3-37048596-G-T

Variant names:

• chr3-37048596-G-T
• rs63749900
• NM_000249.4:c.1976G>T

Variant summary

Our verdict is Pathogenic. The variant received 18 ACMG points: 18P and 0B. PM1 PM2 PM5 PP3_Strong PP5_Very_Strong

The NM_000249.4(MLH1):​c.1976G>T​(p.Arg659Leu) variant causes a missense change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.000000685 in 1,460,632 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 Pathogenic (★★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. R659Q) has been classified as Uncertain significance.

• Frequency:
  • Genomes: not found (cov: 31)
  • Exomes 𝑓: 6.8e-7 (0 hom.)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Pathogenic reviewed by expert panel P:3 U:1
• Conservation: PhyloP100: 9.59
• Publications: 44 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: Pathogenic. The variant received 18 ACMG points.

• PM1: In a hotspot region, there are 11 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 3 benign, 35 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 chr3-37048596-G-C is described in ClinVar as Pathogenic. ClinVar VariationId is 89965.Status of the report is reviewed_by_expert_panel, 3 stars.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.975
• PP5: Variant 3-37048596-G-T is Pathogenic according to our data. Variant chr3-37048596-G-T is described in ClinVar as Pathogenic. ClinVar VariationId is 89966.Status of the report is reviewed_by_expert_panel, 3 stars.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
MLH1	NM_000249.4	c.1976G>T	p.Arg659Leu	missense_variant	Exon 17 of 19	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.1976G>T	p.Arg659Leu	missense_variant	Exon 17 of 19	1	NM_000249.4	ENSP00000231790.3

Frequencies

GnomAD3 genomes Cov.: 31

GnomAD4 exome AF: 6.85e-7 AC: 1 AN: 1460632 Hom.: 0 Cov.: 30 AF XY: 0.00 AC XY: 0 AN XY: 726696

⚠️ The allele balance in gnomAD version 4 Exomes is significantly skewed from the expected value of 0.5.

African (AFR) AF: 0.00 AC: 0 AN: 33444

American (AMR) AF: 0.00 AC: 0 AN: 44724

Ashkenazi Jewish (ASJ) AF: 0.00 AC: 0 AN: 26128

East Asian (EAS) AF: 0.00 AC: 0 AN: 39694

South Asian (SAS) AF: 0.0000116 AC: 1 AN: 86224

European-Finnish (FIN) AF: 0.00 AC: 0 AN: 53334

Middle Eastern (MID) AF: 0.00 AC: 0 AN: 5766

European-Non Finnish (NFE) AF: 0.00 AC: 0 AN: 1110962

Other (OTH) AF: 0.00 AC: 0 AN: 60356

⚠️ The allele balance in gnomAD4 Exomes is highly skewed from 0.5 (p-value = 0), which strongly suggests a high chance of mosaicism in these individuals.

