rs267607840

chr3-37040260-A-G

Variant summary

Our verdict is Uncertain significance. Variant got 3 ACMG points: 3P and 0B. PM1 PP3

The NM_000249.4(MLH1):c.1633A>G(p.Thr545Ala) variant causes a missense change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.0000601 in 1,613,798 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 Uncertain significance (★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. T545S) has been classified as Uncertain significance.

• Frequency:
  • Genomes: 𝑓 0.000033 (0 hom., cov: 32)
  • Exomes 𝑓: 0.000063 (0 hom.)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Uncertain significance criteria provided, multiple submitters, no conflicts U:18
• Conservation: PhyloP100: 9.08

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 Uncertain_significance. Variant got 3 ACMG points.

• PM1: In a hotspot region, there are 6 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 1 benign, 15 uncertain in NM_000249.4
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.821

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.1633A>G	p.Thr545Ala	missense_variant	14/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.1633A>G	p.Thr545Ala	missense_variant	14/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes AF: 0.0000329 AC: 5 AN: 152106 Hom.: 0 Cov.: 32

Gnomad AFR AF: 0.00

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: 0.0000735

Gnomad OTH AF: 0.00

GnomAD3 exomes AF: 0.0000398 AC: 10 AN: 251320 Hom.: 0 AF XY: 0.0000294 AC XY: 4 AN XY: 135834

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00

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: 0.0000880

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.0000629 AC: 92 AN: 1461692 Hom.: 0 Cov.: 30 AF XY: 0.0000550 AC XY: 40 AN XY: 727160

Gnomad4 AFR exome AF: 0.0000598

Gnomad4 AMR exome AF: 0.00

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.00

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.0000782

Gnomad4 OTH exome AF: 0.0000497

GnomAD4 genome AF: 0.0000329 AC: 5 AN: 152106 Hom.: 0 Cov.: 32 AF XY: 0.0000404 AC XY: 3 AN XY: 74316

Gnomad4 AFR AF: 0.00

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: 0.0000735

Gnomad4 OTH AF: 0.00

Alfa AF: 0.0000773 Hom.: 0

Bravo AF: 0.0000151

ExAC AF: 0.0000494 AC: 6

EpiCase AF: 0.0000545

EpiControl AF: 0.00

ClinVar

Significance: Uncertain significance

Submissions summary: Uncertain:18

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

not provided Uncertain:6

Uncertain significance, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	May 17, 2021	- -
Uncertain significance, no assertion criteria provided	clinical testing	Laboratory of Diagnostic Genome Analysis, Leiden University Medical Center (LUMC)	-	- -
Uncertain significance, no assertion criteria provided	clinical testing	Clinical Genetics, Academic Medical Center	-	- -
Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	Apr 10, 2023	Published functional studies demonstrate splicing comparable to wild-type in a minigene assay and in patient RNA analysis, but the impact that this variant has on protein function was not evaluated (van der Klift et al., 2015); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 21404117, 18566915, 26898890, 28259476, 12799449, 20533529, 22753075, 26247049) -
Uncertain significance, criteria provided, single submitter	clinical testing	CeGaT Center for Human Genetics Tuebingen	Aug 01, 2022	MLH1: PM2, BP5 -
Uncertain significance, no assertion criteria provided	clinical testing	Clinical Genetics Laboratory, Department of Pathology, Netherlands Cancer Institute	-	- -

not specified Uncertain:3

Uncertain significance, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Feb 22, 2024	Variant summary: MLH1 c.1633A>G (p.Thr545Ala) results in a non-conservative amino acid change located in the DNA mismatch repair protein Mlh1, C-terminal domain (IPR032189) of the encoded protein sequence. Four of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 4e-05 in 251320 control chromosomes. This frequency is not significantly higher than estimated for a pathogenic variant in MLH1 causing Hereditary Nonpolyposis Colorectal Cancer (4e-05 vs 0.00071), allowing no conclusion about variant significance. c.1633A>G has been reported in the literature in individuals affected with cancer (e.g. Nilbert_2009, Hardt_2011, Caminsky_2016) without strong evidence for causality. These reports do not provide unequivocal conclusions about association of the variant with Lynch Syndrome. A co-occurrence with a pathogenic variant in MSH2 was reported by Hardt_2011 (variant not specified), providing supporting evidence for a benign role. To our knowledge, no experimental evidence demonstrating an impact on protein function has been reported. The following publications have been ascertained in the context of this evaluation (PMID: 18566915, 21404117, 26247049, 26898890). ClinVar contains an entry for this variant (Variation ID: 89812). Based on the evidence outlined above, the variant was classified as VUS-possibly benign. -
Uncertain significance, criteria provided, single submitter	clinical testing	Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital	Aug 15, 2023	- -
Uncertain significance, no assertion criteria provided	clinical testing	Mayo Clinic Laboratories, Mayo Clinic	-	- -

Colorectal cancer, hereditary nonpolyposis, type 2 Uncertain:3

Uncertain significance, criteria provided, single submitter	clinical testing	Baylor Genetics	Aug 13, 2023	- -
Uncertain significance, criteria provided, single submitter	clinical testing	St. Jude Molecular Pathology, St. Jude Children's Research Hospital	Oct 28, 2021	The MLH1 c.1633A>G (p.Thr545Ala) missense change has a maximum subpopulation frequency of 0.0085% in gnomAD v2.1.1 (https://gnomad.broadinstitute.org/variant/3-37081751-A-G). Six of seven in silico tools predict a deleterious effect of this variant on protein function (PP3), but to our knowledge these predictions have not been confirmed by functional assays. This variant has also been observed in an individual with Lynch syndrome who also harbored a pathogenic variant in MSH2 (BP5; PMID: 21404117). It has also been reported in 1/1358 non-cancer control individuals in a study of individuals with multiple primary cancers (PMID: 29641532). In summary, this variant meets criteria to be classified as of uncertain significance based on the ACMG/AMP criteria: PP3, BP5. -
Uncertain significance, no assertion criteria provided	clinical testing	Diagnostic Laboratory, Department of Genetics, University Medical Center Groningen	-	- -

