3-36993633-C-G

Variant names:

• chr3-36993633-C-G
• rs63750216
• NM_000249.4:c.86C>G

Variant summary

Our verdict is Pathogenic. The variant received 14 ACMG points: 14P and 0B. PM1 PM2 PP3_Moderate PP5_Very_Strong

The NM_000249.4(MLH1):​c.86C>G​(p.Ala29Gly) variant causes a missense change involving the alteration of a conserved nucleotide. 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 Pathogenic (★★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. A29S) has been classified as Uncertain significance.

• Frequency: Genomes: not found (cov: 33)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Pathogenic reviewed by expert panel P:5
• Conservation: PhyloP100: 7.52
• Publications: 8 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 14 ACMG points.

• PM1: In a hotspot region, there are 24 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 3 benign, 54 uncertain in NM_000249.4
• PM2: Very rare variant in population databases, with high coverage
• PP3: Splicing predictors support a deleterious effect. Scorers claiming Pathogenic: max_spliceai. No scorers claiming Uncertain. No scorers claiming Benign.
• PP5: Variant 3-36993633-C-G is Pathogenic according to our data. Variant chr3-36993633-C-G is described in ClinVar as Pathogenic. ClinVar VariationId is 90403.Status of the report is reviewed_by_expert_panel, 3 stars.

Variant Effect in Transcripts

ACMG analysis was done for transcript: NM_000249.4. You can select a different transcript below to see updated ACMG assignments.

RefSeq Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	MLH1	NM_000249.4	MANE Select	c.86C>G	p.Ala29Gly	missense	Exon 1 of 19	NP_000240.1
	MLH1	NM_001354628.2		c.86C>G	p.Ala29Gly	missense	Exon 1 of 18	NP_001341557.1
	MLH1	NM_001354629.2		c.86C>G	p.Ala29Gly	missense	Exon 1 of 18	NP_001341558.1

Ensembl Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	MLH1	ENST00000231790.8	TSL:1 MANE Select	c.86C>G	p.Ala29Gly	missense	Exon 1 of 19	ENSP00000231790.3
	MLH1	ENST00000456676.7	TSL:1	c.86C>G	p.Ala29Gly	missense	Exon 1 of 17	ENSP00000416687.3
	MLH1	ENST00000413740.2	TSL:1	c.86C>G	p.Ala29Gly	missense	Exon 1 of 15	ENSP00000416476.2

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 33

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:5

Revision: reviewed by expert panel

Submissions by phenotype

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1

Jul 10, 2023

Myriad Genetics, Inc.

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant is considered likely pathogenic. mRNA analysis has demonstrated abnormal mRNA splicing occurs [PMID: 10734316].

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

Variant causes splicing aberrations: full inactivation of variant allele. In-frame transcript interrupts ATPase domain.

not provided Pathogenic:1

Aug 13, 2020

Quest Diagnostics Nichols Institute San Juan Capistrano

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Not found in the total gnomAD dataset, and the data is high quality. Found in at least one patient with expected phenotype for this gene. Predicted to have a damaging effect on the protein. Assessment of experimental evidence suggests this variant results in abnormal protein function.

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Oct 13, 2023

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This sequence change replaces alanine, which is neutral and non-polar, with glycine, which is neutral and non-polar, at codon 29 of the MLH1 protein (p.Ala29Gly). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individuals with clinical features of Lynch syndrome (PMID: 10734316, 21642682, 28514183, 34178123; Invitae). ClinVar contains an entry for this variant (Variation ID: 90403). 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) performed at Invitae indicates that this missense variant is expected to disrupt MLH1 protein function. Studies have shown that this missense change is associated with altered splicing resulting in unknown protein product impact (Invitae). For these reasons, this variant has been classified as Pathogenic.

Hereditary cancer-predisposing syndrome Pathogenic:1

Sep 29, 2022

Ambry Genetics

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The p.A29G pathogenic mutation (also known as c.86C>G), located in coding exon 1 of the MLH1 gene, results from a C to G substitution at nucleotide position 86. The alanine at codon 29 is replaced by glycine, an amino acid with similar properties.This pathogenic muation was reported in a Japanese family with Turcot syndrome where the 16 year old proband had been diagnosed with a glioblastoma. Authors showed that this mutation created a new cryptic splice donor site resulting in two transcripts of the MLH1 protein which were equally represented. One of the transcripts contained a frameshift leading to a premature stop codon, and the second transcript contained an in-frame deletion of 12 codons (Kanamori M et al. Oncogene. 2000 Mar 16;19(12):1564-71). This alteration has been classified as pathogenic using the following lines of evidence: in silico prediction models, segregation with disease, clinical phenotype including tumor characteristics, mutation co-occurrence, and functional studies (Thompson BA et al. Hum. Mutat. 2013 Jan;34:200-9; Thompson BA et al. Nat. Genet. 2014 Feb;46:107-15; available at [www.insight-group.org/variants/classifications/]). This amino acid position is highly conserved in available vertebrate species. Using the BDGP and ESEfinder splice site prediction tools, this alteration creates a new and highly competitive cryptic donor splice site. In addition, this alteration is predicted to be deleterious by in silico analysis. In addition, this alteration is predicted to be borderline deleterious by MAPP-MMR in silico analyses (Chao EC et al. Hum. Mutat. 2008 Jun;29:852-60). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

Computational scores

Source: dbNSFP v4.9

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Benign	0.26
BayesDel_addAF	Pathogenic	0.50	D
BayesDel_noAF	Pathogenic	0.48
CADD	Pathogenic	36
DANN	Uncertain	1.0
DEOGEN2	Uncertain	0.77	D
Eigen	Pathogenic	0.71
Eigen_PC	Pathogenic	0.73
FATHMM_MKL	Pathogenic	0.98	D
LIST_S2	Pathogenic	0.98	D
M_CAP	Pathogenic	0.66	D
MetaRNN	Pathogenic	0.92	D
MetaSVM	Uncertain	0.50	D
MutationAssessor	Pathogenic	3.1	M
PhyloP100		7.5
PrimateAI	Uncertain	0.67	T
PROVEAN	Uncertain	-2.8	D
REVEL	Pathogenic	0.89
Sift	Uncertain	0.011	D
Sift4G	Uncertain	0.029	D
Polyphen		1.0	D
Vest4		0.67
MutPred		0.71		Loss of stability (P = 0.094)
MVP		0.96
MPC		0.40
ClinPred		0.99	D
GERP RS		6.0
PromoterAI		0.27	Neutral
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.0
Varity_R		0.83
gMVP		0.61
Mutation Taster		=9/91	disease causing (ClinVar)

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.99		Details are displayed if max score is > 0.2
DS_DG_spliceai		0.99		Position offset: -1
DS_DL_spliceai		0.79		Position offset: 30

Find out detailed SpliceAI scores and Pangolin per-transcript scores at spliceailookup.broadinstitute.org

Other links and lift over

dbSNP: rs63750216; hg19: chr3-37035124;