NM_000249.4:c.250A>G

Variant names:

• chr3-37000997-A-G
• rs63750641
• NM_000249.4:c.250A>G

Variant summary

Our verdict is Pathogenic. The variant received 18 ACMG points: 18P and 0B. PM1 PM2 PM5 PP3_Strong PP5_Very_Strong

The NM_000249.4(MLH1):​c.250A>G​(p.Lys84Glu) variant causes a missense change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.00000137 in 1,459,670 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 Likely pathogenic (★★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. K84N) has been classified as Likely pathogenic.

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 0.0000014 (0 hom.)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Likely pathogenic reviewed by expert panel P:8
• Conservation: PhyloP100: 8.79
• Publications: 13 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: Pathogenic. The variant received 18 ACMG points.

• PM1: In a hotspot region, there are 11 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 0 benign, 40 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 chr3-37000999-A-C is described in ClinVar as Conflicting_classifications_of_pathogenicity. ClinVar VariationId is 2034168.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.988
• PP5: Variant 3-37000997-A-G is Pathogenic according to our data. Variant chr3-37000997-A-G is described in ClinVar as Likely_pathogenic. ClinVar VariationId is 90120.Status of the report is reviewed_by_expert_panel, 3 stars.

Variant Effect in Transcripts

ACMG analysis was done for transcript: NM_000249.4. You can select a different transcript below to see updated ACMG assignments.

RefSeq Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	MLH1	NM_000249.4	MANE Select	c.250A>G	p.Lys84Glu	missense	Exon 3 of 19	NP_000240.1
	MLH1	NM_001354628.2		c.250A>G	p.Lys84Glu	missense	Exon 3 of 18	NP_001341557.1
	MLH1	NM_001354630.2		c.250A>G	p.Lys84Glu	missense	Exon 3 of 18	NP_001341559.1

Ensembl Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	MLH1	ENST00000231790.8	TSL:1 MANE Select	c.250A>G	p.Lys84Glu	missense	Exon 3 of 19	ENSP00000231790.3
	MLH1	ENST00000456676.7	TSL:1	c.250A>G	p.Lys84Glu	missense	Exon 3 of 17	ENSP00000416687.3
	MLH1	ENST00000413740.2	TSL:1	c.250A>G	p.Lys84Glu	missense	Exon 3 of 15	ENSP00000416476.2

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome AF: 0.00000137 AC: 2 AN: 1459670 Hom.: 0 Cov.: 29 AF XY: 0.00000138 AC XY: 1 AN XY: 726324

African (AFR) AF: 0.00 AC: 0 AN: 33442

American (AMR) AF: 0.00 AC: 0 AN: 44714

Ashkenazi Jewish (ASJ) AF: 0.00 AC: 0 AN: 26112

East Asian (EAS) AF: 0.00 AC: 0 AN: 39622

South Asian (SAS) AF: 0.00 AC: 0 AN: 86200

European-Finnish (FIN) AF: 0.00 AC: 0 AN: 53408

Middle Eastern (MID) AF: 0.00 AC: 0 AN: 5762

European-Non Finnish (NFE) AF: 0.00000180 AC: 2 AN: 1110082

Other (OTH) AF: 0.00 AC: 0 AN: 60328

GnomAD4 genome Cov.: 32

Alfa AF: 0.00 Hom.: 0

ClinVar

Significance: Likely pathogenic

Submissions summary: Pathogenic:8

Revision: reviewed by expert panel

Submissions by phenotype

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:2

Nov 27, 2024

Molecular Pathology, Peter Maccallum Cancer Centre

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Jul 12, 2023

Myriad Genetics, Inc.

