rs267607760

Positions:

• chr3-37008908-A-G

Variant summary

Our verdict is Pathogenic. Variant got 10 ACMG points: 10P and 0B. PM2 PP5_Very_Strong

The NM_000249.4(MLH1):​c.545+3A>G variant causes a splice region, intron change. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a benign 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: 31)
• Consequence: MLH1 NM_000249.4 splice_region, intron
• Scores: Splicing: ADA: 0.9993
• Clinical Significance: Pathogenic reviewed by expert panel P:14 O:1
• Conservation: PhyloP100: 4.80

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 10 ACMG points.

• PM2: Very rare variant in population databases, with high coverage
• PP5: Variant 3-37008908-A-G is Pathogenic according to our data. Variant chr3-37008908-A-G is described in ClinVar as [Pathogenic]. Clinvar id is 90260.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37008908-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.545+3A>G		splice_region_variant, intron_variant		ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.545+3A>G		splice_region_variant, intron_variant		1	NM_000249.4	ENSP00000231790.3

Frequencies

GnomAD3 genomes Cov.: 31

GnomAD4 exome Cov.: 29

GnomAD4 genome Cov.: 31

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:14 Other:1

Revision: reviewed by expert panel

Submissions by phenotype

not provided Pathogenic:4

Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Nov 10, 2017	This variant is denoted MLH1 c.545+3A>G or IVS6+3A>G and consists of an A>G nucleotide substitution at the +3 position of intron 6 of the MLH1 gene. In-silico analyses, including splice predictors and evolutionary conservation, support a deleterious effect. RNA studies and protein truncation testing confirm this prediction, demonstrating abnormal splicing leading to a frameshift that results in a truncated protein (Pensotti 1997, Thiffault 2004). This variant was observed in multiple individuals with colon cancer, including two who had absent MLH1 on immunohistochemistry, and is considered an Italian pathogenic founder variant (Pensotti 1997, Hendriks 2003, Liu 2004, Thiffault 2004, Mangold 2005, Valentin 2011). This variant has been shown to segregate with disease in two affected siblings in one family and with three affected relatives in another family (Thiffault 2004). MLH1 c.545+3A>G was not observed in large population cohorts (Lek 2016). The adenine (A) nucleotide that is altered is conserved across species. Based on the current evidence, we consider this variant to be pathogenic. -
Pathogenic, no assertion criteria provided	clinical testing	Joint Genome Diagnostic Labs from Nijmegen and Maastricht, Radboudumc and MUMC+	-	- -
Pathogenic, no assertion criteria provided	clinical testing	Clinical Genetics DNA and cytogenetics Diagnostics Lab, Erasmus MC, Erasmus Medical Center	-	- -
Pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Mar 16, 2021	The variant has been reported in individuals and families with Lynch syndrome in the published literature (PMIDs: 28874130 (2017), 26437257 (2015), 21681552 (2011), 15849733 (2005)). In addition, published studies indicate that this variant causes a damaging effect on normal MLH1 splicing (PMIDs: 15253764 (2004), 9218993 (1997)). This variant has not been reported in large, multi-ethnic general populations (http://gnomad.broadinstitute.org). Based on the available information, this variant is classified as pathogenic. -

Lynch syndrome Pathogenic:3

Pathogenic, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Sep 19, 2016	Variant summary: The MLH1 c.545+3A>G variant involves the alteration of a conserved nucleotide located at the intron/exon boundary. One in silico tool predicts a damaging outcome for this variant. 4/4 splice prediction tools predict loss/weakening effect on the canonical splicing donor site. This variant was absent in 121426 control chromosomes but has been reported in multiple affected individuals/families, and some families showed evidence of co-segregation of the variant with disease. In addition, multiple clinical diagnostic laboratories/reputable databases classified this variant as pathogenic. RT-PCR showed the variant lead to an aberrant splicing product (Pensotti_GCC_1997). Taken together, this variant was classified as pathogenic. -
Pathogenic, reviewed by expert panel	research	International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	Sep 05, 2013	Variant causes splicing aberration, >2 MSI-H, co-segregation with disease & absent in 1000 genomes -
Pathogenic, criteria provided, single submitter	research	A.C.Camargo Cancer Center / LGBM, A.C.Camargo Cancer Center	Jan 30, 2019	- -

