3-36993612-G-C

Variant names:

• chr3-36993612-G-C
• rs41295280
• NM_000249.4:c.65G>C

Variant summary

Our verdict is Likely benign. Variant got -6 ACMG points: 2P and 8B. PP3_Moderate BP6_Very_Strong

The NM_000249.4(MLH1):​c.65G>C​(p.Gly22Ala) variant causes a missense change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.000317 in 1,614,116 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a pathogenic outcome for this variant. Variant has been reported in ClinVar as Benign (★★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. G22V) has been classified as Likely benign.

• Frequency:
  • Genomes: 𝑓 0.00014 (0 hom., cov: 33)
  • Exomes 𝑓: 0.00034 (0 hom.)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Benign reviewed by expert panel U:10 B:14
• Conservation: PhyloP100: 9.49

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 Likely_benign. Variant got -6 ACMG points.

• PP3: MetaRNN computational evidence supports a deleterious effect, 0.935
• BP6: Variant 3-36993612-G-C is Benign according to our data. Variant chr3-36993612-G-C is described in ClinVar as [Benign]. Clinvar id is 90306.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-36993612-G-C is described in Lovd as [Benign]. Variant chr3-36993612-G-C is described in Lovd as [Pathogenic]. Variant chr3-36993612-G-C is described in Lovd as [Likely_benign].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
MLH1	NM_000249.4	c.65G>C	p.Gly22Ala	missense_variant	Exon 1 of 19	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.65G>C	p.Gly22Ala	missense_variant	Exon 1 of 19	1	NM_000249.4	ENSP00000231790.3

Frequencies

GnomAD3 genomes AF: 0.000145 AC: 22 AN: 152236 Hom.: 0 Cov.: 33

Gnomad AFR AF: 0.0000241

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.00

Gnomad ASJ AF: 0.00

Gnomad EAS AF: 0.00

Gnomad SAS AF: 0.00

Gnomad FIN AF: 0.00

Gnomad MID AF: 0.00

Gnomad NFE AF: 0.000309

Gnomad OTH AF: 0.00

GnomAD3 exomes AF: 0.000123 AC: 31 AN: 251404 Hom.: 0 AF XY: 0.000118 AC XY: 16 AN XY: 135898

Gnomad AFR exome AF: 0.000123

Gnomad AMR exome AF: 0.0000289

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: 0.000229

Gnomad OTH exome AF: 0.000326

GnomAD4 exome AF: 0.000335 AC: 490 AN: 1461880 Hom.: 0 Cov.: 31 AF XY: 0.000318 AC XY: 231 AN XY: 727240

Gnomad4 AFR exome AF: 0.000119

Gnomad4 AMR exome AF: 0.0000224

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.0000232

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.000423

Gnomad4 OTH exome AF: 0.000215

GnomAD4 genome AF: 0.000145 AC: 22 AN: 152236 Hom.: 0 Cov.: 33 AF XY: 0.000134 AC XY: 10 AN XY: 74380

Gnomad4 AFR AF: 0.0000241

Gnomad4 AMR AF: 0.00

Gnomad4 ASJ AF: 0.00

Gnomad4 EAS AF: 0.00

Gnomad4 SAS AF: 0.00

Gnomad4 FIN AF: 0.00

Gnomad4 NFE AF: 0.000309

Gnomad4 OTH AF: 0.00

Alfa AF: 0.000363 Hom.: 0

Bravo AF: 0.0000945

TwinsUK AF: 0.00 AC: 0

ALSPAC AF: 0.000259 AC: 1

ESP6500AA AF: 0.00 AC: 0

ESP6500EA AF: 0.000116 AC: 1

ExAC AF: 0.0000824 AC: 10

EpiCase AF: 0.000436

EpiControl AF: 0.0000593

ClinVar

Significance: Benign

Submissions summary: Uncertain:10 Benign:14

Revision: reviewed by expert panel

Submissions by phenotype

not provided Uncertain:5 Benign:2

-

Joint Genome Diagnostic Labs from Nijmegen and Maastricht, Radboudumc and MUMC+

Significance: Uncertain significance

Review Status: no assertion criteria provided

Collection Method: clinical testing

- -

Jun 01, 2016

CeGaT Center for Human Genetics Tuebingen

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

-

Clinical Genetics DNA and cytogenetics Diagnostics Lab, Erasmus MC, Erasmus Medical Center

Significance: Uncertain significance

Review Status: no assertion criteria provided

Collection Method: clinical testing

- -

-

Clinical Genetics, Academic Medical Center

Significance: Uncertain significance

Review Status: no assertion criteria provided

Collection Method: clinical testing

- -

-

Genome Diagnostics Laboratory, Amsterdam University Medical Center

Significance: Uncertain significance

Review Status: no assertion criteria provided

Collection Method: clinical testing

- -

Nov 05, 2022

Quest Diagnostics Nichols Institute San Juan Capistrano

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Nov 30, 2020

GeneDx

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant is associated with the following publications: (PMID: 28466842, 25986311, 27930734, 26333163, 17453009, 19224586, 19389263, 22843852, 27150160, 18383312, 16203774, 23729658, 15520370, 22875147, 17117178, 18033691, 25559809, 25871441, 22949387, 30680046) -

