3-37017508-C-T

Position:

• chr3-37017508-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>T​(p.Arg265Cys) variant causes a missense, splice region change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.000000684 in 1,461,428 control chromosomes in the GnomAD database, with no homozygous occurrence. 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. R265P) has been classified as Likely pathogenic.

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 6.8e-7 (0 hom.)
• Consequence: MLH1 NM_000249.4 missense, splice_region
• Clinical Significance: Pathogenic reviewed by expert panel P:20 U:1 O:1
• 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 chr3-37017509-G-C is described in ClinVar as [Likely_pathogenic]. Clinvar id is 433856.Status of the report is reviewed_by_expert_panel, 3 stars.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.986
• PP5: Variant 3-37017508-C-T is Pathogenic according to our data. 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. Variant chr3-37017508-C-T is described in Lovd as [Pathogenic]. Variant chr3-37017508-C-T is described in Lovd as [Benign]. Variant chr3-37017508-C-T is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.793C>T	p.Arg265Cys	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>T	p.Arg265Cys	missense_variant, splice_region_variant	10/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome AF: 6.84e-7 AC: 1 AN: 1461428 Hom.: 0 Cov.: 31 AF XY: 0.00 AC XY: 0 AN XY: 727036

Gnomad4 AFR exome AF: 0.00

Gnomad4 AMR exome AF: 0.00

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.00

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 9.00e-7

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 32

Bravo AF: 0.00000378

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:20 Uncertain:1 Other:1

Revision: reviewed by expert panel

Submissions by phenotype

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:6

Pathogenic, criteria provided, single submitter	clinical testing	Neuberg Centre For Genomic Medicine, NCGM	Feb 14, 2023	The missense c.793C>T(p.Arg265Cys) variant in MLH1 gene has been reported in heterozygous state in multiple individuals affected with colorectal cancer (Tang R, et. al., 2009; Choi YH, et. al., 2009). This variant has been observed to segregate with disease (Tang R, et. al., 2009). Functional studies demonstrate skipping of exon 9-10, aberrant splicing, reduced mRNA and protein expression as well as deceased protein stability, hence proving a damaging effect (Fan Y, et. al., 2012; Casey G, et. al., 2005). The p.Arg265Cys variant is novel (not in any individuals) in both gnomAD Exomes and 1000 Genomes databases. This variant has been reported to the ClinVar database as Uncertain Significance / Pathogenic (multiple submissions). The amino acid change p.Arg265Cys in MLH1 is predicted as conserved by GERP++ and PhyloP across 100 vertebrates. The amino acid Arg at position 265 is changed to a Cys changing protein sequence and it might alter its composition and physico-chemical properties. For these reasons, this variant has been classified as Pathogenic. -
Pathogenic, no assertion criteria provided	literature only	OMIM	Apr 01, 2009	- -
Pathogenic, criteria provided, single submitter	clinical testing	Counsyl	Mar 02, 2017	- -
Pathogenic, criteria provided, single submitter	clinical testing	Baylor Genetics	Nov 19, 2023	- -
Pathogenic, criteria provided, single submitter	clinical testing	Institute of Human Genetics, University of Leipzig Medical Center	May 06, 2019	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	Myriad Genetics, Inc.	Mar 16, 2023	This variant is considered likely pathogenic. This variant is strongly associated with more severe personal and family histories of cancer, typical for individuals with pathogenic variants in this gene [PMID: 25085752]. This variant is expected to disrupt protein structure [Myriad internal data]. -

