rs63751598

chr3-37017598-A-Cchr3-37017598-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.883A>C(p.Ser295Arg) 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. 14/25 in silico tools predict a damaging outcome for this variant. 2/3 splice prediction tools predicting alterations to 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. S295G) has been classified as Pathogenic.

• Frequency: Genomes: not found (cov: 32)
• Consequence: MLH1 NM_000249.4 missense, splice_region
• Scores: Splicing: ADA: 0.9999
• Clinical Significance: Pathogenic reviewed by expert panel P:5
• Conservation: PhyloP100: 8.95

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 3 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 chr3-37017598-A-G is described in ClinVar as [Pathogenic]. Clinvar id is 90409.Status of the report is reviewed_by_expert_panel, 3 stars.
• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF. Scorers claiming Uncertain: max_spliceai. No scorers claiming Benign.
• PP5: Variant 3-37017598-A-C is Pathogenic according to our data. Variant chr3-37017598-A-C is described in ClinVar as [Pathogenic]. Clinvar id is 90408.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37017598-A-C is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.883A>C	p.Ser295Arg	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.883A>C	p.Ser295Arg	missense_variant, splice_region_variant	10/19	1	NM_000249.4	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

Hereditary cancer-predisposing syndrome Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Dec 13, 2021	This missense variant replaces a serine with an arginine in codon 295 of the MLH1 gene. Functional RNA studies have shown that this variant results in exon 10 skipping (PMID: 16830052, 26761715), predicted to lead to a frameshift and premature translation stop signal. This is expected to result in an absent or non-functional protein product. This variant has been reported in individuals affected with Lynch syndrome (PMID: 18618713, http://www.insight-database.org) or colorectal cancer (PMID: 16830052). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Loss of MLH1 function is a known mechanism of disease. Based on the available evidence, this variant is classified as Likely Pathogenic. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	May 06, 2019	The p.S295R variant (also known as c.883A>C), located in coding exon 10 of the MLH1 gene, results from an A to C substitution at nucleotide position 883. The serine at codon 295 is replaced by arginine, an amino acid with dissimilar properties. Using the BDGP and ESEfinder splice site prediction tools, this alteration is predicted to weaken the efficiency of the native splice donor site. RT-PCR and minigene assays indicate that this alteration leads to the skipping of exon 10 and a subsequent frameshift (Kurzawski G et al. Clin. Genet. 2006 Jan;69(1):40-7; Zavodna K et al. Neoplasma 2006;53:269-76; Soukarieh O et al. PLoS Genet. 2016 Jan;12:e1005756). This alteration was reported in an individual with MSI-H, MLH1-absent colorectal cancer diagnosed at age 32 whose family met Amsterdam II criteria (Alemayehu A et al. Genes Chromosomes Cancer 2008 Oct;47:906-14). This alteration has been classified as pathogenic using the following lines of evidence: in silico prediction models, segregation with disease, clinical phenotype including tumor characteristics, mutation co-occurrence, and functional studies (Thompson BA et al. Hum. Mutat. 2013 Jan;34:200-9; Thompson BA et al. Nat. Genet. 2014 Feb;46:107-15; available at [www.insight-group.org/variants/classifications/]). This variant was not reported in population-based 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. In addition, this alteration is predicted to be borderline deleterious by MAPP-MMR in silico analyses (Chao EC et al. Hum. Mutat. 2008 Jun;29:852-60). Based on the majority of available evidence to date, this variant is likely to be pathogenic. -

Carcinoma of colon Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

The p.Ser295Arg variant has been reported in the literature in 3 out of 652 proband chromsomes in individuals who met Amsterdam and Bethesda criteria for HNPCC and was not identified in 60 control chromosomes from these studies (Kurzawski 2006, Alemayehu 2008, Bujalkova 2008). In addition, this variant has been previously reported by our laboratory in two families with a strong family history of colon and endometrial cancer. In one individual tested, a tumor was MSI-H and deficient for MLH1 by immunohistochemistry suggesting absent protein product and a functional consequence of this variant. Furthermore, another variant at the same position (c.883A>G, p.Ser295Gly) has been identified by our laboratory in one family and met our criteria for pathogenicity, increasing the likelihood that this variant also has clinical significance. The p.Ser295Arg variant occurs in the second last base of the exon and this position has been shown to be part of the splicing consensus sequence and variants involving this position sometimes affect splicing further supporting a pathogenic role for this variant. In summary, based on the above information this variant is classified as Pathogenic. -

Lynch syndrome Pathogenic:1

Pathogenic, reviewed by expert panel

research

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Sep 05, 2013

Variant allele results in splicing abberation leading to premature stop codon: full inactivation of variant allele -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Invitae

May 06, 2022

For these reasons, this variant has been classified as Pathogenic. Other variant(s) that result in skipping of exon 10 have been determined to be pathogenic (PMID: 15713769, 15849752, 26247049, 26761715; Invitae). This suggests that this variant may also be clinically significant and likely to be disease-causing. Studies have shown that this missense change is associated with altered splicing resulting in exon 10 skipping and introducing a premature termination codon (Invitae). Algorithms developed to predict the effect of missense changes on protein structure and function (SIFT, PolyPhen-2, Align-GVGD) all suggest that this variant is likely to be disruptive. ClinVar contains an entry for this variant (Variation ID: 90408). This missense change has been observed in individual(s) with clinical features of Lynch syndrome (PMID: 16451135, 16830052, 18618713, 18772310, 20223024). This variant is not present in population databases (gnomAD no frequency). This sequence change replaces serine, which is neutral and polar, with arginine, which is basic and polar, at codon 295 of the MLH1 protein (p.Ser295Arg). -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.93
BayesDel_addAF	Pathogenic	0.44	D
BayesDel_noAF	Pathogenic	0.40
Cadd	Pathogenic	30
Dann	Uncertain	1.0
DEOGEN2	Uncertain	0.75	D;.;.;.;.;.;.
Eigen	Pathogenic	0.71
Eigen_PC	Pathogenic	0.69
FATHMM_MKL	Pathogenic	0.99	D
LIST_S2	Pathogenic	0.98	D;D;.;.;.;D;D
M_CAP	Pathogenic	0.40	D
MetaRNN	Pathogenic	0.93	D;D;D;D;D;D;D
MetaSVM	Uncertain	0.72	D
MutationAssessor	Pathogenic	2.9	M;.;.;.;.;.;.
MutationTaster	Benign	1.0	D;D;D;D;D;D
PrimateAI	Pathogenic	0.81	D
PROVEAN	Uncertain	-4.1	D;D;D;D;D;D;D
REVEL	Pathogenic	0.94
Sift	Uncertain	0.0010	D;D;D;D;D;D;D
Sift4G	Benign	0.10	T;D;D;D;D;T;D
Polyphen		0.78	P;.;.;.;.;.;.
Vest4		0.95
MutPred		0.78		Gain of catalytic residue at S295 (P = 0.1516);.;.;.;.;.;.;
MVP		0.98
MPC		0.29
ClinPred		1.0	D
GERP RS		5.3
Varity_R		0.95
gMVP		0.79

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	1.0
SpliceAI score (max)		0.26		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.26		Position offset: 1

Find out detailed SpliceAI scores and Pangolin per-transcript scores at spliceailookup.broadinstitute.org

Publications

LitVar

Other links and lift over

dbSNP: rs63751598; hg19: chr3-37059089;