chr3-37047531-C-T

Variant names:

• chr3-37047531-C-T
• rs63751713
• NM_000249.4:c.1744C>T

Variant summary

Our verdict is Likely pathogenic. Variant got 6 ACMG points: 6P and 0B. PM2 PM5 PP3_Moderate

The NM_000249.4(MLH1):​c.1744C>T​(p.Leu582Phe) variant causes a missense change. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a pathogenic outcome for this variant. Variant has been reported in ClinVar as Conflicting classifications of pathogenicity (no stars). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. L582P) has been classified as Pathogenic.

• Frequency: Genomes: not found (cov: 31)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:5 U:1
• Conservation: PhyloP100: 4.88

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_pathogenic. Variant got 6 ACMG points.

• PM2: Very rare variant in population databases, with high coverage
• PM5: Other missense variant is known to change same aminoacid residue: Variant chr3-37047532-T-C is described in Lovd as [Pathogenic].
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.909

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
MLH1	NM_000249.4	c.1744C>T	p.Leu582Phe	missense_variant	Exon 16 of 19	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.1744C>T	p.Leu582Phe	missense_variant	Exon 16 of 19	1	NM_000249.4	ENSP00000231790.3

Frequencies

GnomAD3 genomes Cov.: 31

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 31

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:5 Uncertain:1

Revision: criteria provided, conflicting classifications

Submissions by phenotype

not provided Pathogenic:2 Uncertain:1

May 01, 2019

CeGaT Center for Human Genetics Tuebingen

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Jan 04, 2017

GeneDx

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant is denoted MLH1 c.1744C>T at the cDNA level, p.Leu582Phe (L582F) at the protein level, and results in the change of a Leucine to a Phenylalanine (CTC>TTC). This variant has been reported in individuals with early onset colorectal cancer, whose tumor testing demonstrated microsatellite instability and loss of MLH1, and also loss of PMS2 when performed, on mismatch repair immunohistochemistry (IHC) (Hendriks 2003, van Puijenbroek 2008, van der Klift 2016). Functional assays demonstrate MLH1 Leu582Phe to have reduced mismatch repair activity compared to wild-type, while other assays show defective MLH1-PMS2 dimerization (Drost 2010, Andersen 2012). MLH1 Leu582Phe was not observed in approximately 6,500 individuals of European and African American ancestry in the NHLBI Exome Sequencing Project, suggesting it is not a common benign variant in these populations. Since Leucine and Phenylalanine share similar properties, this is considered a conservative amino acid substitution. MLH1 Leu582Phe occurs at a position that is conserved across species and is located in the PMS2/MLH3/PMS1 interaction domain and region of interaction with EXO1 (Pang 1997, Raevaara 2005, Hardt 2011, UniProt). In silico analyses are inconsistent regarding the effect this variant may have on protein structure and function. Based on currently available evidence, it is unclear whether MLH1 Leu582Phe is a pathogenic or benign variant. We consider it to be a variant of uncertain significance. -

Apr 22, 2020

Quest Diagnostics Nichols Institute San Juan Capistrano

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Not found in the total gnomAD dataset, and the data is high quality. Located in potentially critical domain of the protein. Assessment of experimental evidence suggests this variant results in abnormal protein function. -

Hereditary nonpolyposis colon cancer Pathogenic:1

Dec 26, 2024

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

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Variant summary: MLH1 c.1744C>T (p.Leu582Phe) results in a non-conservative amino acid change located in the DNA mismatch repair protein Mlh1 C-terminus (IPR032189) 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 251262 control chromosomes. c.1744C>T has been reported in the literature in individuals affected with Hereditary Nonpolyposis Colorectal Cancer (e.g. Hendriks_2003, van der Klift_2016, van Puijenbroek_2008, Bouvet_2019; Labcorp, formerly Invitae). These data indicate that the variant may be associated with disease. At least one publication reports experimental evidence evaluating an impact on protein function (Drost_2009, Andersen_2012). The most pronounced variant effect results in significantly reduced MMR activity and abrogated interaction with PMS2. The following publications have been ascertained in the context of this evaluation (PMID: 22753075, 30998989, 20020535, 12547705, 17192056, 22949387, 18415027, 27435373). ClinVar contains an entry for this variant (Variation ID: 89870). Based on the evidence outlined above, the variant was classified as likely pathogenic. -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Jan 19, 2025