not provided Pathogenic:5 Uncertain:1

Pathogenic, no assertion criteria provided	clinical testing	Clinical Genetics DNA and cytogenetics Diagnostics Lab, Erasmus MC, Erasmus Medical Center	-	- -
Pathogenic, criteria provided, single submitter	clinical testing	Institute for Clinical Genetics, University Hospital TU Dresden, University Hospital TU Dresden	Nov 03, 2021	- -
Pathogenic, no assertion criteria provided	clinical testing	Joint Genome Diagnostic Labs from Nijmegen and Maastricht, Radboudumc and MUMC+	-	- -
Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	May 30, 2023	Published functional studies demonstrate a damaging effect: aberrant splicing and reduced mRNA and protein expression as well as deceased protein stability and reduced binding of MLH1 to PMS2 (Yuen et al., 2002; Casey et al., 2005; Perera et al., 2008; Perera et al., 2010; Andersen et al., 2012; Fan et al., 2012; Soukarieh et al., 2016); Observed in individuals with personal or family histories consistent with pathogenic variants in MLH1 and concordant tumor studies (Wahlberg et al., 1999; Hutter et al., 2002; Yuen et al., 2002; Hendriks et al., 2003; Mangold et al., 2005; Wolf et al., 2005; Lagerstedt Robinson et al., 2007; Berginc et al., 2009; Choi et al., 2009; Tang et al., 2009; Chang et al., 2010; Sjursen et al., 2010; Woods et al., 2010; Bonadona et al., 2011; Hardt et al., 2011; Fan et al., 2012; Liu et al., 2014; Guindalini et al., 2015; Win et al., 2015; Jiang W et al., 2019; Chang et al., 2020); Not observed at significant frequency in large population cohorts (gnomAD); This variant is associated with the following publications: (PMID: 17210669, 20020535, 18561205, 22824075, 20045164, 21404117, 20682701, 19224586, 21681552, 20864636, 28127413, 15731775, 21952876, 11781295, 11555625, 17594722, 22736432, 22949387, 22753075, 17135187, 23760103, 10495924, 16995940, 15849733, 12547705, 19698169, 26247049, 22843852, 24710284, 21642682, 16216036, 19526325, 20587412, 25345868, 17312306, 21155023, 26053027, 26248088, 17192056, 15926618, 12011148, 26202870, 23741719, 16830052, 15713769, 12386821, 17510385, 26761715, 25081409, 11948175, 28445943, 30274973, 31589614, 32849802, 31332305, 19419416, 18205192, 33260537, 29758216, 24362816, 31447099, 30787465, 30521064, 31830689) -
Pathogenic, criteria provided, single submitter	clinical testing	Clinical Genetics and Genomics, Karolinska University Hospital	Sep 22, 2014	- -
Uncertain significance, no assertion criteria provided	research	Biesecker Lab/Clinical Genomics Section, National Institutes of Health	Jul 13, 2012	- -

Lynch syndrome Pathogenic:3

Pathogenic, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	Nov 17, 2016	The p.Arg265Cys variant in MLH1 is absent from large population studies but has been reported in >30 individuals with Lynch Syndrome (Casey 2005, Hardt 2011, Ta ng 2009, InSiGHT database: http://chromium.lovd.nl/LOVD2/colon_cancer/variants.p hp). It segregated with disease in at least 12 family members (Hardt 2011, Tang 2009). Functional studies using patient mRNA showed that the variant leads to ex on skipping and protein truncation (Casey 2005). Additionally, this variant has been classified as pathogenic on Sept 5, 2013 by the ClinGen-approved InSiGHT pa nel (ClinVar SCV000106886.2). In summary, this variant meets criteria to be clas sified as pathogenic for Lynch Syndrome in an autosomal dominant manner based up on segregation studies and the impact of the variant on the protein. -
Pathogenic, reviewed by expert panel	research	International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	Sep 05, 2013	Variant causes aberrant splicing leading to truncated protein: full inactivation of variant allele. -
Pathogenic, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Apr 21, 2016	Variant summary: Variant affects a conserved nucleotide and results in a replacement of a large size and basic Arginine (R) with a medium size and polar Cysteine (C). 4/4 in silico tools predict the variant to be disease causing (SNPs&GO was not capture due to low reliability index). The variant is absent from the large and broad cohorts of the ExAC project while it was observed in several HNPCC patients indicating pathogenicity. Moreover, the variant is considered to be a founder mutation in populations of Chinese/Taiwanese origin. Functional studies report the variant to result in exon skipping and a partial MMR defect which in combination likely result in a pathogenic impact. Furthermore, a clinical laboratory and databases cite variant as Pathogenic. Considering all evidence, the variant was classified as a Pathogenic. -

Carcinoma of colon Pathogenic:2

Pathogenic, no assertion criteria provided	clinical testing	Department of Pathology and Laboratory Medicine, Sinai Health System	-	The p.Arg265Cys variant has been previously reported in the literature and by our laboratory. It is also reported in dbSNP from a clinical source (dbSNP#:rs63751194). In one study, this variant was reported in 2 different families and was associated with skipping of exons 9 and 10 from cDNA (Casey 2005). In another report, including probands who met Amsterdam criteria for HNPCC or who had a family history, this variant was found to reduce exon inclusion. This variant has been tested in different functional assays at the protein level, with results that were not always consistent; the p.Arg265Cys was associated with a mild reduction of mismatch repair efficiency in four studies (Plotz 2006; Ellison 2001; Wanat 2007; Takahashi 2007) but not in a fifth one (Trojan 2002) (Tournier 2007). One large study of Tawainese patients demonstrated that the p.Arg265Cys variant was identified in 13 out of 93 unrelated families. In total, 93 cancers were noted in these 13 families including 66 with cases of colon cancer, 6 cases of rectal cancer, 3 cases of endometrial cancer, 6 cases of gastric cancer, 1 case of ovarian cancer and 11 cases of other types of cancer (Tang 2009). In another report, deficient MLH1 IHC status and MSI-H tumor was noted in an individual with this variant who developed colorectal cancer at age 55 (Perera 2010). In summary, based on the above information this variant is classified as pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	3DMed Clinical Laboratory Inc	Feb 10, 2018	- -

