3-37014430-A-G

Position:

• chr3-37014430-A-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.678-2A>G variant causes a splice acceptor 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 Likely pathogenic (★★★).

• Frequency: Genomes: not found (cov: 33)
• Consequence: MLH1 NM_000249.4 splice_acceptor
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Likely pathogenic reviewed by expert panel P:5
• Conservation: PhyloP100: 8.88

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, No cryptic splice site detected. Exon removal results in frameshift change.
• PM2: Very rare variant in population databases, with high coverage
• PP5: Variant 3-37014430-A-G is Pathogenic according to our data. Variant chr3-37014430-A-G is described in ClinVar as [Likely_pathogenic]. Clinvar id is 90324.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37014430-A-G is described in Lovd as [Likely_pathogenic]. Variant chr3-37014430-A-G is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
MLH1	NM_000249.4	c.678-2A>G		splice_acceptor_variant		ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.678-2A>G		splice_acceptor_variant		1	NM_000249.4	ENSP00000231790	P1

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD4 exome Cov.: 24

GnomAD4 genome Cov.: 33

ClinVar

Significance: Likely pathogenic

Submissions summary: Pathogenic:5

Revision: reviewed by expert panel

Submissions by phenotype

Hereditary cancer-predisposing syndrome Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Jun 03, 2020	This variant causes an A to G nucleotide substitution at the -2 position of intron 8 of the MLH1 gene. Splice site prediction tools suggest that this variant may have a significant impact on RNA splicing. Although this prediction has not been confirmed in published RNA studies, this variant is expected to result in an absent or disrupted protein product. To our knowledge, this variant has not been reported in individuals affected with hereditary cancer in the literature. 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. -
Pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Dec 19, 2022	The c.678-2A>G intronic pathogenic mutation results from an A to G substitution two nucleotides upstream from coding exon 9 in the MLH1 gene. This variant has been identified in a proband who met Amsterdam II criteria for Lynch syndrome and tumor demonstrated loss of MLH1/PMS2 expression by immunohistochemistry (Ambry internal data). An external study reported that this alteration results in skipping of coding exon 9, leading to an out-of-frame transcript (Thompson BA et al. Hum Mutat, 2013 Jan;34:200-9). This nucleotide position is highly conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice acceptor site; however, direct evidence is insufficient at this time (Ambry internal data). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Myriad Genetics, Inc.

Jul 17, 2023

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. -

Lynch syndrome Pathogenic:1

Likely pathogenic, reviewed by expert panel

curation

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Jun 21, 2019

Interrupts canonical donor splice site -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Mar 01, 2020

This sequence change affects an acceptor splice site in intron 8 of the MLH1 gene. It is expected to disrupt RNA splicing and likely results in an absent or disrupted protein product. For these reasons, this variant has been classified as Pathogenic. Donor and acceptor splice site variants 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). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site, but this prediction has not been confirmed by published transcriptional studies. Disruption of this splice site has been observed in individual(s) with colorectal cancer (PMID: RQ1138090). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 90324). This variant is not present in population databases (ExAC no frequency). -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.63	D
BayesDel_noAF	Pathogenic	0.33
CADD	Pathogenic	28
DANN	Uncertain	0.98
Eigen	Pathogenic	1.2
Eigen_PC	Pathogenic	1.0
FATHMM_MKL	Pathogenic	1.0	D
MutationTaster	Benign	1.0	D;D;D;D;D;D
GERP RS		5.5

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.92
SpliceAI score (max)		0.85		Details are displayed if max score is > 0.2
DS_AL_spliceai		0.85		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: rs587779035; hg19: chr3-37055921;