GnomAD4 genome Cov.: 31

Alfa AF: 0.00 Hom.: 0

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:3 Uncertain:1

Revision: reviewed by expert panel

Submissions by phenotype

Hereditary cancer-predisposing syndrome Pathogenic:2

Jan 08, 2025

Ambry Genetics

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The p.R659L pathogenic mutation (also known as c.1976G>T), located in coding exon 17 of the MLH1 gene, results from a G to T substitution at nucleotide position 1976. The arginine at codon 659 is replaced by leucine, an amino acid with dissimilar properties. This alteration has been reported in multiple individuals either meeting classic ‘Amsterdam’ criteria for HNPCC, or a modified set of criteria (Cravo M et al. Gut 2002 Mar; 50(3):405-12; Fidalgo P et al. Eur. J. Hum. Genet. 2000 Jan; 8(1):49-53; Lage PA et al. Cancer 2004 Jul; 101(1):172-7; Nyström-Lahti M et al. Genes Chromosomes Cancer 1999 Dec; 26(4):372-5). Further, this alteration has been shown to segregate with disease in one family, as two affected members were shown to carry this alteration while one unaffected member did not (Cravo M et al. Gut 2002 Mar; 50(3):405-12). This alteration is located in the c-terminal ATP binding and hydrolysis domain of the MLH1 protein, which is responsible for constitutive dimerization with PMS2. In a study examining pathogenic effects due to interference with dimerization with PMS2, this alteration was reported to results in statistically significant reduction of MLH1 expression by multiple independent Western blot transfection experiments (Kosinski J et al. Hum. Mutat. 2010 Aug; 31(8):975-82). This alteration has also been classified as pathogenic by multifactorial analysis, which integrates the following lines of evidence to produce a quantitative likelihood of pathogenicity: in silico prediction models, segregation with disease, clinical phenotype including tumor characteristics, mutation co-occurrence, and functional assay results (Thompson B et al. Nat Genet. 2014 Feb;46(2):107-15; available at [www.insight-group.org/variants/classifications/]). Functional analysis of this alteration using patient RNA sequencing via RT-PCR revealed a deletion of 93 base pairs due to skipping of exon 17; authors note that no sequence alterations of the intron-exon boundaries were identified suggesting this alteration will thereby lead to a non-functional protein (Nyström-Lahti M et al. Genes Chromosomes Cancer 1999 Dec; 26(4):372-5). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). Based on the supporting evidence, this variant is interpreted as a disease-causing mutation.

Apr 23, 2020

Color Diagnostics, LLC DBA Color Health

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This missense variant replaces arginine with leucine at codon 659 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). Functional studies have shown that this variant reduces MLH1 protein expression, disrupts nuclear localization and interaction with PMS2, and results in reduced DNA mismatch repair activity (PMID: 11839723, 20533529, 23403630, 30504929). An RNA study using cells from a heterozygous carrier has detected a low level of an alternate splicing transcript due to out-of-frame exon 17 skipping (PMID: 10534773). This variant has been reported in two related individuals affected with Lynch syndrome (PMID: 10534773, 10713887, 11839723, 15222003) and in an unrelated individual affected with Lynch syndrome (PMID: 22776989). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Loss of MLH1 function is a known mechanism of disease (clinicalgenome.org). Based on the available evidence, this variant is classified as Likely Pathogenic.

Lynch syndrome Pathogenic:1

Sep 05, 2013

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Significance: Pathogenic

Review Status: reviewed by expert panel

Collection Method: research

Multifactorial likelihood analysis posterior probability >0.99

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

May 01, 2024

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This sequence change replaces arginine, which is basic and polar, with leucine, which is neutral and non-polar, at codon 659 of the MLH1 protein (p.Arg659Leu). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with Lynch syndrome (PMID: 10713887, 11839723). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 89966). 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. Studies have shown that this missense change alters MLH1 gene expression (PMID: 20533529, 23403630). 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	0.91
BayesDel_addAF	Pathogenic	0.53	D
BayesDel_noAF	Pathogenic	0.52
CADD	Pathogenic	31
DANN	Uncertain	1.0
DEOGEN2	Pathogenic	0.92	D;.;.;.;.;.
Eigen	Pathogenic	1.1
Eigen_PC	Pathogenic	0.99
FATHMM_MKL	Pathogenic	0.99	D
LIST_S2	Pathogenic	0.98	D;D;.;.;.;D
M_CAP	Pathogenic	0.56	D
MetaRNN	Pathogenic	0.98	D;D;D;D;D;D
MetaSVM	Pathogenic	1.1	D
MutationAssessor	Pathogenic	3.7	H;.;.;.;.;.
PhyloP100		9.6
PrimateAI	Uncertain	0.66	T
PROVEAN	Pathogenic	-6.5	D;D;D;D;D;D
REVEL	Pathogenic	0.96
Sift	Uncertain	0.0010	D;D;D;D;D;D
Sift4G	Uncertain	0.0030	D;D;D;D;D;D
Vest4		0.92
ClinPred		1.0	D
GERP RS		5.5
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.1
Varity_R		0.97
gMVP		0.90
Mutation Taster		=0/100	disease causing (ClinVar)

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: rs63749900; hg19: chr3-37090087;