rs63750206

chr3-36996701-G-Achr3-36996701-G-Cchr3-36996701-G-T

Variant summary

Our verdict is Pathogenic. Variant got 26 ACMG points: 26P and 0B. PS1_Very_Strong PM1 PM2 PM5 PP3_Strong PP5_Very_Strong

The NM_000249.4(MLH1):c.199G>A(p.Gly67Arg) variant causes a missense change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.00000137 in 1,459,462 control chromosomes in the GnomAD database, with no homozygous occurrence. 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 Pathogenic (★★★). Another nucleotide change resulting in same amino acid change has been previously reported as Pathogenicin ClinVar. Another variant affecting the same amino acid position, but resulting in a different missense (i.e. G67E) has been classified as Pathogenic.

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 0.0000014 (0 hom.)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Pathogenic reviewed by expert panel P:21 U:1
• 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]

ACMG classification

Verdict is Pathogenic. Variant got 26 ACMG points.

• PS1: Transcript NM_000249.4 (MLH1) is affected with MISSENSE_VARIANT having same AA change as one Pathogenic present in ClinVar as 495763
• PM1: In a hotspot region, there are 5 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 0 benign, 16 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 chr3-36996702-G-A is described in ClinVar as [Pathogenic]. Clinvar id is 17106.Status of the report is reviewed_by_expert_panel, 3 stars.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.977
• PP5: Variant 3-36996701-G-A is Pathogenic according to our data. Variant chr3-36996701-G-A is described in ClinVar as [Pathogenic]. Clinvar id is 89992.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-36996701-G-A is described in Lovd as [Pathogenic]. Variant chr3-36996701-G-A is described in Lovd as [Benign]. Variant chr3-36996701-G-A is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.199G>A	p.Gly67Arg	missense_variant	2/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.199G>A	p.Gly67Arg	missense_variant	2/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome AF: 0.00000137 AC: 2 AN: 1459462 Hom.: 0 Cov.: 30 AF XY: 0.00000275 AC XY: 2 AN XY: 726230

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: 0.00000180

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:21 Uncertain:1

Revision: reviewed by expert panel

Submissions by phenotype

not provided Pathogenic:6

Pathogenic, criteria provided, single submitter	clinical testing	PreventionGenetics, part of Exact Sciences	Jan 08, 2018	- -
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	Quest Diagnostics Nichols Institute San Juan Capistrano	Apr 18, 2023	This variant has not been reported in large, multi-ethnic general populations (http://gnomad.broadinstitute.org). In the published literature, the variant has been reported in individuals and families with Lynch syndrome (PMIDs: 32490589 (2020), 28874130 (2017), 26681312 (2015), 21239990 (2011), 18383312 (2008), 17312306 (2007), 15613555 (2004), 12419761 (2002), 8521398 (1995), 12658575(2003)). In published functional studies, this variant was demonstrated to have a damaging effect on MLH1 function and cause defective mismatch repair activity (PMIDs: 18337503 (2008), 18094436 (2007), 16083711 (2005), 12810663 (2003), 9697702 (1998), 32849802(2020), 30504929 (2018)). Based on the available information, this variant is classified as pathogenic. -
Pathogenic, no assertion criteria provided	clinical testing	Diagnostic Laboratory, Department of Genetics, University Medical Center Groningen	-	- -
Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Mar 24, 2022	Observed in multiple individuals with Lynch syndrome-associated cancers and tumor studies consistent with pathogenic variants in MLH1 (Tannergard 1995, Yuan 2004, Alazzouzi 2005, Casey 2005, Halvarsson 2005, Auclair 2006, Ewald 2007, Lagerstedt Robinson 2007, Takahashi 2007, Drost 2010, Hardt 2011, Pastrello 2011); Published functional studies demonstrate a damaging effect: loss of dominant mutator effect, deficient nuclear localization, reduced protein expression, increased mutation rate, and defective mismatch repair activity (Shimodaira 1998, Shcherbakova 1999, Ellison 2001, Raevaara 2005, Blasi 2006, Takahashi 2007, Avdievich 2008, Drost 2010, Kansikas 2011, Andersen 2012, Borras 2013); Not observed in large population cohorts (gnomAD); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 18561205, 22753075, 23760103, 31447099, 12810663, 16083711, 9833759, 21056691, 15713769, 16395668, 16995940, 8521398, 21239990, 10970186, 12070261, 14961575, 17312306, 15731775, 8993979, 16982745, 19072991, 21404117, 17440950, 12555990, 9067757, 10375096, 22949379, 15340264, 15613555, 17074586, 18094436, 18337503, 10082584, 11555625, 9697702, 17510385, 22736432, 21120944, 10495924, 26895986, 20020535, 26708047, 26681312, 27601186, 27739435, 26720728, 24362816, 27413734, 17594722, 28874130, 29887214, 31159747, 30998989, 32490589, 30787465, 33087929, 34178123) -
Pathogenic, no assertion criteria provided	clinical testing	Mayo Clinic Laboratories, Mayo Clinic	-	- -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:4 Uncertain:1