Hereditary cancer-predisposing syndrome Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Jan 01, 2022	This missense variant replaces arginine with cysteine at codon 265 in the MutS interacting domain 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 this variant significantly decreases MLH1 protein stability (PMID: 18205192) and the ability of MLH1 protein to bind PMS2 (PMID: 21952876). This variant has also been shown to affect mRNA splicing (PMID: 12386821, 15713769, 18561205). This variant has been reported in individuals affected with Lynch syndrome or colorectal cancer (PMID: 12386821, 15713769, 19419416, 20587412, 20864636). 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 Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Jul 01, 2021	The p.R265C pathogenic mutation (also known as c.793C>T), located in coding exon 10 of the MLH1 gene, results from a C to T substitution at nucleotide position 793. The arginine at codon 265 is replaced by cysteine, an amino acid with highly dissimilar properties. This mutation has been identified in many individuals with early-onset colorectal cancer with concordant tumor results and family histories which meet Amsterdam criteria (Trojan J et al. Gastroenterology. 2002 Jan;122:211-9; Wolf B et al. Wien. Klin. Wochenschr. 2005 Apr;117:269-77; Woods MO et al. Gut. 2010 Oct;59:1369-77; Hardt K et al. Fam. Cancer. 2011 Jun;10:273-84; Ambry internal data). This mutation has been associated with exon-skipping, premature protein truncation, and low expression of the variant allele (Casey G et al. JAMA. 2005 Feb;293:799-809; Tournier I et al. Hum. Mut. 2008 Dec;29:1412-24; van der Klift HM et al. Mol. Genet. Genomic Med. 2015 Jul;3:327-45). This mutation has also been shown to segregate with disease in several families meeting Amsterdam criteria (De Jong AE et al. Gastroenterology. 2004 Jan;126:42-8; Tang R et al. Clin. Genet. 2009 Apr;75:334-45; Sjursen W et al. J. Med. Genet. 2010 Sep;47:579-85). 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 BA et al. Nat. Genet. 2014 Feb;46:107-15; available at [www.insight-group.org/variants/classifications/]). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. -

Lynch syndrome 1 Pathogenic:1 Other:1

Pathogenic, no assertion criteria provided	clinical testing	Ding PR Lab, Sun Yat-sen University Cancer Center	-	- -
not provided, no classification provided	literature only	GeneReviews	-	- -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Invitae

Jan 09, 2024

This sequence change replaces arginine, which is basic and polar, with cysteine, which is neutral and slightly polar, at codon 265 of the MLH1 protein (p.Arg265Cys). RNA analysis indicates that this missense change induces altered splicing and may result in an absent or disrupted protein product. This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with colorectal cancer or Lynch syndrome (PMID: 12386821, 15713769, 19419416, 20587412, 20864636). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 29654). An algorithm developed to predict the effect of missense changes on protein structure and function (PolyPhen-2) suggests that this variant is likely to be disruptive. Experimental studies are conflicting or provide insufficient evidence to determine the effect of this variant on MLH1 function (PMID: 11555625, 11781295, 17135187, 17510385). Studies have shown that this missense change alters mRNA splicing and is expected to lead to the loss of protein expression (PMID: 18561205, 22736432, 26247049; Invitae). For these reasons, this variant has been classified as Pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.99
BayesDel_addAF	Pathogenic	0.57	D
BayesDel_noAF	Pathogenic	0.59
CADD	Pathogenic	33
DANN	Pathogenic	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	0.99	D;D;.;.;.;D;D
M_CAP	Pathogenic	0.72	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	0.72	T
PROVEAN	Pathogenic	-7.8	D;D;D;D;D;D;D
REVEL	Pathogenic	0.97
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		0.88		Loss of disorder (P = 0.0477);.;.;.;.;.;.;
MVP		0.99
MPC		0.47
ClinPred		1.0	D
GERP RS		5.7
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7
Varity_R		0.96
gMVP		0.88

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.15		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; COSMIC: COSV51616044; COSMIC: COSV51616044;