NM_000249.4:c.1693A>T

Variant names:

• chr3-37042293-A-T
• rs63750062
• NM_000249.4:c.1693A>T

Variant summary

Our verdict is Uncertain significance. The variant received 3 ACMG points: 4P and 1B. PM1 PP3_Moderate BP6

The NM_000249.4(MLH1):​c.1693A>T​(p.Ile565Phe) variant causes a missense change. The variant allele was found at a frequency of 0.00000617 in 1,458,388 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 Conflicting classifications of pathogenicity (no stars). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. I565V) has been classified as Uncertain significance.

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 0.0000062 (0 hom.)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications U:6 B:2
• Conservation: PhyloP100: 3.70
• Publications: 6 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 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 2 benign, 31 uncertain in NM_000249.4
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.927
• BP6: Variant 3-37042293-A-T is Benign according to our data. Variant chr3-37042293-A-T is described in ClinVar as Conflicting_classifications_of_pathogenicity. ClinVar VariationId is 89842.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
MLH1	NM_000249.4	c.1693A>T	p.Ile565Phe	missense_variant	Exon 15 of 19	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.1693A>T	p.Ile565Phe	missense_variant	Exon 15 of 19	1	NM_000249.4	ENSP00000231790.3

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD2 exomes AF: 0.00000796 AC: 2 AN: 251154 AF XY: 0.00000737

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 FIN exome AF: 0.00

Gnomad NFE exome AF: 0.0000176

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.00000617 AC: 9 AN: 1458388 Hom.: 0 Cov.: 29 AF XY: 0.00000413 AC XY: 3 AN XY: 725834

African (AFR) AF: 0.00 AC: 0 AN: 33408

American (AMR) AF: 0.00 AC: 0 AN: 44718

Ashkenazi Jewish (ASJ) AF: 0.00 AC: 0 AN: 26106

East Asian (EAS) AF: 0.00 AC: 0 AN: 39678

South Asian (SAS) AF: 0.00 AC: 0 AN: 86190

European-Finnish (FIN) AF: 0.00 AC: 0 AN: 53388

Middle Eastern (MID) AF: 0.000173 AC: 1 AN: 5768

European-Non Finnish (NFE) AF: 0.00000721 AC: 8 AN: 1108834

Other (OTH) AF: 0.00 AC: 0 AN: 60298

GnomAD4 genome Cov.: 32

Alfa AF: 0.000111 Hom.: 0

ExAC AF: 0.00000824 AC: 1

EpiCase AF: 0.000109

EpiControl AF: 0.00

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Uncertain:6 Benign:2

Revision: criteria provided, conflicting classifications

Submissions by phenotype

Lynch syndrome Uncertain:2

Jan 11, 2024

All of Us Research Program, National Institutes of Health

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This missense variant replaces isoleucine with phenylalanine at codon 565 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). A functional study has shown that this variant causes a partial loss of mismatch repair activity of MLH1 protein (PMID: 17510385). This variant has been observed in several individuals affected with Lynch syndrome (PMID: 9833759, 15943554, 21404117, 21901500). However, a tumor sample from one of these individuals showed normal MLH1 protein expression but loss of MSH2 and MSH6 protein expression, indicating the presence of a pathogenic variant in the MSH2 gene (PMID: 15943554). In addition, one of the affected individuals carried a pathogenic MSH2 variant that could explain the observed phenotype (PMID: 21404117). This variant has been identified in 2/251154 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. -

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Department of Pathology and Laboratory Medicine, Sinai Health System

Significance: Uncertain significance

Review Status: no assertion criteria provided

Collection Method: clinical testing

The MLH1 p.Ile565Phe variant was identified in 2 of 424 proband chromosomes (frequency: 0.005) from Swiss and German individuals or families with HNPCC (Hardt 2011, Hutter 1998). The variant was also identified in dbSNP (ID: rs63750062) as “With Uncertain significance allele” and ClinVar (classified as uncertain significance by an InSiGHT expert panel (2013), GeneDx, Color, Invitae and Ambry Genetics). The variant was not identified in UMD-LSDB. The variant was identified in control databases in 2 of 245948 chromosomes at a frequency of 0.000008 (Genome Aggregation Database Feb 27, 2017). It was observed in the following population: European Non-Finnish in 2 of 111450 chromosomes (freq: 0.00002) but not in the African, Other, Latino, Ashkenazi Jewish, East Asian, European Finnish, or South Asian populations. The p.Ile565 residue is conserved in mammals but not in more distantly related organisms, and four out of five computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) suggest that the Phe variant may impact the protein; however, this information is not predictive enough to assume pathogenicity. The variant occurs outside of the splicing consensus sequence and in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) do not predict a difference in splicing. One study using yeast assays, the variant showed decreased MLH1 expression at approximately 75% of wildtype and 53% in vitro MMR activity relative to wild type, but did not show a dominant mutator effect (Takahashi 2007). This variant was identified by our laboratory in a patient with an MLH1-deficient pancreatic tumour. In summary, based on the above information, the clinical significance of this variant cannot be determined with certainty at this time. This variant is classified as a variant of uncertain significance. -

not provided Uncertain:2

Jul 22, 2022

GeneDx

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Not observed at significant frequency in large population cohorts (gnomAD); 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: 11093816, 21404117, 18383312, 19015241, 9833759, 15943554, 17510385, 21901500, 23741719, 34426522, 22753075, 12799449, 20533529, 31697235) -