Hereditary cancer-predisposing syndrome Uncertain:1 Benign:4

Jul 13, 2018

True Health Diagnostics

Significance: Uncertain significance

Review Status: no assertion criteria provided

Collection Method: clinical testing

- -

Dec 09, 2015

Color Diagnostics, LLC DBA Color Health

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

May 18, 2018

Ambry Genetics

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This alteration is classified as likely benign based on a combination of the following: seen in unaffected individuals, population frequency, intact protein function, lack of segregation with disease, co-occurrence, RNA analysis, in silico models, amino acid conservation, lack of disease association in case-control studies, and/or the mechanism of disease or impacted region is inconsistent with a known cause of pathogenicity. -

Apr 20, 2021

Sema4, Sema4

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: curation

- -

Sep 02, 2024

Molecular Diagnostics Laboratory, Catalan Institute of Oncology

Significance: Benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

BS1, BS4, BP5, PP3_Moderate c.65G>C located in exon 1 of the MLH1 gene, is predicted to result in the substitution of glycine by alanine at codon 22, p.(Gly22Ala). This variant is found in 29/118108 at a filtered allele frequency of 0.018% in in the gnomAD v2.1.1 database, European non-Finnish non-cancer data set (BS1). Computational tools for this variant predict a deleterious effect of the variant on protein function (MAPP+PolyPhen-2 prior probability for pathogenicity: 0.888) (PP3_Moderate). This variant has been identified in patients with suspected Lynch Syndrome with inconsistent tumor IHC patterns (PMID: 18033691, PMID: 17117178)(BP5). The variant was classified as benign in the Insight database as benign based on multifactorial likelihood analysis posterior probability (<0.0001) and does not cosegregate with disease (LR 5E-8) (BS4). In addition, the variant was also identified in the ClinVar database (10x uncertain significance, 7x likely benign, 5x benign) and LOVD (9x VUS, 2x likely benign, 5x benign, 4x pathogenic, 2x not provided) databases. Based on currently available information, the variant c.65G>C is classified as a benign variant according to ACMG guidelines. -

not specified Uncertain:1 Benign:2

Oct 18, 2021

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

Significance: Benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Variant summary: MLH1 c.65G>C (p.Gly22Ala) results in a non-conservative amino acid change located in the DNA mismatch repair protein family, 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 allele was found at a frequency of 0.00013 in 254472 control chromosomes. This frequency is not significantly higher than estimated for a pathogenic variant in MLH1 causing Hereditary Nonpolyposis Colorectal Cancer (0.00013 vs 0.00071), allowing no conclusion about variant significance. c.65G>C has been reported in the literature in individuals affected with colorectal cancer as well as unaffected controls (example, Fearnhead_2004, Woods_2005, Kets_2006, Barneston_2008, Rouleau_2009, Li_2020). A recent large scale study estimating the likelihood ratios used to compute a tumor characteristic likelihood ratio (TCLR) in combination with in-silico predictors and a multifactorial variant prediction model classified this variant as benign (Li_2020). Multiple co-occurrences with other pathogenic variant(s) have been reported in the UMD database and in the literature (examples, MSH2 c.571_573delCTC, p.Leu191del; MSH2 c.1585delG, p.Glu529LysfsX14; MSH6 c.814G>T, p.Glu272X), providing supporting evidence for a benign role. Multiple clinical diagnostic laboratories and an expert panel (InSiGHT) have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. Multiple submitters reported the variant with conflicting assessments but an emerging consensus towards a benign/likely benign outcome (B/LB, n=5; VUS, n=6). The expert panel has assessed this as a benign variant citing a multifactorial likelihood analysis posterior probability of <0.001. Based on the evidence outlined above, the variant was classified as benign. -

Mar 04, 2025

Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital

Significance: Benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Jun 23, 2016

Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Variant identified in a genome or exome case(s) and assessed due to predicted null impact of the variant or pathogenic assertions in the literature or databases. Disclaimer: This variant has not undergone full assessment. The following are preliminary notes: ClinVar: 1 LB, 3 VUS (includes expert panel), no new evidence since expert classification -

Colorectal cancer, hereditary nonpolyposis, type 2 Uncertain:1 Benign:1

Jan 24, 2016

Counsyl

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Nov 14, 2023

Myriad Genetics, Inc.

