3-37048596-G-A

Position:

chr3-37048596-G-A

Variant summary

Our verdict is Uncertain significance. Variant got 4 ACMG points: 4P and 0B. PM5PP3_Moderate

The NM_000249.4(MLH1):c.1976G>A(p.Arg659Gln) variant causes a missense change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.0000279 in 1,612,460 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. R659L) has been classified as Pathogenic.

Frequency

Genomes: 𝑓 0.000033 ( 0 hom., cov: 31)

Exomes 𝑓: 0.000027 ( 0 hom. )

Consequence

MLH1
NM_000249.4 missense

Scores

Clinical Significance

Conflicting classifications of pathogenicity criteria provided, conflicting classifications U:7B:2O:1

Conservation

PhyloP100: 9.59

Variant links:

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 links:

Genome browser will be placed here

ACMG classification

Classification made for transcript

Verdict is Uncertain_significance. Variant got 4 ACMG points.

PM5

Other missense variant is known to change same aminoacid residue: Variant chr3-37048596-G-T is described in Lovd as [Pathogenic].

PP3

MetaRNN computational evidence supports a deleterious effect, 0.885

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
MLH1	NM_000249.4 linkuse as main transcript	c.1976G>A	p.Arg659Gln	missense_variant	17/19	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8 linkuse as main transcript	c.1976G>A	p.Arg659Gln	missense_variant	17/19	1	NM_000249.4	ENSP00000231790	P1	P40692-1

Frequencies

GnomAD3 genomes

AF:

0.0000329

AC:

AN:

151826

Hom.:

Cov.:

show subpopulations

Gnomad AFR

AF:

0.0000726

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:

0.0000294

Gnomad OTH

AF:

0.00

GnomAD3 exomes

AF:

0.0000159

AC:

AN:

251070

Hom.:

AF XY:

0.00000737

AC XY:

AN XY:

135700

show subpopulations

Gnomad AFR exome

AF:

0.000123

Gnomad AMR exome

AF:

0.00

Gnomad ASJ exome

AF:

0.00

Gnomad EAS exome

AF:

0.00

Gnomad SAS exome

AF:

0.0000327

Gnomad FIN exome

AF:

0.00

Gnomad NFE exome

AF:

0.00000881

Gnomad OTH exome

AF:

0.00

GnomAD4 exome

AF:

0.0000274

AC:

AN:

1460634

Hom.:

Cov.:

AF XY:

0.0000261

AC XY:

AN XY:

726698

show subpopulations

Gnomad4 AFR exome

AF:

0.00

Gnomad4 AMR exome

AF:

0.00

Gnomad4 ASJ exome

AF:

0.00

Gnomad4 EAS exome

AF:

0.0000252

Gnomad4 SAS exome

AF:

0.0000464

Gnomad4 FIN exome

AF:

0.00

Gnomad4 NFE exome

AF:

0.0000306

Gnomad4 OTH exome

AF:

0.0000166

GnomAD4 genome

AF:

0.0000329

AC:

AN:

151826

Hom.:

Cov.:

AF XY:

0.0000405

AC XY:

AN XY:

74124

show subpopulations

Gnomad4 AFR

AF:

0.0000726

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:

0.0000294

Gnomad4 OTH

AF:

0.00

Bravo

AF:

0.0000227

TwinsUK

AF:

0.000270

AC:

ALSPAC

AF:

0.00

AC:

ExAC

AF:

0.0000165

AC:

Asia WGS

AF:

0.000289

AC:

AN:

