3-37047531-C-T

Position:

• chr3-37047531-C-T

Variant summary

Our verdict is Likely pathogenic. Variant got 8 ACMG points: 8P and 0B. PM1 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:4 U:2
• 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 8 ACMG points.

• PM1: In a hotspot region, there are 5 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 3 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 chr3-37047532-T-C is described in ClinVar as [Pathogenic]. Clinvar id is 89871.Status of the report is reviewed_by_expert_panel, 3 stars.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.909

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.1744C>T	p.Leu582Phe	missense_variant	16/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.1744C>T	p.Leu582Phe	missense_variant	16/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 31

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 31

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:4 Uncertain:2

Revision: criteria provided, conflicting classifications

Submissions by phenotype

not provided Pathogenic:2 Uncertain:1

Likely pathogenic, criteria provided, single submitter	clinical testing	CeGaT Center for Human Genetics Tuebingen	May 01, 2019	- -
Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	Jan 04, 2017	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. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Apr 22, 2020	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 colorectal neoplasms Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Invitae

Apr 02, 2021

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 has been reported to affect MLH1 protein function (PMID: 20020535, 22753075, 30998989). This variant has been observed in individual(s) with Lynch syndrome (PMID: 18415027, 12547705, 27435373, 30998989, Invitae). ClinVar contains an entry for this variant (Variation ID: 89870). This variant is not present in population databases (ExAC no frequency). This sequence change replaces leucine with phenylalanine at codon 582 of the MLH1 protein (p.Leu582Phe). The leucine residue is moderately conserved and there is a small physicochemical difference between leucine and phenylalanine. -

Hereditary cancer-predisposing syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

May 01, 2024

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. -

not specified Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

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

Oct 20, 2017

Variant summary: The MLH1 c.1744C>T (p.L582F) variant involves the alteration of a highly conserved nucleotide and 3/4 in silico tools predict a damaging outcome for this variant. The variant is located within the PMS2/MLH3/PMS1 interaction domain and the abrogated interactions between MLH1 and PMS2 partners in addition to the variant being deficient in an MMR complementation assay were confirmed by functional studies (Drost_2009; Andersen_2012). Tumors for carriers of this variant showed MSH-H and loss of MLH1-PMS2 staining. The variant is absent from the large control datasets of ExAC and gnomAD (121282 and 246040, chrs tested respectively). The variant was identified in several affected individuals (Hendriks_2003; van Puijenbroek_2008; van der Klift_2016). Although several clinical diagnostic laboratories/reputable databases cite the variant with a classification of VUS, the collective evidence points towards a deleterious effect. Taken together, the variant was classified as VUS-Possibly Pathogenic, until more data becomes available. -

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;.;.;.;.;.
MutationTaster	Benign	1.0	D;D;D;D;D;D
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;