3-37017603-A-G

Variant names:

• chr3-37017603-A-G
• rs267607777
• NM_000249.4:c.884+4A>G

Variant summary

Our verdict is Pathogenic. The variant received 11 ACMG points: 11P and 0B. PM2 PP3 PP5_Very_Strong

The NM_000249.4(MLH1):​c.884+4A>G variant causes a splice region, intron change. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a benign outcome for this variant. 2/3 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_region, intron
• Scores: Splicing: ADA: 0.9903
• Clinical Significance: Pathogenic reviewed by expert panel P:8
• Conservation: PhyloP100: 4.51
• 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: Pathogenic. The variant received 11 ACMG points.

• PM2: Very rare variant in population databases, with high coverage
• PP3: Splicing predictors support a deleterious effect. Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF. Scorers claiming Uncertain: max_spliceai. No scorers claiming Benign.
• PP5: Variant 3-37017603-A-G is Pathogenic according to our data. Variant chr3-37017603-A-G is described in ClinVar as Pathogenic. ClinVar VariationId is 90414.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.884+4A>G		splice_region intron	N/A	NP_000240.1
	MLH1	NM_001354628.2		c.884+4A>G		splice_region intron	N/A	NP_001341557.1
	MLH1	NM_001354629.2		c.785+4A>G		splice_region intron	N/A	NP_001341558.1

Ensembl Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	MLH1	ENST00000231790.8	TSL:1 MANE Select	c.884+4A>G		splice_region intron	N/A	ENSP00000231790.3
	MLH1	ENST00000456676.7	TSL:1	c.884+4A>G		splice_region intron	N/A	ENSP00000416687.3
	MLH1	ENST00000413740.2	TSL:1	c.884+4A>G		splice_region intron	N/A	ENSP00000416476.2

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 30

GnomAD4 genome Cov.: 32

Alfa AF: 0.00 Hom.: 0

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:8

Revision: reviewed by expert panel

Submissions by phenotype

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:2

Jun 16, 2021

Baylor Genetics

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Jul 18, 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: 18561205, 17653898].

not provided Pathogenic:2

Sep 20, 2024

GeneDx

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Observed in patients with Lynch-related and other cancers as well as tumor studies consistent with pathogenic variants in this gene (PMID: 17653898, 20459533, 21034533, 21239990, 25980754, 33436027, 38355628); Not observed at significant frequency in large population cohorts (gnomAD); This variant is associated with the following publications: (PMID: 21239990, 25525159, 18561205, 21034533, 20459533, 17653898, 19339519, 24333619, 33436027, 25980754, 38355628)

Dec 13, 2023

Quest Diagnostics Nichols Institute San Juan Capistrano

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The MLH1 c.884+4A>G variant has been reported in the published literature in individuals/families affected with Lynch syndrome associated cancers and/or polyps (PMIDs: 20459533 (2010), 21034533 (2010), 21239990 (2011), 21642682 (2011), 25980754 (2015), 33436027 (2021)). In vitro studies have shown that this variant causes a splicing defect that results in exon 10 skipping (PMIDs: 17653898 (2007), 18561205 (2008)). 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.

Hereditary cancer-predisposing syndrome Pathogenic:2

Jun 10, 2024

Ambry Genetics

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The c.884+4A>G intronic pathogenic mutation results from an A to G substitution four nucleotides after coding exon 10 in the MLH1 gene. In one study, this variant was observed in two individuals fulfilling Bethesda criteria, whose tumors showed high microsatellite instability (MSI-H) and absence of MLH1 staining on immunohistochemistry. Furthermore, the variant was observed to segregate with colon cancer in the father and sister of one proband (Rahner N et al. Acta Oncol. 2007;46(6):763-9). This variant was also identified in an Italian proband whose tumor was MSI-H (Pastrello C et al. Genet Med, 2011 Feb;13:115-24). In silico splice site analysis for this alteration is inconclusive. Functional splicing analysis by RT-PCR using patient samples and in a mini gene assay demonstrated exon 10 skipping was associated with this variant (Rahner N et al. Acta Oncol. 2007;46(6):763-9; Tournier I et al. Hum. Mutat. 2008;29(12):1412-24; Ambry internal data). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

Oct 21, 2024

Molecular Diagnostics Laboratory, Catalan Institute of Oncology

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

PVS1, PM2_Supporting, PP1_Strong c.884+4A>G is located close to a canonical splice site of MLH1 exon 10. Functional RNA studies have demonstrated complete abnormal splicing for this variant and deletion of exon 10 (r.791_884del, p.His264Leufs*2)(PVS1)(PMID: 22736432). It is not present in the population database gnomAD v2.1.1, non cancer dataset (PM2_supporting). The variant co-segregates in several families with clinical features of Lynch syndrome (PMID: 17653898 and internal data) (PP1_Strong). Also, this variant has been identified in many individuals with studied tumors demonstrating microsatellite instability and loss or conserved MLH1 expression on immunohistochemistry (PMID: 17653898 and internal data). In adition, this variant has been reported in the ClinVar database (1x as likely pathogenic, 4x pathogenic) and in the LOVD database (4x pathogenic, 1x likely pathogenic, 1x uncertain significance). In addition, the variant has been also reported in InSiGHT database 2013/09/05 v1.9 as pathogenic variant (Summary Justification Variant causes splicing aberration: full inactivation of variant allele). Based on currently available information, the variant c.884+4A>G is classified as a pathogenic variant according to ACMG guidelines.

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 allele results in splicing abberation leading to premature stop codon: full inactivation of variant allele

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Nov 12, 2024

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This sequence change falls in intron 10 of the MLH1 gene. It does not directly change the encoded amino acid sequence of the MLH1 protein. RNA analysis indicates that this variant induces altered splicing and may result in an absent or altered protein product. This variant is not present in population databases (gnomAD no frequency). This variant has been observed in individual(s) with MLH1-related conditions (PMID: 17653898, 18561205, 21239990, 25980754). It has also been observed to segregate with disease in related individuals. 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 IVS10+4A>G. ClinVar contains an entry for this variant (Variation ID: 90414). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant results in exon 10 skipping, and produces a non-functional protein and/or introduces a premature termination codon (PMID: 1856120, 17653898). This variant disrupts the p.Arg265 amino acid residue in MLH1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 24344984, 28874130). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic.

Computational scores

Source: dbNSFP v4.9

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.23
CADD	Benign	23
DANN	Benign	0.96
PhyloP100		4.5
Mutation Taster		=1/99	disease causing (ClinVar)

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	0.99
dbscSNV1_RF	Pathogenic	0.80
Splicevardb		3.0
SpliceAI score (max)		0.29		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.29		Position offset: -4

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

Other links and lift over

dbSNP: rs267607777; hg19: chr3-37059094;