3-37040296-T-C

Position:

• chr3-37040296-T-C

Variant summary

Our verdict is Pathogenic. Variant got 19 ACMG points: 19P and 0B. PVS1 PM2 PP3 PP5_Very_Strong

The NM_000249.4(MLH1):​c.1667+2T>C variant causes a splice donor 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. 2/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Likely pathogenic (★★).

• Frequency: Genomes: not found (cov: 32)
• Consequence: MLH1 NM_000249.4 splice_donor
• Scores: Splicing: ADA: 0.9717
• Clinical Significance: Pathogenic/Likely pathogenic criteria provided, multiple submitters, no conflicts P:5
• Conservation: PhyloP100: 7.81

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 19 ACMG points.

• PVS1: Splicing 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, max_spliceai. No scorers claiming Uncertain. Scorers claiming Benign: dbscSNV1_RF.
• PP5: Variant 3-37040296-T-C is Pathogenic according to our data. Variant chr3-37040296-T-C is described in ClinVar as [Likely_pathogenic]. Clinvar id is 237317.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.1667+2T>C		splice_donor_variant		ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.1667+2T>C		splice_donor_variant		1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 28

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic/Likely pathogenic

Submissions summary: Pathogenic:5

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Mendelics	May 28, 2019	- -
Pathogenic, criteria provided, single submitter	clinical testing	Myriad Genetics, Inc.	Jul 21, 2023	This variant is considered 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. -

not provided Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

The MLH1 c.1667+2T>C substitution has been previously reported in a patient (1/390 proband chromosomes; frequency: 0.003) with early age of onset who met Amsterdam Criteria II (Limburg PJ et al. 2011); however, no normal control chromosomes were examined in this study. The c.1667+2T>C variant is predicted to cause abnormal splicing because the nucleotide substitution occurs in the invariant +1 and +2 positions of the splice consensus sequence. Loss of function of the MLH1 gene is an established disease mechanism in patients with colorectal cancer, which makes it highly likely that the c.1667+2T>C variant is pathogenic. -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Invitae

Feb 14, 2021

In summary, the currently available evidence indicates that the variant is pathogenic, but additional data are needed to prove that conclusively. Therefore, this variant has been classified as Likely 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). Disruption of this splice site has been observed in individuals with colorectal cancer (PMID: 21056691, Invitae). ClinVar contains an entry for this variant (Variation ID: 237317). This variant is not present in population databases (ExAC no frequency). This sequence change affects a donor splice site in intron 14 of the MLH1 gene. It is expected to disrupt RNA splicing and likely results in an absent or disrupted protein product. -

Hereditary cancer-predisposing syndrome Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Apr 06, 2022

The c.1667+2T>C intronic variant results from a T to C substitution two nucleotides after coding exon 14 in the MLH1 gene. This variant has been reported in a patient diagnosed with colon cancer at age 47 whose family met Amsterdam II criteria (Limburg et al. Clin Gastroenterol Hepatol. 2011 Jun;9(6):497-502). This nucleotide position is highly conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice donor site. Based on the majority of available evidence to date, this variant is likely to be pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.63	D
BayesDel_noAF	Uncertain	0.040
CADD	Pathogenic	27
DANN	Benign	0.96
Eigen	Pathogenic	0.96
Eigen_PC	Pathogenic	0.76
FATHMM_MKL	Pathogenic	0.99	D
MutationTaster	Benign	1.0	D;D;D;D;D;D
GERP RS		5.4
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.1

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	0.97
dbscSNV1_RF	Benign	0.62
SpliceAI score (max)		0.98		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.98		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: rs878853780; hg19: chr3-37081787;