Pathogenic, no assertion criteria provided	clinical testing	Zotz-Klimas Genetics Lab, MVZ Zotz Klimas	Oct 30, 2023	- -
Uncertain significance, criteria provided, single submitter	clinical testing	Neuberg Centre For Genomic Medicine, NCGM	-	The missense variant p.G67R in MLH1 (NM_000249.4) has been previously reported in individuals affected with Hereditary non-polyposis colorectal cancer (HNPCC) (Alazzouzi et al, 2005). Experimental studies have shown a significant reduction of mismatch repair activity of the mutated protein in both in vitro and in vivo assays, as well as deleterious effects on other aspects of protein function (Avdievich et al, 2008; Ellison et al, 2001). The p.G67R variant is novel (not in any individuals) in gnomAD Exomes. There is a moderate physicochemical difference between glycine and arginine. The p.G67R missense variant is predicted to be damaging by both SIFT and PolyPhen2. The glycine residue at codon 67 of MLH1 is conserved in all mammalian species. The nucleotide c.199 in MLH1 is predicted conserved by GERP++ and PhyloP across 100 vertebrates. For these reasons, this variant has been classified as Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Counsyl	Aug 16, 2017	- -
Pathogenic, criteria provided, single submitter	clinical testing	Myriad Genetics, Inc.	Jul 10, 2023	This variant is considered pathogenic. Functional studies indicate this variant impacts protein function [PMID: 16083711, 16982745, 20020535, 22736432]. This variant is strongly associated with more severe personal and family histories of cancer, typical for individuals with pathogenic variants in this gene [PMID: 27363726]. -
Pathogenic, criteria provided, single submitter	clinical testing	Baylor Genetics	Apr 14, 2023	- -

Lynch syndrome Pathogenic:3

Pathogenic, reviewed by expert panel	research	International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	Sep 05, 2013	Abrogated function, >2 MSI-H tumours, co-segregation with disease & MAF 0.00. Multifactorial likelihood analysis posterior probability >0.99 -
Pathogenic, criteria provided, single submitter	clinical testing	University of Washington Department of Laboratory Medicine, University of Washington	Nov 20, 2015	- -
Pathogenic, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	Apr 19, 2019	The p.Gly67Arg variant in MLH1 has been identified in a large number of individuals with Lynch syndrome and segregated with disease in at least 4 affected family members in 2 families (Tannergard 1995, Mitchell 2002, Alazzouzi 2005, Lagerstedt Robinson 2007, InSiGHT database: http://chromium.lovd.nl/LOVD2/colon_cancer/variants.php). This variant was absent from large population studies. Mice carrying the p.Gly67Arg variant have a strong cancer predisposition phenotype (Avdievich 2008). Additionally, this variant has been classified as pathogenic on Sep 5, 2013 by the ClinGen-approved InSiGHT panel (ClinVar SCV000106471.2). In summary, this variant meets criteria to be classified as pathogenic for Lynch syndrome in an autosomal dominant manner. ACMG/AMP criteria applied: PM2, PS4, PP1_Supporting, PS3. -

