3-37048955-G-C

Variant names:

• chr3-37048955-G-C
• rs63750217
• NM_000249.4:c.2041G>C

Variant summary

Our verdict is Uncertain significance. The variant received 2 ACMG points: 2P and 0B. PP4 PM2_Supporting

This summary comes from the ClinGen Evidence Repository: The NM_000249.4: c.2041G>C variant in MLH1 is a missense variant predicted to cause substitution of Alanin by Prolin at amino acid 681 (p.Ala681Pro). The variant is not reported in gnomAD (PM2_supporting). The variant was detected in one CRC/Endometrial MSI-H tumour using a standard panel of 5-10 markers and/or loss of MMR protein expression consistent with the variant location (PP4). In summary, this variant meets the criteria to be classified as a variant of uncertain significance (VUS) for Lynch-Syndrome based on the ACMG/AMP criteria applied, as specified by the ClinGen InSiGHT Hereditary Colorectal Cancer/ Polyposis VCEP: PM2_SUP, PP4 (VCEP specifications version 1) LINK:https://erepo.genome.network/evrepo/ui/classification/CA10578272/MONDO:0007356/115

• Frequency: Genomes: not found (cov: 32)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Pathogenic/Likely pathogenic criteria provided, multiple submitters, no conflicts P:3
• Conservation: PhyloP100: 4.68
• Publications: 70 publications found

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]

MLH1 Gene-Disease associations (from GenCC):

• Lynch syndrome

  Inheritance: AD Classification: DEFINITIVE, SUPPORTIVE Submitted by: G2P, ClinGen, Orphanet
• Lynch syndrome 2

  Inheritance: AD Classification: DEFINITIVE, STRONG Submitted by: Ambry Genetics, Genomics England PanelApp
• Muir-Torre syndrome

  Inheritance: AD Classification: DEFINITIVE, STRONG, MODERATE, SUPPORTIVE Submitted by: Genomics England PanelApp, Ambry Genetics, G2P, Orphanet
• mismatch repair cancer syndrome 1

  Inheritance: AR Classification: DEFINITIVE, STRONG, SUPPORTIVE Submitted by: Ambry Genetics, G2P, Labcorp Genetics (formerly Invitae), Orphanet, ClinGen
• Lynch syndrome 1

  Inheritance: AD Classification: STRONG Submitted by: Labcorp Genetics (formerly Invitae)
• ovarian cancer

  Inheritance: AD Classification: STRONG Submitted by: Genomics England PanelApp
• malignant pancreatic neoplasm

  Inheritance: AD Classification: MODERATE Submitted by: Genomics England PanelApp
• rhabdomyosarcoma

  Inheritance: AR Classification: MODERATE Submitted by: Genomics England PanelApp
• prostate cancer

  Inheritance: AD Classification: LIMITED Submitted by: Ambry Genetics
• breast cancer

  Inheritance: AD Classification: NO_KNOWN Submitted by: Ambry Genetics
• hereditary breast carcinoma

  Inheritance: AD Classification: NO_KNOWN Submitted by: ClinGen

ACMG classification

Our verdict: Uncertain_significance. The variant received 2 ACMG points.

• PM2: For more information check the summary or visit ClinGen Evidence Repository.
• PP4: For more information check the summary or visit ClinGen Evidence Repository.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
MLH1	NM_000249.4	c.2041G>C	p.Ala681Pro	missense_variant	Exon 18 of 19	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.2041G>C	p.Ala681Pro	missense_variant	Exon 18 of 19	1	NM_000249.4	ENSP00000231790.3

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic/Likely pathogenic

Submissions summary: Pathogenic:3

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Hereditary cancer-predisposing syndrome Pathogenic:2

