NM_000249.4:c.117-2A>G

Variant names:

• chr3-36996617-A-G
• rs267607712
• NM_000249.4:c.117-2A>G

Variant summary

Our verdict is Pathogenic. Variant got 18 ACMG points: 18P and 0B. PVS1 PM2 PP5_Very_Strong

The NM_000249.4(MLH1):​c.117-2A>G variant causes a splice acceptor, intron 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. 3/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Pathogenic (★★★).

• Frequency: Genomes: not found (cov: 32)
• Consequence: MLH1 NM_000249.4 splice_acceptor, intron
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Pathogenic reviewed by expert panel P:7
• Conservation: PhyloP100: 8.77

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 Pathogenic. Variant got 18 ACMG points.

• PVS1: Splicing +-2 bp (donor or acceptor) variant, LoF is a know mechanism of disease, Cryptic splice site detected, with MaxEntScore 7.4, offset of 5, new splice context is: tgtttgatttgccggtttAGatg. Cryptic site results in frameshift change. If cryptic site found is not functional and variant results in exon loss, it results in frameshift change.
• PM2: Very rare variant in population databases, with high coverage
• PP5: Variant 3-36996617-A-G is Pathogenic according to our data. Variant chr3-36996617-A-G is described in ClinVar as [Pathogenic]. Clinvar id is 142856.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-36996617-A-G is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
MLH1	NM_000249.4	c.117-2A>G		splice_acceptor_variant, intron_variant	Intron 1 of 18	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.117-2A>G		splice_acceptor_variant, intron_variant	Intron 1 of 18	1	NM_000249.4	ENSP00000231790.3

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 30

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:7

Revision: reviewed by expert panel

Submissions by phenotype

not provided Pathogenic:2

Nov 19, 2024

Quest Diagnostics Nichols Institute San Juan Capistrano

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The MLH1 c.117-2A>G variant disrupts a canonical splice-acceptor site and interferes with normal MLH1 mRNA splicing. This variant has been reported in the published literature in individuals with prostate cancer (PMID: 25117503 (2014)) and colorectal cancer whose tumors showed loss of MLH1 expression by immunohistochemistry (PMID: 15713769 (2005), 25117503 (2014), 27978560 (2016), 30917047 (2019)). In-vitro splicing studies showed that this variant causes altered splicing due to deletion of 5 nucleotides at the beginning of exon 2 and a premature stop signal resulting in a truncated protein (PMID: 15713769 (2005), 32849802 (2020)). This variant has not been reported in large, multi-ethnic general populations (Genome Aggregation Database, http://gnomad.broadinstitute.org). Based on the available information, this variant is classified as pathogenic. -

Mar 06, 2017

GeneDx

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant is denoted MLH1 c.117-2A>G or IVS1-2A>G and consists of an A>G nucleotide substitution at the -2 position of intron 1 of the MLH1 gene. This variant destroys a canonical splice acceptor site and is predicted to cause abnormal gene splicing, leading to either an abnormal message that is subject to nonsense-mediated mRNA decay or to an abnormal protein product. This variant has been reported in mulitple individuals with colorectal cancer, including at least three with personal and family histories fulfilling clinical Lynch syndrome criteria (Casey 2005, Rosty 2014, Guindalini 2015, Pearlman 2016). Based on the current evidence, we consider this variant to be pathogenic. -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1

Jul 11, 2023

Myriad Genetics, Inc.

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant is considered likely pathogenic. This variant occurs within a consensus splice junction and is predicted to result in abnormal mRNA splicing of either an out-of-frame exon or an in-frame exon necessary for protein stability and/or normal function. -

Hereditary nonpolyposis colon cancer Pathogenic:1

Aug 06, 2020

Women's Health and Genetics/Laboratory Corporation of America, LabCorp

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Variant summary: MLH1 c.117-2A>G is located in a canonical splice-site and is predicted to affect mRNA splicing resulting in a significantly altered protein due to either exon skipping, shortening, or inclusion of intronic material. Several computational tools predict a significant impact on normal splicing: Three predict the variant abolishes a 3 acceptor site. Three predict the variant strengthens a cryptic exonic 3 acceptor site. However, these predictions have yet to be confirmed by functional studies. The variant was absent in 251426 control chromosomes (gnomAD). c.117-2A>G has been reported in the literature in multiple individuals affected with Hereditary Nonpolyposis Colorectal Cancer (Espenschied_2017, Pearlman_2017, Guindalini_2015, Casey_2005, Sarode_2019). These data indicate that the variant is very likely to be associated with disease. To our knowledge, no experimental evidence demonstrating an impact on protein function has been reported. Five other ClinVar submitters including an expert panel (InSiGHT) cite the variant as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic. -

