rs267607720

chr3-37000952-C-Gchr3-37000952-C-T

Variant summary

Our verdict is Pathogenic. Variant got 11 ACMG points: 11P and 0B. PM2 PP3 PP5_Very_Strong

The NM_000249.4(MLH1):c.208-3C>G variant causes a splice region, splice polypyrimidine tract, intron change involving the alteration of a non-conserved nucleotide. 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 Likely pathogenic (★★★).

• Frequency: Genomes: not found (cov: 32)
• Consequence: MLH1 NM_000249.4 splice_region, splice_polypyrimidine_tract, intron
• Scores: Splicing: ADA: 0.9989
• Clinical Significance: Likely pathogenic reviewed by expert panel P:8
• Conservation: PhyloP100: 0.562

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 11 ACMG points.

• 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 3-37000952-C-G is Pathogenic according to our data. Variant chr3-37000952-C-G is described in ClinVar as [Likely_pathogenic]. Clinvar id is 90032.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37000952-C-G is described in Lovd as [Likely_pathogenic]. Variant chr3-37000952-C-G is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.208-3C>G		splice_region_variant, splice_polypyrimidine_tract_variant, intron_variant		ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.208-3C>G		splice_region_variant, splice_polypyrimidine_tract_variant, intron_variant		1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 26

GnomAD4 genome Cov.: 32

ClinVar

Significance: Likely pathogenic

Submissions summary: Pathogenic:8

Revision: reviewed by expert panel

Submissions by phenotype

Lynch syndrome Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	University of Washington Department of Laboratory Medicine, University of Washington	May 01, 2018	MLH1 NM_000249.3:c.208-3C>G has a 97.4% probability of pathogenicity based on combining prior probability from public data with a likelihood ratio of 1.56 to 1, generated from evidence of seeing this as a somatic mutation in a tumor without loss of heterozygosity at the MLH1 locus. See Shirts et al 2018, PMID 29887214. -
Likely pathogenic, reviewed by expert panel	curation	International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	Jun 21, 2019	Variant causes splicing aberration (not quantified) & 3 MSI-H tumours. Multifactorial likelihood analysis posterior probability 0.95-0.99. -

not provided Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	GeneDx	May 30, 2023	Intronic variant demonstrated to result in an in-frame deletion of exon 3 (Arnold et al., 2009); Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports a deleterious effect on splicing; This variant is associated with the following publications: (PMID: 25525159, 26681312, 25980754, 28765196, 30787465, 29345684, 29887214, 31997046, 19267393, 15991306) -
Likely pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	May 19, 2023	The MLH1 c.208-3C>G variant (also known as IVS2-3C>G) has been reported in the published literature in individuals and families affected with a Lynch syndrome associated cancer and/or polyps including colorectal cancer (PMID: 15991306 (2005), 19267393 (2009), 25980754 (2015), 26681312 (2015)). Functional studies report this variant results in the in-frame deletion of exon 3 (PMID: 15991306 (2005), 19267393 (2009)). 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 likely pathogenic. -

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 is strongly associated with more severe personal and family histories of cancer, typical for individuals with pathogenic variants in this gene [PMID: 27363726]. -

Muir-Torré syndrome;C1333991:Colorectal cancer, hereditary nonpolyposis, type 2;C5399763:Mismatch repair cancer syndrome 1 Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Fulgent Genetics, Fulgent Genetics

Oct 31, 2018

- -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Invitae

May 25, 2023

For these reasons, this variant has been classified as Pathogenic. This variant disrupts a region of the MLH1 protein in which other variant(s) (Deletion of Exon 3) have been determined to be pathogenic (Invitae). This suggests that this is a clinically significant region of the protein, and that variants that disrupt it are likely to be disease-causing. 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 skipping of exon 3, but is expected to preserve the integrity of the reading-frame (PMID: 15991306, 19267393, 25980754). ClinVar contains an entry for this variant (Variation ID: 90032). This variant is also known as IVS2-3C>G. This variant has been observed in individual(s) with clinical features of Lynch syndrome (PMID: 15991306, 19267393, 26681312). It has also been observed to segregate with disease in related individuals. This variant is not present in population databases (gnomAD no frequency). This sequence change falls in intron 2 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 likely results in a shortened protein product. -

Hereditary cancer-predisposing syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

May 30, 2023

The c.208-3C>G intronic variant results from a C to G substitution 3 nucleotides upstream from coding exon 3 in the MLH1 gene. This alteration has been reported in multiple individuals with MLH1/PMS2-absent, microsatellite unstable, HNPCC-related tumors (Arnold S, Hum. Mutat. 2009 May; 30(5):757-70; Ambry internal data). In silico splice site analysis predicts that this alteration will weaken the native splice acceptor site and analysis of patient-derived RNA found that this alteration results in aberrant splicing leading to transcripts lacking coding exon 3 (Otway R, Hum. Genet. 2005 May; 116(6):535; Arnold S, Hum. Mutat. 2009 May; 30(5):757-70). This alteration also co-segregates with HNPCC-related tumors in multiple families (Arnold S, Hum. Mutat. 2009 May; 30(5):757-70; Ambry internal data). This nucleotide position is poorly conserved in available vertebrate species. In addition, 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. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.46
Cadd	Benign	17
Dann	Benign	0.67
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Benign	0.70
SpliceAI score (max)		0.97		Details are displayed if max score is > 0.2
DS_AL_spliceai		0.97		Position offset: 3

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

Publications

LitVar

Other links and lift over

dbSNP: rs267607720; hg19: chr3-37042443;