Sep 02, 2025

Ambry Genetics

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The p.A681P pathogenic mutation (also known as c.2041G>C), located in coding exon 18 of the MLH1 gene, results from a G to C substitution at nucleotide position 2041. The alanine at codon 681 is replaced by proline, an amino acid with highly similar properties. This variant has been identified in a proband(s) who met Amsterdam I/II criteria for Lynch syndrome and tumor demonstrated loss of at least MLH1 expression by immunohistochemistry (Ambry internal data). A similar variant, MLH1 p.A681V, was reported in one family that met Bethesda Criteria from a cohort of 98 probands (Wei W et al. BMB Rep 2011 May; 44(5):317-22). Further, a well-characterized mutation at the same codon, p.A681T (c.2041G>A), has been identified in multiple families either meeting Amsterdam II criteria or whose family history was suggestive of Lynch syndrome and has also been shown to co-segregate with disease (Froggatt et al. J Med Genet. 1996 Sep; 33(9): 762-30; Shimodaira et al. Nat Genet 1998 Aug; 19(4): 384-9; Kurzawski G et al. Clin. Genet. 2006 Jan; 69(1):40-7; Barnetson RA et al. Hum. Mutat. 2008 Mar; 29(3):367-74). Based on internal structural analysis, this variant is anticipated to result in a significant decrease in structural stability (Berman HM et al. Nucleic Acids Res. 2000 Jan;28:235-42 (3RBN unpublished structure)). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). Based on the supporting evidence, this variant is interpreted as a disease-causing mutation.

Jun 17, 2024

Color Diagnostics, LLC DBA Color Health

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This missense variant replaces alanine with proline at codon 681 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 has normal mismatch repair in vitro but reduced protein expression and stability, 41-53% of wild type (PMID: 21404117, 22736432, 23403630). This variant has been reported in multiple individuals and families affected with Lynch syndrome (PMID: 8880570, 16083711, 16142001, 16341804, 17054581, 17473388, 18033691, 19731080, 20007843, 20305446, 21404117, 21615986, 21642682, 22034109, 22736432; ClinVar SCV001575707.3, SCV000277914.7). Tumor data from affected individuals has demonstrated microsatellite instability and/or loss of MLH1 expression via immunohistochemistry (PMID: 16083711, 18033691, 22736432; ClinVar SCV000277914.7). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). A different variant affecting the same codon, c.2041G>A (p.Ala681Thr), is considered to be disease-causing (ClinVar variation ID: 17099), suggesting that this position is important for the protein function. Based on the available evidence, this variant is classified as Likely Pathogenic.

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Jul 22, 2022

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

In summary, the currently available evidence indicates that the variant is pathogenic, but additional data are needed to prove that conclusively. Therefore, this variant has been classified as Likely Pathogenic. This variant disrupts the p.Ala681 amino acid residue in MLH1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 2362047, 8880570, 18033691, 21642682, 22736432). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. 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. ClinVar contains an entry for this variant (Variation ID: 233523). This missense change has been observed in individual(s) with clinical features of Lynch syndrome (PMID: 21520333; Invitae). This variant is not present in population databases (gnomAD no frequency). This sequence change replaces alanine, which is neutral and non-polar, with proline, which is neutral and non-polar, at codon 681 of the MLH1 protein (p.Ala681Pro).

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.99
BayesDel_addAF	Pathogenic	0.46	D
BayesDel_noAF	Pathogenic	0.42
CADD	Pathogenic	27
DANN	Uncertain	1.0
DEOGEN2	Pathogenic	0.93	D;.;.;.;.;.
Eigen	Pathogenic	0.88
Eigen_PC	Pathogenic	0.84
FATHMM_MKL	Uncertain	0.92	D
LIST_S2	Uncertain	0.93	D;D;.;.;.;D
M_CAP	Pathogenic	0.41	D
MetaRNN	Pathogenic	0.94	D;D;D;D;D;D
MetaSVM	Pathogenic	0.99	D
MutationAssessor	Pathogenic	3.3	M;.;.;.;.;.
PhyloP100		4.7
PrimateAI	Uncertain	0.55	T
PROVEAN	Uncertain	-3.5	D;D;D;D;D;D
REVEL	Pathogenic	0.84
Sift	Uncertain	0.0050	D;D;D;D;D;D
Sift4G	Uncertain	0.010	D;D;D;D;D;D
Vest4		0.88
ClinPred		0.98	D
GERP RS		5.9
Varity_R		0.97
gMVP		0.95
Mutation Taster		=12/88	disease causing (ClinVar)

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.010		Details are displayed if max score is > 0.2

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

Other links and lift over

dbSNP: rs63750217; hg19: chr3-37090446;