Lynch syndrome Pathogenic:1

Oct 18, 2018

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Significance: Pathogenic

Review Status: reviewed by expert panel

Collection Method: curation

Multifactorial likelihood analysis posterior probability > 0.99 (1.0) -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Dec 17, 2024

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This sequence change affects an acceptor splice site in intron 1 of the MLH1 gene. It is expected to disrupt RNA splicing. Variants that disrupt the donor or acceptor splice site typically lead to a loss of protein function (PMID: 16199547), and loss-of-function variants in MLH1 are known to be pathogenic (PMID: 15713769, 24362816). This variant is not present in population databases (gnomAD no frequency). Disruption of this splice site has been observed in individuals with clinical features of Lynch syndrome (PMID: 15713769, 25117503, 26248088, 27978560; internal data). Invitae Evidence Modeling of clinical and family history, age, sex, and reported ancestry of multiple individuals with this MLH1 variant has been performed. This variant is expected to be pathogenic with a positive predictive value of at least 99%. This is a validated machine learning model that incorporates the clinical features of 1,370,736 individuals referred to our laboratory for MLH1 testing. This variant is also known as IVS01-2A>G. ClinVar contains an entry for this variant (Variation ID: 142856). Studies have shown that disruption of this splice site is associated with inconclusive levels of altered splicing (PMID: 15713769, 24811117; internal data). For these reasons, this variant has been classified as Pathogenic. -

Hereditary cancer-predisposing syndrome Pathogenic:1

Dec 20, 2021

Ambry Genetics

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The c.117-2A>G intronic pathogenic mutation results from an A to G substitution two nucleotides upstream from coding exon 2 in the MLH1 gene. This mutation has been reported in numerous colorectal cancer patients whose tumors demonstrated loss of MLH1 expression and/or microsatellite instability and whose family histories met Amsterdam criteria (Casey G et al. JAMA. 2005 Feb;293(7):799-809; Rosty C et al. Fam. Cancer 2014 Dec;13(4):573-82; Guindalini RS et al. Gastroenterology 2015 Nov;149:1446-53; Pearlman R et al. JAMA Oncol. 2017 Apr;3(4):464-471; Sarode VR et al. Arch Pathol Lab Med, 2019 10;143:1225-1233). This variant was also identified in a cohort of 3,579 African males diagnosed with prostate cancer who underwent multi-gene panel testing (Matejcic M et al. JCO Precis Oncol, 2020 Jan;4:32-43). In two studies, cDNA splicing analysis showed that this pathogenic mutation is associated with aberrant splicing causing a deletion of 5 bases at the beginning of exon 2 resulting in a premature stop codon (Casey G et al. JAMA. 2005 Feb;293(7):799-809; Thompson BA et al. Front Genet. 2020 Jul;11:798). In silico splice site analysis predicts that this alteration will weaken the native splice acceptor site. RNA studies have demonstrated that this alteration results in abnormal splicing in the set of samples tested (Ambry internal data). In addition to the clinical data presented in the literature, alterations that disrupt the canonical splice site are expected to cause aberrant splicing, resulting in an abnormal protein or a transcript that is subject to nonsense-mediated mRNA decay. As such, this alteration is classified as a disease-causing mutation. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.63	D
BayesDel_noAF	Pathogenic	0.37
CADD	Pathogenic	30
DANN	Uncertain	0.99
Eigen	Pathogenic	1.2
Eigen_PC	Pathogenic	1.0
FATHMM_MKL	Pathogenic	0.99	D
GERP RS		5.5

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.93
SpliceAI score (max)		0.99		Details are displayed if max score is > 0.2
DS_AL_spliceai		0.99		Position offset: 2

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

Publications

LitVar

Other links and lift over

dbSNP: rs267607712; hg19: chr3-37038108;