Hereditary cancer-predisposing syndrome Pathogenic:3

Pathogenic, criteria provided, single submitter	curation	Sema4, Sema4	Apr 11, 2021	- -
Pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Nov 03, 2023	The c.545+3A>G intronic pathogenic mutation results from an A to G substitution 3 nucleotides after coding exon 6 in the MLH1 gene. This mutation has been identified in probands whose Lynch syndrome-associated tumor demonstrated high microsatellite instability and/or loss of MLH1 or MLH1/PMS2 expression by immunohistochemistry (Hendriks Y et al. Am. J. Pathol., 2003 Feb;162:469-77; Thiffault I et al. Clin. Genet., 2004 Aug;66:137-43; Carneiro da Silva F et al. PLoS ONE, 2015 Oct;10:e0139753; Ambry internal data). This alteration has been shown to cause a deleterious impact on splicing by RT-PCR and cDNA analyses (Pensotti V et al. Genes Chromosomes Cancer, 1997 Jul;19:135-42; Morak M et al. Eur J Hum Genet, 2019 Dec;27:1808-1820). In addition, RNA studies have demonstrated that this alteration results in abnormal splicing in the set of samples tested (Ambry internal data). 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. 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. -
Pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	May 26, 2020	This variant causes an A to G nucleotide substitution at the +3 position of intron 6 of the MLH1 gene. A functional RNA study has shown that this variant causes aberrant splicing and a significant truncation of exon 6 due to the use of a cryptic donor site leaving all but the first 3 nucleotides of exon 6 (PMID: 9218993). This variant has been reported in multiple individuals affected with hereditary nonpolyposis colorectal cancer (PMID: 9218993, 12547705, 15253764, 15309712) and shown to segregate with disease in family studies (PMID: 9218993, 15253764). 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. -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Neuberg Centre For Genomic Medicine, NCGM	Jun 02, 2023	The splice site c.545+3A>G variant has been reported in multiple individuals affected with hereditary nonpolyposis colorectal cancer (Rossi BM. et al., 2017, Pensotti V. et. al., 1997). A functional RNA study has shown that this variant causes aberrant splicing and a significant truncation of exon 6 due to the use of a cryptic donor site leaving all but the first 3 nucleotides of exon 6 (Pensotti V. et. al., 1997). This variant has been reported to the ClinVar database as Pathogenic with a status of reviewed by expert panel. The c.545+3A>G variant is novel (not in any individuals) in gnomAD Exomes and 1000 Genomes. Loss of function variants have been previously reported to be disease causing. For these reasons, this variant has been classified as Pathogenic. -
Pathogenic, no assertion criteria provided	clinical testing	Diagnostic Laboratory, Department of Genetics, University Medical Center Groningen	-	- -

Lynch-like syndrome Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Constitutional Genetics Lab, Leon Berard Cancer Center

Jul 01, 2019

- -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Oct 28, 2023

This sequence change falls in intron 6 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 may result in an absent or disrupted protein product. This variant is not present in population databases (gnomAD no frequency). This variant has been observed in individuals with Lynch syndrome (PMID: 9218993, 12547705, 15253764, 15309712, 15849733, 26437257, 28874130). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 90260). 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 activation of a cryptic splice site and introduces a premature termination codon (PMID: 9218993). 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_noAF	Benign	-0.21
CADD	Benign	15
DANN	Benign	0.96
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.1

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.99
SpliceAI score (max)		0.91		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.91		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: rs267607760; hg19: chr3-37050399;