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This sequence change replaces leucine, which is neutral and non-polar, with phenylalanine, which is neutral and non-polar, at codon 582 of the MLH1 protein (p.Leu582Phe). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individuals with Lynch syndrome (PMID: 12547705, 18415027, 27435373, 30998989; internal data). ClinVar contains an entry for this variant (Variation ID: 89870). Invitae Evidence Modeling incorporating data from in vitro experimental studies (internal data) indicates that this missense variant is not expected to disrupt MLH1 function with a negative predictive value of 95%. Experimental studies have shown that this missense change affects MLH1 function (PMID: 20020535, 22753075, 30998989). 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

May 01, 2024

Ambry Genetics

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The p.L582F pathogenic mutation (also known as c.1744C>T), located in coding exon 16 of the MLH1 gene, results from a C to T substitution at nucleotide position 1744. The leucine at codon 582 is replaced by phenylalanine, an amino acid with highly similar properties. This alteration showed moderate segregation with disease in a French family that met Amsterdam criteria where the colorectal tumor of the proband displayed loss of MLH1 protein expression on immunohistochemistry (personal communication with Universal Mutation Database depositor). This variant was identified as somatic in conjunction with a second somatic MLH1 pathogenic mutation in an MSI-H colorectal tumor with loss of MLH1 and PMS2 by IHC (Rigter LS et al. Gut, 2018 03;67:447-455). In a cell-free system designed to measure mismatch repair function, the MLH1 protein with the p.L582F variant was determined to have 26.5% relative repair activity compared to wild type (100%) (Drost M et al. Hum Mutat, 2010 Mar;31:247-53). In another in vitro assay, the binding capacity of the MLH1 p.L582F protein variant with PMS2 was measured in a GST pull-down assay and no binding was detected (Andersen SD et al. Hum. Mutat., 2012 Dec;33:1647-55). The p.L582F variant was also classified as potentially damaging in a methylation tolerance-based functional assay (Bouvet D et al. Gastroenterology, 2019 08;157:421-431). Based on internal structural analysis, L582F is more disruptive than an internally pathogenic variant in the same domain (Gueneau E et al. Nat Struct Mol Biol., 2013 Apr;20(4):461-8). 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 variant is interpreted as a disease-causing mutation. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.66
BayesDel_addAF	Pathogenic	0.48	D
BayesDel_noAF	Pathogenic	0.46
CADD	Pathogenic	26
DANN	Uncertain	1.0
DEOGEN2	Pathogenic	0.91	D;.;.;.;.;.
Eigen	Pathogenic	0.79
Eigen_PC	Pathogenic	0.70
FATHMM_MKL	Uncertain	0.91	D
LIST_S2	Pathogenic	0.98	D;D;.;.;.;D
M_CAP	Pathogenic	0.38	D
MetaRNN	Pathogenic	0.91	D;D;D;D;D;D
MetaSVM	Pathogenic	1.0	D
MutationAssessor	Pathogenic	3.5	H;.;.;.;.;.
PrimateAI	Uncertain	0.66	T
PROVEAN	Uncertain	-3.1	D;D;D;D;D;D
REVEL	Pathogenic	0.86
Sift	Uncertain	0.0040	D;D;D;D;D;D
Sift4G	Uncertain	0.010	D;D;D;D;D;D
Polyphen		1.0	D;.;.;.;.;.
Vest4		0.68
MutPred		0.73		Loss of loop (P = 0.0603);.;.;.;.;.;
MVP		0.96
MPC		0.36
ClinPred		0.95	D
GERP RS		5.6
Varity_R		0.93
gMVP		0.77

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.060		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: rs63751713; hg19: chr3-37089022;