Mar 01, 2019

CeGaT Center for Human Genetics Tuebingen

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Hereditary cancer-predisposing syndrome Uncertain:2

Feb 10, 2022

Color Diagnostics, LLC DBA Color Health

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This missense variant replaces isoleucine with phenylalanine at codon 565 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). A functional study has shown that this variant causes a partial loss of mismatch repair activity of MLH1 protein (PMID: 17510385). This variant has been observed in several individuals affected with Lynch syndrome (PMID: 9833759, 15943554, 21404117, 21901500). However, a tumor sample from one of these individuals showed normal MLH1 protein expression but loss of MSH2 and MSH6 protein expression, indicating the presence of a pathogenic variant in the MSH2 gene (PMID: 15943554). In addition, one of the affected individuals carried a pathogenic MSH2 variant that could explain the observed phenotype (PMID: 21404117). This variant has been identified in 2/251154 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. -

Nov 16, 2023

Ambry Genetics

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The p.I565F variant (also known as c.1693A>T), located in coding exon 15 of the MLH1 gene, results from an A to T substitution at nucleotide position 1693. The isoleucine at codon 565 is replaced by phenylalanine, an amino acid with highly similar properties. This alteration was detected in a proband with colorectal cancer diagnosed at age 44 as well as in another relative with colorectal cancer in a family meeting Amsterdam criteria (Hutter P et al. Int. J. Cancer. 1998 Dec;78:680-4). This alteration was detected in another individual who also carried a deletion in MSH2, and that proband's tumor that showed intact MLH1 protein expression but absent MSH2 and MSH6 proteins by IHC analysis (Hardt K et al. Fam. Cancer. 2011 Jun;10:273-84). Another study concluded that this variant was most likely not disease causing when it was detected in a Lynch-related tumor showing intact MLH1 and absent MSH2 and MSH6 proteins on IHC (Grabowski M et al. Genet. Test. 2005;9:138-46). Two Iranian patients with colorectal cancer meeting Amsterdam criteria were found to have this alteration; however, they were not tested for mutations in other MMR genes, and no IHC or MSI data was available on these tumors (Shahmoradi S et al. Fam. Cancer. 2012 Mar;11:13-7). In a study assessing the function of various MLH1 alterations, p.I565F demonstrated 52.5% of in vitro mismatch repair activity and >75% of relative MLH1 protein expression (Takahashi M et al. Cancer Res. 2007 May;67:4595-604). This amino acid position is well 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. -

Colorectal cancer, hereditary nonpolyposis, type 2 Benign:1

Nov 25, 2024

Myriad Genetics, Inc.

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant is considered likely benign. This variant is strongly associated with less severe personal and family histories of cancer, typical for individuals without pathogenic variants in this gene [PMID: 27363726]. -

Hereditary nonpolyposis colorectal neoplasms Benign:1

Jan 28, 2025

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Benign	0.32
BayesDel_addAF	Pathogenic	0.40	D
BayesDel_noAF	Pathogenic	0.35
CADD	Benign	22
DANN	Uncertain	0.98
DEOGEN2	Pathogenic	0.80	D;.;.;.;.;.
Eigen	Uncertain	0.24
Eigen_PC	Uncertain	0.26
FATHMM_MKL	Uncertain	0.97	D
LIST_S2	Uncertain	0.96	D;D;.;.;.;D
M_CAP	Uncertain	0.10	D
MetaRNN	Pathogenic	0.93	D;D;D;D;D;D
MetaSVM	Uncertain	0.26	D
MutationAssessor	Benign	1.8	L;.;.;.;.;.
PhyloP100		3.7
PrimateAI	Uncertain	0.73	T
PROVEAN	Uncertain	-2.6	D;D;D;D;D;D
REVEL	Pathogenic	0.79
Sift	Uncertain	0.010	D;D;D;D;D;D
Sift4G	Uncertain	0.013	D;D;D;D;D;D
Polyphen		0.81	P;.;.;.;.;.
Vest4		0.91
MutPred		0.83		Gain of sheet (P = 0.0827);.;.;.;.;.;
MVP		0.99
MPC		0.20
ClinPred		0.68	D
GERP RS		3.1
Varity_R		0.57
gMVP		0.80
Mutation Taster		=0/100	disease causing

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: rs63750062; hg19: chr3-37083784;