Significance: Benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant is considered benign. This variant is strongly associated with less severe personal and family histories of cancer, typical for individuals without pathogenic variants in this gene [PMID: 27363726]. Homozygosity for this variant has been confirmed in one or more individuals lacking clinical features consistent with gene-specific recessive disease, indicating that this variant is unlikely to be pathogenic. -

Breast and/or ovarian cancer Uncertain:1

May 06, 2022

CHEO Genetics Diagnostic Laboratory, Children's Hospital of Eastern Ontario

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Muir-Torré syndrome;C1333991:Colorectal cancer, hereditary nonpolyposis, type 2;C5399763:Mismatch repair cancer syndrome 1 Uncertain:1

Oct 31, 2018

Fulgent Genetics, Fulgent Genetics

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Hereditary nonpolyposis colon cancer Benign:1

Aug 22, 2023

Mendelics

Significance: Benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Lynch syndrome Benign:1

Oct 18, 2018

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Significance: Benign

Review Status: reviewed by expert panel

Collection Method: curation

Multifactorial likelihood analysis posterior probability <0.001 -

MLH1-related disorder Benign:1

Feb 01, 2023

PreventionGenetics, part of Exact Sciences

Significance: Likely benign

Review Status: no assertion criteria provided

Collection Method: clinical testing

This variant is classified as likely benign based on ACMG/AMP sequence variant interpretation guidelines (Richards et al. 2015 PMID: 25741868, with internal and published modifications). -

Hereditary nonpolyposis colorectal neoplasms Benign:1

Jan 26, 2025

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Malignant tumor of breast Benign:1

-

Department of Pathology and Laboratory Medicine, Sinai Health System

Significance: Likely benign

Review Status: no assertion criteria provided

Collection Method: clinical testing

MLH1, EXON1, c.65G>C, p.Gly22Ala, Heterozygous, Likely Benign The MLH1 p.Gly22Ala variant was identified in 4 of 2434 proband chromosomes (frequency: 0.002) from individuals with Lynch syndrome, colorectal cancer, or synchronous or metachronous adenomatous polyps, and was present in 3 of 3068 control chromosomes (frequency: 0.001) from healthy individuals (Fearnhead 2004, Woods 2005, Overbeek 2007, Barnetson 2008). The variant was identified in dbSNP (rs41295280) as “with uncertain significance allele”, ClinVar (classified as benign by an InSiGHT expert panel and Invitae; as likely benign by Ambry Genetics and Color; and as uncertain significance by GeneDx and 5 other submitters), UMD-LSDB (observed 5x as neutral and co-occurring with pathogenic mutations: MLH1, c.571-573delCTC, p.Leu191del and MLH1, c.1585delG, p.Glu529LysfsX14). The variant was identified in control databases in 35 of 277,150 chromosomes at a frequency of 0.0001 (Genome Aggregation Database Feb 27, 2017). The variant was observed in the following populations: African in 4 of 24,034 chromosomes (freq: 0.0002), Other in 2 of 6466 chromosomes (freq: 0.0003), Latino in 1 of 34,420 chromosomes (freq: 0.00003), European in 28 of 126,644 chromosomes (freq: 0.0002), but it was not observed in the Ashkenazi Jewish, East Asian, Finnish, or South Asian populations. The variant was also identified in a patient with a pathogenic variant in MSH6 (c.814G>T, p.Glu272X) and an MSH6-deficient endometrial tumour (Overbeek 2007). The p.Gly22 residue is conserved across mammals and other organisms, and four out of five computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) suggest that the variant may impact the protein; however, this information is not predictive enough to assume pathogenicity. The variant occurs outside of the splicing consensus sequence and in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) do not predict a difference in splicing. In summary, based on the above information, the clinical significance of this variant cannot be determined with certainty at this time although we would lean towards a more benign role for this variant. This variant is classified as likely benign. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.96
BayesDel_addAF	Pathogenic	0.29	D
BayesDel_noAF	Pathogenic	0.47
CADD	Pathogenic	32
DANN	Uncertain	1.0
DEOGEN2	Pathogenic	0.81	D
Eigen	Uncertain	0.64
Eigen_PC	Uncertain	0.66
FATHMM_MKL	Pathogenic	0.99	D
LIST_S2	Pathogenic	0.99	D
M_CAP	Pathogenic	0.63	D
MetaRNN	Pathogenic	0.94	D
MetaSVM	Pathogenic	0.93	D
MutationAssessor	Pathogenic	3.5	H
PrimateAI	Pathogenic	0.83	D
PROVEAN	Pathogenic	-4.9	D
REVEL	Pathogenic	0.91
Sift	Pathogenic	0.0	D
Sift4G	Uncertain	0.014	D
Polyphen		1.0	D
Vest4		0.83
MVP		0.98
MPC		0.40
ClinPred		0.91	D
GERP RS		5.1
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		2.0
Varity_R		0.96
gMVP		0.85

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: rs41295280; hg19: chr3-37035103; COSMIC: COSV51625627; COSMIC: COSV51625627;