3-37012100-G-T

Position:

• chr3-37012100-G-T

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.677+1G>T variant causes a splice donor change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.000000689 in 1,451,290 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: 6.9e-7 (0 hom.)
• Consequence: MLH1 NM_000249.4 splice_donor
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Pathogenic reviewed by expert panel P:8
• Conservation: PhyloP100: 7.32

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 3-37012100-G-T is Pathogenic according to our data. Variant chr3-37012100-G-T is described in ClinVar as [Pathogenic]. Clinvar id is 90312.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37012100-G-T is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.677+1G>T		splice_donor_variant		ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.677+1G>T		splice_donor_variant		1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome AF: 6.89e-7 AC: 1 AN: 1451290 Hom.: 0 Cov.: 30 AF XY: 0.00000138 AC XY: 1 AN XY: 722580

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: 0.0000187

Gnomad4 NFE exome AF: 0.00

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:8

Revision: reviewed by expert panel

Submissions by phenotype

Lynch syndrome Pathogenic:2

Pathogenic, no assertion criteria provided	clinical testing	Department of Pathology and Laboratory Medicine, Sinai Health System	-	The MLH1 c.677+1G>A variant was identified in 4 of 1350 proband chromosomes (frequency: 0.003) from individuals or families with Lynch Syndrome (Parc 2003, Domingo 2004, Lagerstedt Robinson 2007, Schneider 2018). The variant was identified in dbSNP (rs267607778) as “with pathogenic allele”, ClinVar (classified as pathogenic by GeneDx and InSiGHT and likely pathogenic by Invitae) and UMD-LSDB (observed 6x). The variant was not identified in the following control databases: the Exome Aggregation Consortium (August 8th 2016), or the Genome Aggregation Database (Feb 27, 2017). The c.677+1G>A variant is predicted to cause abnormal splicing because the nucleotide substitution occurs in the invariant region of the splice consensus sequence. In addition, in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) predict a loss of the 5’ splice site. In summary, based on the above information this variant meets our laboratory’s criteria to be classified as pathogenic. -
Pathogenic, reviewed by expert panel	research	International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	Sep 05, 2013	Multifactorial likelihood analysis posterior probability >0.99 -

not provided Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Jul 16, 2015	This pathogenic variant is denoted MLH1 c.677+1G>T or IVS8+1G>T and consists of a G>T nucleotide substitution at the +1 position of intron 8 of the MLH1 gene. The variant destroys a canonical splice donor 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 individuals with familial and/or early-onset colorectal cancer, including one individual whose colorectal tumor demonstrated microsatellite instability (Parc 2003, Domingo 2004, Lagerstedt Robinson 2007). The International Society for Gastrointestinal Hereditary Tumours Incorporated (InSiGHT) classifies this variant as pathogenic based on their multifactorial likelihood analysis (Thompson 2014). Based on the current evidence, we consider MLH1 c.677+1G>T to be pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Jul 12, 2022	This variant disrupts a canonical splice-donor 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 or at high risk of colorectal cancer (PMIDs: 27601186 (2016), 23544471 (2013), 17312306 (2007), 15342696 (2004), 12624141 (2003)). Based on the available information, this variant is classified as pathogenic. -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Myriad Genetics, Inc.

Jul 17, 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. Functional studies indicate this variant impacts protein function [PMID: 12655562]. -

Hereditary nonpolyposis colon cancer Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

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

Jun 20, 2022

Variant summary: MLH1 c.677+1G>T 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. 4 computational tools predict a significant impact on normal splicing and abolishes a 5' splicing donor site. However, these predictions have yet to be confirmed by functional studies. The variant was absent in 250982 control chromosomes (gnomAD). The variant, c.677+1G>T, has been reported in the literature in individuals affected with Lynch Syndrome and early-onset colorectal cancer (examples: Parc_2003, Domingo_2004, Tanskanen_2013 and FerrerAvargues_2021). These data indicate that the variant is likely to be associated with disease. Two 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 -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Invitae

Apr 19, 2023

For these reasons, this variant has been classified as Pathogenic. Studies have shown that disruption of this splice site results in skipping of exon 8 and introduces a premature termination codon (Invitae). The resulting mRNA is expected to undergo nonsense-mediated decay. Based on a multifactorial likelihood algorithm using genetic, in silico, and/or statistical data, this variant has been determined to have a high probability of being pathogenic (PMID: 24362816). ClinVar contains an entry for this variant (Variation ID: 90312). This variant is also known as IVS8+1G>T. Disruption of this splice site has been observed in individuals with a MLH1-related disease (PMID: 12624141, 15342696, 17312306, 27601186; Invitae). This variant is not present in population databases (gnomAD no frequency). This sequence change affects a donor splice site in intron 8 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. -

Hereditary cancer-predisposing syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Jun 20, 2022

The c.677+1G>T intronic pathogenic mutation results from a G to T substitution one nucleotide after coding exon 8 of the MLH1 gene. This mutation, also referred to as IVS8+1G>T in published literature, has been reported in a family that met modified Amsterdam criteria (Parc Y et al. J Med Genet. 2003 Mar;40(3):208-13). This mutation was also detected in several individuals whose tumors showed high microsatellite instability and negative BRAF V600E analyses (Domingo E et al. J Med Genet. 2004 Sep;41(9):664-8; Lagerstedt Robinson K et al. J Natl Cancer Inst. 2007 Feb 21;99(4):291-9). This mutation has been identified in at least one proband who met Amsterdam I/II criteria for Lynch syndrome and tumor demonstrated high microsatellite instability with loss of MLH1 expression by immunohistochemistry (IHC; Ambry internal data). Furthermore, this mutation has been identified in probands whose Lynch syndrome-associated tumor demonstrated loss of MLH1/PMS2 expression by IHC (Ambry internal data). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). 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 and may result in the creation or strengthening of a novel splice donor site; however, direct evidence is insufficient at this time (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	0.43
CADD	Pathogenic	34
DANN	Uncertain	0.99
Eigen	Pathogenic	1.2
Eigen_PC	Pathogenic	1.1
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	0.94
SpliceAI score (max)		0.99		Details are displayed if max score is > 0.2
DS_DG_spliceai		0.37		Position offset: -9
DS_DL_spliceai		0.99		Position offset: -1

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

Publications

LitVar

Other links and lift over

dbSNP: rs267607778; hg19: chr3-37053591;