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant is considered likely pathogenic. Functional studies indicate this variant impacts protein function [PMID: 16083711].

not provided Pathogenic:2

Mar 04, 2025

Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Feb 14, 2017

Clinical Genetics and Genomics, Karolinska University Hospital

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Carcinoma of colon Pathogenic:1

Department of Pathology and Laboratory Medicine, Sinai Health System

Significance: Likely pathogenic

Review Status: no assertion criteria provided

Collection Method: clinical testing

The p.Lys84Glu variant has been reported in the literature in 5 out of 2140 proband chromosomes (frequency of 0.002) from patients with HNPCC; the variant was not reported in any of at least 600 control chromosomes, increasing the likelihood this variant may be a low frequency variant with clinical significance (Ellison 2004, Raevaara 2005, Takahashi 2007, Wanat 2007, Lamberti 1999, Ou 2007, Quaresima 2003, Martinez 2010, Tang 2009, Overbeek 2007). The p.Lys84 residue is conserved across mammals and lower species and computational analyses (PolyPhen2, SIFT, AlignGVGD) suggest that the p.Lys84Glu variant may impact the protein. However, this information is not predictive enough to assume pathogenicity. It is listed in the dbSNP database as coming from a "clinical source" (ID#: rs63750641) but no frequency information was provided so this was not informative for assessing the population frequency. Functional assays using site-directed mutagenesis and in vitro mismatch repair (MMR) assays found the p.Lys84Glu variant to be deficient in MMR function, increasing the likelihood that an alteration to this residue might have clinical significance (Ellison 2004, Raevaara 2005, Takahashi 2007, Wanat 2007, Quaresima 2003). 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 pathogenic role for this variant. This variant is classified as Predicted Pathogenic.

Lynch syndrome Pathogenic:1

Jun 21, 2019

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Significance: Likely pathogenic

Review Status: reviewed by expert panel

Collection Method: curation

Abrogated function & >2 MSI-H tumours

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Apr 29, 2024

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This sequence change replaces lysine, which is basic and polar, with glutamic acid, which is acidic and polar, at codon 84 of the MLH1 protein (p.Lys84Glu). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with clinical features of Lynch syndrome (PMID: 10323887, 17453009, 19419416, 21404117, 26053027). ClinVar contains an entry for this variant (Variation ID: 90120). Advanced modeling performed at Invitae incorporating data from internal and/or published experimental studies (Invitae) indicates that this missense variant is expected to disrupt MLH1 function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects MLH1 function (PMID: 12513688, 15475387, 16083711, 17510385, 21404117, 23403630, 31784484). 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.

Hereditary cancer-predisposing syndrome Pathogenic:1

Jul 02, 2025

Ambry Genetics

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The p.K84E variant (also known as c.250A>G), located in coding exon 3 of the MLH1 gene, results from an A to G substitution at nucleotide position 250. The lysine at codon 84 is replaced by glutamic acid, an amino acid with similar properties. This alteration was detected in 1 of 93 unrelated families from Taiwan who met Amsterdam Criteria II for a clinical diagnosis of Lynch syndrome (Tang R et al. Clin Genet. 2009 Apr;75(4):334-45). This variant was also detected in an individual whose tumor exhibited high microsatellite instability (Lamberti et al. Gut. 1999 Jun; 44(6): 839-43). This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). Based on the majority of available evidence to date, this variant is likely to be pathogenic.

Computational scores

Source: dbNSFP v4.9

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	1.0
BayesDel_addAF	Pathogenic	0.53	D
BayesDel_noAF	Pathogenic	0.52
CADD	Pathogenic	30
DANN	Uncertain	1.0
DEOGEN2	Pathogenic	0.96	D
Eigen	Pathogenic	1.1
Eigen_PC	Pathogenic	1.0
FATHMM_MKL	Pathogenic	0.98	D
LIST_S2	Pathogenic	1.0	D
M_CAP	Pathogenic	0.33	D
MetaRNN	Pathogenic	0.99	D
MetaSVM	Pathogenic	1.1	D
MutationAssessor	Pathogenic	5.0	H
PhyloP100		8.8
PrimateAI	Uncertain	0.73	T
PROVEAN	Uncertain	-3.7	D
REVEL	Pathogenic	0.95
Sift	Pathogenic	0.0	D
Sift4G	Pathogenic	0.0	D
Polyphen		1.0	D
Vest4		0.95
MutPred		0.92		Loss of ubiquitination at K84 (P = 0.0161)
MVP		0.99
MPC		0.47
ClinPred		1.0	D
GERP RS		5.9
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7
Varity_R		0.96
gMVP		0.95
Mutation Taster		=6/94	disease causing (ClinVar)

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

Other links and lift over

dbSNP: rs63750641; hg19: chr3-37042488;