Hereditary cancer-predisposing syndrome Pathogenic:3

Likely pathogenic, criteria provided, single submitter	clinical testing	GeneKor MSA	Jan 01, 2020	This variant is a point mutation that substitutes Glycine with Arginine in the position 67 of the MLH1 protein (p.Gly67Arg). This particular Glycine is highly conserved and in a functional domain, the "ATP-binding and hydrolysis domain" (PMID: 16083711 ). Additionally there is a large physicochemical difference between Glycine and Arginine. This finding has been described in international literature in patients with Lynch syndrome (PMID: 8521398, JPMID: 16810763). Furthermore, functional and in silico analysis (PMID: 17510385, PMID: 17510385) have indicated that this mutation is responsible of Lynch Syndrome occurrence in carriers. The mutation database ClinVar contains entries for this variant (Variation ID: 89992). -
Pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Feb 01, 2022	This missense variant replaces glycine with arginine at codon 67 in the ATPase 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 causes a significant reduction in protein expression and DNA mismatch repair activity (PMID: 24362816) and that mice transgenic for the mutant gene develop tumors and show a severely reduced survival rate (PMID: 18337503). This variant has been reported in individuals affected with Lynch syndrome (PMID: 8521398, 12419761, 15563510, 15613555, 17312306, 18383312, 21239990). Based on a multifactorial likelihood algorithm using genetic, in silico, and statistical data, this variant has been determined to have a high probability of being pathogenic (PMID: 22949379). 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	Oct 29, 2021	The p.G67R pathogenic mutation (also known as c.199G>A), located in coding exon 2 of the MLH1 gene, results from a G to A substitution at nucleotide position 199. The glycine at codon 67 is replaced by arginine, an amino acid with dissimilar properties. This mutation has been reported in multiple individuals and families affected with hereditary non-polyposis colorectal cancer (HNPCC)/Lynch syndrome; several with tumors demonstrating loss of MLH1 by immunohistochemistry (IHC) (Tannerg&aring;rd P et al. Cancer Res. 1995 Dec;55(24):6092-6; Palicio M et al. J. Med. Genet. 2002 Jun;39(6):E29; Wagner A et al. Am. J. Hum. Genet. 2003 May;72(5):1088-100; Rosty C et al. BMJ Open. 2016 Feb;6:e010293; Rossi BM et al. BMC Cancer, 2017 Sep;17:623; Li F et al. Mol Genet Genomic Med, 2020 08;8:e1295; Gonz&aacute;lez-Acosta M et al. J Med Genet, 2020 04;57:269-273; Ambry internal data). In addition, functional studies have demonstrated that this variant results in low (5.9%) in vitro mismatch repair activity and decreased (<25%) relative MLH1 expression, and demonstrated no dominant mutator effect in reporter assays in yeast, which is consistent with pathogenicity (Takahashi M et al. Cancer Res. 2007 May;67(10):4595-604; Shimodaira H et al. Nat Genet, 1998 Aug;19:384-9; Shcherbakova PV et al. Mol Cell Biol 1999 Apr;19(4):3177-83). In a yeast two-hybrid assay, G67R displayed low levels of beta-galactosidase activity similar to known deleterious alterations (Kondo E et al. Cancer Res, 2003 Jun;63:3302-8). Using a Bayesian analysis that incorporates tumor mutation data, this variant was classified as pathogenic (Shirts BH et al. Am J Hum Genet, 2018 07;103:19-29). 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. -

