodna_2006_16830052, Alemayehu_2008_18618713, Niessen_2006_16636019). It was identified in 3 out of 572 proband chromosomes (frequency 0.005) in individuals with colorectal cancer, however, no normal population controls were included in these studies. In addition, this variant has been previously reported by our laboratory in two families. In one family the variant was shown to segregate with disease in 4 affected individuals with Lynch syndrome (two were obligate carriers), and did not segregate in at least two unaffected individuals, increasing the likelihood this variant is pathogenic. The p.Arg265 residue is conserved across mammals/species and computational analyses (PolyPhen, SIFT, AlignGVGD, BLOSUM, MAPP-MMR) suggest that the p.Arg265Ser variant may impact the protein. However, this information is not predictive enough to assume pathogenicity. However, functional studies including in vitro MMR complementation assays, protein expression assays, promoter methylation studies, LOH analysis, and a yeast-based reversion rate assay, suggests this variant is likely pathogenic (Drost_2010_20020535, Alemayehu_2008_18618713, Wanat_2007_17210669). Of note, another variant at the same nucleotide position (c.793C>T) causing a different missense substitution (p.Arg265Cys) has been previously identified in Lynch Syndrome families in literature and by our laboratory, has been well-characterized by similar functional studies as the p.Arg265Ser variant, and has been classified as pathogenic, further suggesting that p.Arg265Ser may also have clinical significance. In summary, based on the above information this variant meets our laboratory's criteria to be classified as Pathogenic. -

Lynch-like syndrome Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Constitutional Genetics Lab, Leon Berard Cancer Center

Jul 01, 2019

- -

not provided Pathogenic:1

Pathogenic, no assertion criteria provided

research

Mayo Clinic Laboratories, Mayo Clinic

-

- -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Invitae

Aug 16, 2023

For these reasons, this variant has been classified as Pathogenic. Studies have shown that this missense change results in skipping of exon 10 and introduces a premature termination codon (PMID: 26247049; Invitae). The resulting mRNA is expected to undergo nonsense-mediated decay. Based on a multifactorial likelihood algorithm using genetic, in silico, and/or statistical data, this variant has been determined to have a high probability of being pathogenic (PMID: 24362816). ClinVar contains an entry for this variant (Variation ID: 90380). This missense change has been observed in individuals with clinical features of Lynch syndrome (PMID: 21404117, 25081409, 27435373). This variant is present in population databases (rs63751194, gnomAD 0.0009%). This sequence change replaces arginine, which is basic and polar, with serine, which is neutral and polar, at codon 265 of the MLH1 protein (p.Arg265Ser). RNA analysis indicates that this missense change induces altered splicing and may result in an absent or disrupted protein product. -

Hereditary cancer-predisposing syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Feb 10, 2022

The c.793C>A pathogenic mutation (also known as p.R265S), located in coding exon 10 of the MLH1 gene, results from a C to A substitution at nucleotide position 793. The arginine at codon 265 is replaced by serine, an amino acid with dissimilar properties. This mutation has been identified as germline in multiple individuals with Lynch syndrome, several whose tumors demonstrated high microsatellite instability and/or loss of MLH1/PMS2 proteins on immunohistochemistry (Zavodna K et al. Neoplasma. 2006;53:269-76; Niessen RC et al. Gut, 2006 Dec;55:1781-8; Alemayehu A et al. Genes Chromosomes Cancer. 2008 Oct;47:906-14; Hardt K et al. Fam. Cancer. 2011 Jun;10:273-84; Ferguson SE et al. Cancer. 2014 Dec;120:3932-9; van der Klift HM et al. Hum Mutat, 2016 11;37:1162-1179; Latham A et al. J Clin Oncol, 2019 02;37:286-295; Ambry internal data). This alteration is located in the ATP-binding domain of the MLH1 protein and has been demonstrated to reduce mismatch repair proficiency in two different yeast strains when compared to wildtype (Wanat JJ et al. Hum. Mol. Genet. 2007 Feb;16:445-52). In an in vitro complementation assay, this variant was determined to be functionally deficient (Drost M et al. Genet Med, 2019 07;21:1486-1496). Additionally, minigene and RNA assays have shown that this alteration results in a transcript with near-complete skipping of coding exon 10 (van der Klift HM et al. Mol Genet Genomic Med. 2015 Jul;3:327-45; Soukarieh O et al. PLoS Genet. 2016 Jan;12:e1005756). This nucleotide position is highly conserved in available vertebrate species. In silico splice site analysis for this alteration is inconclusive. RNA studies have demonstrated that this alteration results in abnormal splicing in the set of samples tested (Ambry internal data). 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	1.0
BayesDel_addAF	Pathogenic	0.57	D
BayesDel_noAF	Pathogenic	0.59
CADD	Pathogenic	32
DANN	Uncertain	1.0
DEOGEN2	Pathogenic	0.90	D;.;.;.;.;.;.
Eigen	Pathogenic	1.1
Eigen_PC	Pathogenic	1.0
FATHMM_MKL	Pathogenic	1.0	D
LIST_S2	Pathogenic	0.98	D;D;.;.;.;D;D
M_CAP	Pathogenic	0.77	D
MetaRNN	Pathogenic	0.99	D;D;D;D;D;D;D
MetaSVM	Pathogenic	0.94	D
MutationAssessor	Pathogenic	4.8	H;.;.;.;.;.;.
MutationTaster	Benign	1.0	D;D;D;D;D;D
PrimateAI	Uncertain	0.66	T
PROVEAN	Pathogenic	-5.9	D;D;D;D;D;D;D
REVEL	Pathogenic	0.98
Sift	Pathogenic	0.0	D;D;D;D;D;D;D
Sift4G	Pathogenic	0.0	D;D;D;D;D;D;D
Polyphen		1.0	D;.;.;.;.;.;.
Vest4		0.99
MutPred		0.94		Gain of disorder (P = 0.0801);.;.;.;.;.;.;
MVP		0.99
MPC		0.43
ClinPred		1.0	D
GERP RS		5.7
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7
Varity_R		0.99
gMVP		0.91

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.19		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: rs63751194; hg19: chr3-37058999;