Hereditary cancer-predisposing syndrome Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	Ambry Genetics	Nov 03, 2022	The p.T545A variant (also known as c.1633A>G), located in coding exon 14 of the MLH1 gene, results from an A to G substitution at nucleotide position 1633. The threonine at codon 545 is replaced by alanine, an amino acid with similar properties. This variant was reported in conjunction with a pathogenic mutation in MSH2 in an individual diagnosed with breast and colon cancer whose family met Amsterdam I criteria (Hardt K et al. Fam Cancer. 2011 Jun;10:273-84). This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Since supporting evidence is limited at this time, the clinical significance of this alteration remains unclear. -
Uncertain significance, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Feb 14, 2023	This missense variant replaces threonine with alanine at codon 545 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). To our knowledge, functional studies have not been reported for this variant. This variant has been reported in two suspected Lynch syndrome families (PMID: 18566915, 26247049) and one diagnosed Lynch syndrome family (Amsterdam II criteria) that has three colon cancer affected members, one of whom also has a pathogenic MSH2 covariant (PMID: 21404117). This variant has also been observed in an individual affected with familial breast cancer (PMID: 26898890). This variant has been identified in 11/282688 chromosomes in the general population by the Genome Aggregation Database (gnomAD). The available evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance. -

Breast and/or ovarian cancer Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

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

Apr 25, 2023

- -

Lynch syndrome Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

All of Us Research Program, National Institutes of Health

Nov 28, 2023

This missense variant replaces threonine with alanine at codon 545 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). Splice site prediction tools suggest that this variant may not impact RNA splicing. To our knowledge, functional studies have not been reported for this variant. This variant has been reported in two suspected Lynch syndrome families (PMID: 18566915, 26247049) and one diagnosed Lynch syndrome family (Amsterdam II criteria) that has three colon cancer affected members, one of whom also has a pathogenic MSH2 covariant (PMID: 21404117). This variant also has been observed in an individual affected with familial breast cancer (PMID: 26898890). This variant has been identified in 11/282688 chromosomes in the general population by the Genome Aggregation Database (gnomAD). The available evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance. -

MLH1-related disorder Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

PreventionGenetics, part of Exact Sciences

Jan 17, 2024

The MLH1 c.1633A>G variant is predicted to result in the amino acid substitution p.Thr545Ala. This variant has been reported as a variant of uncertain significance in an individual with Lynch syndrome (Nilbert et al. 2009. PubMed ID: 18566915) and in an individual with Lynch syndrome that harbored a pathogenic variant in MSH2 (Hardt et al. 2011. PubMed ID: 21404117, Table S1). Results from a minigene study indicates this variant does not impact splicing (Tables 1 and 2, van der Klift et al. 2015. PubMed ID: 26247049). This variant is reported in 0.0085% of alleles in individuals of European (Non-Finnish) descent in gnomAD. It is interpreted as uncertain in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/variation/89812/). At this time, the clinical significance of this variant is uncertain due to the absence of conclusive functional and genetic evidence. -

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Invitae

Jan 31, 2024

This sequence change replaces threonine, which is neutral and polar, with alanine, which is neutral and non-polar, at codon 545 of the MLH1 protein (p.Thr545Ala). This variant is present in population databases (rs267607840, gnomAD 0.009%). This missense change has been observed in individual(s) with breast and/or ovarian cancer and/or hereditary non-polyposis colorectal cancer (PMID: 18566915, 21404117, 26898890). ClinVar contains an entry for this variant (Variation ID: 89812). 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) has been performed at Invitae for this missense variant, however the output from this modeling did not meet the statistical confidence thresholds required to predict the impact of this variant on MLH1 protein function. RNA analysis performed to evaluate the impact of this missense change on mRNA splicing indicates it does not significantly alter splicing (PMID: 26247049; Invitae). In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.57
BayesDel_addAF	Pathogenic	0.35	D
BayesDel_noAF	Pathogenic	0.46
Cadd	Uncertain	25
Dann	Uncertain	1.0
DEOGEN2	Pathogenic	0.91	D;.;.;.;.;.;D
Eigen	Pathogenic	0.70
Eigen_PC	Uncertain	0.62
FATHMM_MKL	Pathogenic	0.99	D
LIST_S2	Uncertain	0.96	D;D;.;.;.;D;D
M_CAP	Pathogenic	0.51	D
MetaRNN	Pathogenic	0.82	D;D;D;D;D;D;D
MetaSVM	Pathogenic	1.0	D
MutationAssessor	Pathogenic	3.5	M;.;.;.;.;.;.
MutationTaster	Benign	1.0	D;D;D;D;D;D
PrimateAI	Uncertain	0.71	T
PROVEAN	Uncertain	-3.5	D;D;D;D;D;D;D
REVEL	Pathogenic	0.77
Sift	Benign	0.042	D;D;D;D;D;D;D
Sift4G	Benign	0.062	T;D;D;D;D;T;D
Polyphen		0.97	D;.;.;.;.;.;.
Vest4		0.72
MVP		0.96
MPC		0.35
ClinPred		0.77	D
GERP RS		5.7
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7
Varity_R		0.85
gMVP		0.77

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: rs267607840; hg19: chr3-37081751;