Carcinoma of colon Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

The p.Gly67Arg variant has been previously reported in the literature in 12 out of 726 proband chromosomes from individuals meeting the Amsterdam or Bethesda criteria for HNPCC/Lynch syndrome, and was absent in 1024 control chromosomes tested (Selected publications: Lastella 2006, Cederquist 2004, Alazzouzi 2005, Avdievich 2008, Blasi 2006, Casey 2005, Shimodaira 1998, Yuan 2004, Wang 2006, Wagner 2003, Clyne 2009, Ewald 2007). Several of these studies have reported loss of expression of the MLH1 protein product and others reported microsatellite instability for this variant. It is listed in the dbSNP database as coming from a "clinical source" (ID#: rs63750206) but no frequency information was provided and so the population frequency is not known. The p.Gly67 residue is conserved across mammals and computational analyses (SIFT, AlignGVGD) suggest that the p.Gly67Arg variant may impact the protein. However, this information is not predictive enough to assume pathogenicity. Functional studies have shown that compared to the wild-type protein, the p.Gly67Arg variant showed a severe reduction in repair capacity, and mice studies showed that the variant affected protein stability and led to a strong cancer predisposition phenotype (Blasi 2006, Shimodaira 1998, Clyne 2009, Avdievich 2008). In summary, based on the above information, this variant is classified as pathogenic. -

Hereditary nonpolyposis colon cancer Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

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

Jan 11, 2021

Variant summary: MLH1 c.199G>A (p.Gly67Arg) results in a non-conservative amino acid change located in the N-terminal domain (IPR002099) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 251400 control chromosomes (gnomAD). c.199G>A has been reported in the literature in multiple individuals affected with Lynch Syndrome, including families that fulfilled the Amsterdam I criteria (e.g. Alazzouzi_2005, Hardt_2011, Bonadona_2011, Gonzalez-Acosta_2020 and in the InSiGHT database); in several cases a microsatellite instable tumor and the loss of the MLH1 (with or without the loss of PMS2) protein was noted. These data indicate that the variant is very likely to be associated with disease. Experimental evidence demonstrated that variant had no impact on splicing (e.g. Auclair_2006), and the variant protein had normal interaction with PMS2, however it had decreased expression, impaired subcellular localization, and decreased repair activity compared to wild type (e.g. Raevaara_2005, Andersen_2012, Drost_2019). In addition, knock-in mice carrying the variant in homozygous state had a strong cancer predisposition phenotype (Avdievich 2008). Ten submitters, including an expert panel (InSiGHT), have provided clinical-significance assessments for this variant in ClinVar after 2013, and all of them classified the variant as pathogenic (n=9; including the expert panel) / likely pathogenic (n=1). Based on the evidence outlined above, the variant was classified as pathogenic. -

Lynch syndrome 1 Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Department of Molecular Diagnostics, Institute of Oncology Ljubljana

Apr 02, 2020

- -

Lynch-like syndrome Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Constitutional Genetics Lab, Leon Berard Cancer Center

Jul 01, 2019

- -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Invitae

Jan 24, 2024

This sequence change replaces glycine, which is neutral and non-polar, with arginine, which is basic and polar, at codon 67 of the MLH1 protein (p.Gly67Arg). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with clinical features of MLH1-related conditions (PMID: 8521398, 12419761, 15563510, 15613555, 17312306, 18383312, 21239990). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 89992). 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 with a positive predictive value of 95%. Experimental studies have shown that this missense change affects MLH1 function (PMID: 9697702, 11555625, 12810663, 16083711, 18094436, 18337503, 22949379). For these reasons, this variant has been classified as Pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	1.0
BayesDel_addAF	Pathogenic	0.59	D
BayesDel_noAF	Pathogenic	0.62
Cadd	Pathogenic	33
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	0.60	D
MetaRNN	Pathogenic	0.98	D
MetaSVM	Pathogenic	0.90	D
MutationAssessor	Pathogenic	5.0	H
MutationTaster	Benign	1.0	D;D;D;D;D;D
PrimateAI	Pathogenic	0.82	D
PROVEAN	Pathogenic	-7.3	D
REVEL	Pathogenic	0.99
Sift	Pathogenic	0.0	D
Sift4G	Pathogenic	0.0	D
Polyphen		1.0	D
Vest4		0.98
MutPred		1.0		Gain of MoRF binding (P = 0.089);
MVP		0.99
MPC		0.44
ClinPred		1.0	D
GERP RS		5.8
RBP_binding_hub_radar		0.92
RBP_regulation_power_radar		2.6
Varity_R		0.98
gMVP		0.95

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: rs63750206; hg19: chr3-37038192; COSMIC: COSV51614574; COSMIC: COSV51614574;