3478

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Uncertain:7Benign:2Other:1

Revision: criteria provided, conflicting classifications

LINK: link

Submissions by phenotype

Hereditary cancer-predisposing syndrome

Uncertain:2Benign:1

Likely benign, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Dec 16, 2016	- -
Uncertain significance, criteria provided, single submitter	clinical testing	Ambry Genetics	Mar 25, 2024	The p.R659Q variant (also known as c.1976G>A) is located in coding exon 17 of the MLH1 gene. This alteration results from a G to A substitution at nucleotide position 1976. The arginine at codon 659 is replaced by glutamine, an amino acid with highly similar properties. This alteration has been identified in three probands families with tumor testing demonstrating high microsatellite instability and/or absent MLH1 staining on immunohistochemistry (IHC) and two of the three had family histories that met Amsterdam I/II criteria for Lynch syndrome (Raevaara TE et al. Gastroenterology. 2005 Aug;129(2):537-49; Hampel H et al. Cancer Res., 2006 Aug;66:7810-7; Yurgelun MB et al. J Clin Oncol. 2017 Apr;35(10):1086-1095; Ambry internal data). However, multiple in vitro functional studies including protein expression/stability, subcellular localization, protein-protein interaction, and MMR repair efficiency indicated that this alteration was not likely to be pathogenic (Raevaara TE et al. Gastroenterology. 2005 Aug;129(2):537-49; Kansikas M et al. Hum Mutat. 2011 Jan;32(1):107-15; Abildgaard AB et al. Elife, 2019 11;8). Several other functional studies also indicated that this variant was unlikely to be pathogenic based on in vitro mismatch repair and expression assays that were comparable to wild type (Takahashi M et al. Cancer Res. 2007 May 15;67(10):4595-604; Ou J et al. Hum Mutat. 2007 Nov;28(11):1047-54; Hinrichsen I et al. Clin Cancer Res. 2013 May 1;19(9):2432-41). In contrast, this variant was reported as a pathogenic alteration in an in vivo yeast assay (Vogelsang M et al. BMC Cancer. 2009 Oct 28;9:382). 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 available evidence, the clinical significance of this alteration remains unclear. -
Uncertain significance, criteria provided, single submitter	curation	Sema4, Sema4	Oct 07, 2021	- -

not specified

Uncertain:2Other:1

not provided, no classification provided	reference population	ITMI	Sep 19, 2013	- -
Uncertain significance, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	Mar 31, 2016	Variant identified in a genome or exome case(s) and assessed due to predicted null impact of the variant or pathogenic assertions in the literature or databases. Disclaimer: This variant has not undergone full assessment. The following are preliminary notes: Several papers describe as non-pathogenic; ExAC: 2/10400 African; ClinVar: VUS by expert panel -
Uncertain significance, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	May 01, 2023	Variant summary: MLH1 c.1976G>A (p.Arg659Gln) results in a conservative amino acid change located in the 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 1.6e-05 in 251070 control chromosomes (gnomAD). The available data on variant occurrences in the general population are insufficient to allow any conclusion about variant significance. c.1976G>A has been reported in the literature in individuals/families fulfilling Amsterdam and/or Bethesda criteria for HNPCC, with corresponding tumors showing high microsatellite instability and absent MLH1 following immunohistochemistry (e.g. Hampel_2006 (no PMID), Raevaara_2005, Yurgelun_2017). The variant has also been reported in healthy controls (e.g. Mizukami_2020, Fujita_2022). These reports do not provide unequivocal conclusions about association of the variant with Hereditary Nonpolyposis Colorectal Cancer. In one of the reported CRC patients a KRAS variant (G13D, classified as pathogenic somatic variant) was detected following tumor testing (Yurgelun_2017). Co-occurrences with pathogenic variants have been reported (MLH1 c.2T>C, p.Met1?; BRIP1 c.2010dupT, p.Glu671X; UMD and internal testing), providing supporting evidence for a benign role. Multiple functional studies demonstrated that even though the expression of the variant protein is reduced, there is mild to no deficiency in MMR assays and the protein is stable exhibiting normal cellular localization and normal protein interaction (e.g. Abildgaard_2019, Hinrichsen_2013, Ou_2017, Raevaara_2005, Takahashi_2007). Yeast assays examining the dominant mutator effect and reversion rate were conflicting in their outcomes with some showing a negative effect of the variant while others did not or showed a low mutator phenotype (e.g. Takahashi_2007, Wanat_2007). p.Arg659Gln affects a codon that has been linked to aberrant splicing and subsequent skipping of exon 17, and some publications suggest it could have potential impact on splicing (Nystrom-Lahti_1999 and Raevaara_2005). However, 4/4 computational tools predict no significant impact of the variant on normal splicing and RNA studies did not reveal any aberrant splicing (Hampel_2019). The following publications have been ascertained in the context of this evaluation (PMID: 31697235, 33309985, 16885385, 23403630, 21120944, 32980694, 33558524, 17594722, 16083711, 17510385, 22949387, 27629256, 19863800, 17210669, 28135145, 10534773). Seven submitters have provided clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. Based on the evidence outlined above, the variant was classified as uncertain significance. -

