3-37012009-A-G

Position:

• chr3-37012009-A-G

Variant summary

Our verdict is Pathogenic. Variant got 22 ACMG points: 22P and 0B. PVS1 PM2 PP3_Strong PP5_Very_Strong

The NM_000249.4(MLH1):​c.589-2A>G variant causes a splice acceptor change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.000000685 in 1,460,462 control chromosomes in the GnomAD database, with no homozygous occurrence. 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)
  • Exomes 𝑓: 6.8e-7 (0 hom.)
• Consequence: MLH1 NM_000249.4 splice_acceptor
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Pathogenic reviewed by expert panel P:12 O:1
• Conservation: PhyloP100: 8.78

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 22 ACMG points.

• PVS1: Splicing +-2 bp (donor or acceptor) variant, LoF is a know mechanism of disease, No cryptic splice site detected. Exon removal results in frameshift change.
• PM2: Very rare variant in population databases, with high coverage
• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF, max_spliceai. No scorers claiming Uncertain. No scorers claiming Benign.
• PP5: Variant 3-37012009-A-G is Pathogenic according to our data. Variant chr3-37012009-A-G is described in ClinVar as [Pathogenic]. Clinvar id is 90291.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37012009-A-G is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.589-2A>G		splice_acceptor_variant		ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.589-2A>G		splice_acceptor_variant		1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome AF: 6.85e-7 AC: 1 AN: 1460462 Hom.: 0 Cov.: 30 AF XY: 0.00000138 AC XY: 1 AN XY: 726702

Gnomad4 AFR exome AF: 0.00

Gnomad4 AMR exome AF: 0.00

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.00

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 9.00e-7

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:12 Other:1

Revision: reviewed by expert panel

Submissions by phenotype

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:3

Pathogenic, criteria provided, single submitter	clinical testing	Baylor Genetics	Jun 04, 2023	- -
Pathogenic, criteria provided, single submitter	clinical testing	Counsyl	Dec 13, 2016	- -
Pathogenic, criteria provided, single submitter	clinical testing	Myriad Genetics, Inc.	Mar 17, 2023	This variant is considered pathogenic. Functional studies indicate this variant impacts protein function [PMID: 15713769]. This variant is strongly associated with more severe personal and family histories of cancer, typical for individuals with pathogenic variants in this gene [PMID: 25085752]. -

Lynch syndrome Pathogenic:3

Pathogenic, reviewed by expert panel	research	International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	Sep 05, 2013	Variant causes splicing aberrations: full inactivation of variant allele. Multifactorial likelihood analysis posterior probability >0.99. -
Pathogenic, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Apr 17, 2018	Variant summary: MLH1 c.589-2A>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: Five predict the variant abolishes a 3 acceptor site. At least one publication reports experimental evidence that this variant affects mRNA splicing (Thompson_2013). The variant was absent in 246208 control chromosomes (gnomAD). The variant, c.589-2A>G has been reported in the literature in individuals affected with Lynch Syndrome (Syngal_1999, Susswein_2016, Tannergard_1995, Capozzi_1999). These data indicate that the variant is likely to be associated with disease. Multiple ClinVar submissions from clinical diagnostic laboratories (evaluation after 2014) cite the variant as "pathogenic." Based on the evidence outlined above, the variant was classified as pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	Mar 18, 2019	The c.589-2A>G variant in MLH1 has been reported in >15 individuals with Lynch syndrome and segregated with disease in >15 affected relatives from multiple families (Luce 1995, Casey 2005, Tomsic 2012, DeRycke 2017). This variant is considered a founder variant (Tomsic 2015). This variant was absent from large population studies, but has been reported in ClinVar (Variation ID: 90291). This variant occurs within the canonical splice site (+/- 1,2) and is predicted to cause altered splicing leading to an abnormal or absent protein. In vitro studies demonstrate that this variant causes activation of a cryptic splice site, leading to a frameshift variant (Luce 1995, Arnold 2009). Loss of function of the MLH1 gene is an established disease mechanism in autosomal dominant Lynch syndrome. In summary, this variant meets criteria to be classified as pathogenic for autosomal dominant Lynch syndrome. ACMG/AMP Criteria applied: PVS1, PS4, PM2, PP1_Strong, PS3_Moderate. -