not provided

Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Oct 22, 2022	The frequency of this variant in the general population, 0.00012 (3/24942 chromosomes, http://gnomad.broadinstitute.org), is uninformative in assessment of its pathogenicity. In the published literature, the variant has been reported in a hereditary non-polyposis colorectal cancer family with 10 affected members and fulfilled Amsterdam criteria (PMID: 16083711 (2005)). Published functional studies have reported conflicting results on the effect of this variant on MLH1 protein activity (PMID: 16083711 (2005), 17510385 (2009), 20533529 (2010), 19863800 (2009)). Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded conflicting predictions that this variant is benign or damaging. Based on the available information, we are unable to determine the clinical significance of this variant. -
Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	Feb 01, 2021	Observed in families meeting Amsterdam or Bethesda criteria, with corresponding colon and endometrial tumors showing microsatellite instability (MSI-H) and loss of MLH1 staining by immunohistochemistry (Raevaara 2005, Hampel 2006, Yurgelun 2017); Published functional studies are inconclusive: some demonstrate protein expression, mismatch repair activity, nuclear localization, and PMS2 interaction similar to wild type, while others show reduced protein expression, increased mutation rate, and inconsistent dominant mutator effect, and some suggest this variant may cause a splice defect (Raevaara 2005, Takahashi 2007, Wanat 2007, Vogelsang 2009, Kosinski 2010, Hinrichsen 2013, Xiong 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: 17210669, 16885385, 17510385, 20533529, 23403630, 19863800, 25525159, 22949387, 21120944, 25871441, 17192056, 17370310, 18383312, 17594722, 27629256, 23741719, 24728327, 28135145, 16083711, 31697235, 30212499, 32980694, 33309985) -

Hereditary nonpolyposis colorectal neoplasms

Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Dec 01, 2023

This sequence change replaces arginine, which is basic and polar, with glutamine, which is neutral and polar, at codon 659 of the MLH1 protein (p.Arg659Gln). This variant is present in population databases (rs63749900, gnomAD 0.01%). This missense change has been observed in individual(s) with clinical features of Lynch syndrome (PMID: 16083711, 16885385, 28135145). This variant can co-occur with the variant c.1852_1853delinsGC (p.Lys618Ala) in cis (on the same chromosome), as the haplotype c.[1976G>A;1852_1853delinsGC] (p.[Arg659Gln;Lys618Ala]). This haplotype has been observed in individuals with Lynch syndrome (external communication). ClinVar contains an entry for this variant (Variation ID: 89964). 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. Experimental studies are conflicting or provide insufficient evidence to determine the effect of this variant on MLH1 function (PMID: 16083711, 17210669, 17510385, 19863800, 20533529, 23403630, 31697235). This variant disrupts the p.Arg659 amino acid residue in MLH1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 8776590, 9697702, 10037723, 11555625, 11601928, 11793442, 16083711). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. 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. -

Lynch syndrome

Benign:1

Likely benign, criteria provided, single submitter

clinical testing

All of Us Research Program, National Institutes of Health

Aug 15, 2023

- -

Computational scores

Source: dbNSFP v4.3

Name

Calibrated prediction

Score

Prediction

AlphaMissense

Uncertain

0.41

BayesDel_addAF

Pathogenic

0.36

BayesDel_noAF

Pathogenic

0.52

CADD

Pathogenic

DANN

Pathogenic

1.0

DEOGEN2

Pathogenic

0.85

D;.;.;.;.;.

Eigen

Pathogenic

1.0

Eigen_PC

Pathogenic

0.96

FATHMM_MKL

Pathogenic

0.98

LIST_S2

Pathogenic

0.99

D;D;.;.;.;D

M_CAP

Pathogenic

0.41

MetaRNN

Pathogenic

0.88

D;D;D;D;D;D

MetaSVM

Pathogenic

1.0

MutationAssessor

Pathogenic

3.3

M;.;.;.;.;.

MutationTaster

Benign

1.0

D;D;D;D;D;D

PrimateAI

Uncertain

0.58

PROVEAN

Uncertain

-3.5

D;D;D;D;D;D

REVEL

Pathogenic

0.80

Sift

Uncertain

0.0020

D;D;D;D;D;D

Sift4G

Uncertain

0.0070

D;D;D;D;D;D

Polyphen

1.0

D;.;.;.;.;.

Vest4

0.80

MVP

0.96

MPC

0.42

ClinPred

0.98

GERP RS

5.5

Varity_R

0.92

gMVP

0.85

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

Below is the list of publications found by LitVar. It may be empty.

Variant summary

Frequency

Consequence

Scores

Clinical Significance

Conservation

ACMG classification

Transcripts

RefSeq

Ensembl

Frequencies

ClinVar

Submissions by phenotype

Computational scores

Splicing

Publications

LitVar

Other links and lift over