not provided Pathogenic:3

Pathogenic, criteria provided, single submitter	clinical testing	Revvity Omics, Revvity	Feb 09, 2022	- -
Pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Jul 01, 2022	This variant disrupts a canonical splice-acceptor site and interferes with normal MLH1 mRNA splicing. It has not been reported in large, multi-ethnic general populations (http://gnomad.broadinstitute.org). In the published literature, the variant has been reported in individuals with Lynch syndrome (PMIDs: 30019097 (2019), 28944238 (2017), 25980754 (2015), 21671475 (2012)). Based on the available information, this variant is classified as pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Dec 28, 2022	Canonical splice site variant predicted to result in a null allele in a gene for which loss of function is a known mechanism of disease; Not observed at significant frequency in large population cohorts (gnomAD); Truncating variants in this gene are considered pathogenic by a well-established clinical consortium and/or database; This variant is associated with the following publications: (PMID: 10422993, 19690142, 25345868, 28888541, 15713769, 25530820, 22949379, 7557107, 21671475, 22624972, 19267393, 12067992, 14970868, 10448273, 17192056, 26681312, 28944238, 29288294, 29228462, 25525159, 25980754, 30787465) -

Hereditary cancer-predisposing syndrome Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Dec 11, 2023	The c.589-2A>G intronic pathogenic mutation results from an A to G substitution two nucleotides upstream from coding exon 8 of the MLH1 gene. This mutation has been reported in numerous Lynch syndrome families to date (Luce MC et al. Gastroenterology. 1995 Oct;109(4):1368-74; Viel A et al. Community Genet. 1998;1(4):229-36; Capozzi E et al. Eur. J. Cancer. 1999 Feb;35(2):289-95; Syngal S et al. JAMA. 1999 Jul 21;282(3):247-53; Casey G et al. JAMA. 2005 Feb 16;293(7):799-809; DeRycke MS et al. Mol Genet Genomic Med. 2017 Sep;5(5):553-569). In addition, this alteration has been described as an American founder mutation (Tomsic J et al. Int. J. Cancer. 2012 May 1;130(9):2088-95). RNA studies have demonstrated that this alteration activates a cryptic acceptor site resulting in a transcript with a 4 base pair deletion, which is predicted to lead to a translational frameshift (Ambry internal data; Arnold S et al. Hum. Mutat. 2009 May;30(5):757-70). 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. Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. -
Pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Oct 17, 2023	This variant causes an A to G nucleotide substitution at the -2 position of intron 7 of the MLH1 gene. Functional RNA studies have shown that this variant causes use of a cryptic splice site and skipping of exon 8, resulting in a frameshift and premature truncation (PMID: 7557107, 19267393, 22949379). This variant has been reported in multiple individuals affected with Lynch syndrome or Lynch syndrome-associated disease (PMID: 7557107, 8521398, 19267393, 21671475, 26681312, 29228462, 30019097). 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 Pathogenic. -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Jan 04, 2024

This sequence change affects an acceptor splice site in intron 7 of the MLH1 gene. RNA analysis indicates that disruption of this splice site induces altered splicing and may result in an absent or disrupted protein product. This variant is not present in population databases (gnomAD no frequency). Disruption of this splice site has been observed in individual(s) with Lynch syndrome (PMID: 7557107, 10422993, 21671475). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 90291). Studies have shown that disruption of this splice site results in activation of a cryptic splice site and introduces a premature termination codon (PMID: 7557107; Invitae). The resulting mRNA is expected to undergo nonsense-mediated decay. For these reasons, this variant has been classified as Pathogenic. -

Lynch syndrome 1 Other:1

not provided, no classification provided

literature only

GeneReviews

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Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.63	D
BayesDel_noAF	Pathogenic	0.23
CADD	Pathogenic	34
DANN	Uncertain	0.98
Eigen	Pathogenic	1.1
Eigen_PC	Pathogenic	0.93
FATHMM_MKL	Pathogenic	1.0	D
MutationTaster	Benign	1.0	D;D;D;D;D;D
GERP RS		4.2

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.94
SpliceAI score (max)		0.99		Details are displayed if max score is > 0.2
DS_AG_spliceai		0.57		Position offset: 6
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: rs267607767; hg19: chr3-37053500;