rs63751632

chr3-37047683-G-Achr3-37047683-G-C

Variant summary

Our verdict is Pathogenic. Variant got 14 ACMG points: 14P and 0B. PM2 PP3_Strong PP5_Very_Strong

The NM_000249.4(MLH1):c.1896G>A(p.Glu632=) variant causes a splice region, synonymous 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, synonymous
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Pathogenic reviewed by expert panel P:13 U:1
• Conservation: PhyloP100: 6.64

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 14 ACMG points.

• 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 3-37047683-G-A is Pathogenic according to our data. Variant chr3-37047683-G-A is described in ClinVar as [Pathogenic]. Clinvar id is 89930.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37047683-G-A is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.1896G>A	p.Glu632=	splice_region_variant, synonymous_variant	16/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.1896G>A	p.Glu632=	splice_region_variant, synonymous_variant	16/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 31

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 31

Bravo AF: 0.00000378

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:13 Uncertain:1

Revision: reviewed by expert panel

Submissions by phenotype

not provided Pathogenic:5 Uncertain:1

Pathogenic, no assertion criteria provided	clinical testing	Genome Diagnostics Laboratory, University Medical Center Utrecht	-	- -
Uncertain significance, no assertion criteria provided	clinical testing	Department of Pathology and Laboratory Medicine, Sinai Health System	-	- -
Pathogenic, no assertion criteria provided	clinical testing	Clinical Genetics DNA and cytogenetics Diagnostics Lab, Erasmus MC, Erasmus Medical Center	-	- -
Pathogenic, no assertion criteria provided	clinical testing	Joint Genome Diagnostic Labs from Nijmegen and Maastricht, Radboudumc and MUMC+	-	- -
Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Sep 04, 2019	Exonic splice site variant resulting in the production of full-length transcript as well as a transcript with an in-frame deletion of exon 16 (Wijnen 1996); Not observed in large population cohorts (Lek 2016); In silico analysis, which includes splice predictors and evolutionary conservation, supports a deleterious effect; This variant is associated with the following publications: (PMID: 12414824, 12373605, 22081473, 24278394, 25525159, 18625694, 9311737, 8571956) -
Pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Jun 02, 2021	The variant is located at the last nucleotide of exon 16 and has been reported to result in aberrant RNA splicing in vitro (PMIDs: 8571956 (1996)). This variant has been reported in multiple individuals/ families affected with Lynch syndrome or colorectal cancer in the published literature (PMIDs: 8571956 (1996), 12414824 (2002), 18625694 (2008), 22081473 (2012), 24278394 (2013)). In addition, loss of MLH1 and PMS2 expression by tumor immunohistochemistry (IHC) has been reported in tumor specimens from individuals carrying this variant (PMIDs: 22081473 (2012) and 29758216 (2018)). Therefore, the variant is classified as pathogenic. -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Myriad Genetics, Inc.	Jul 24, 2023	This variant is considered likely pathogenic. mRNA analysis has demonstrated abnormal mRNA splicing occurs [PMID: 8571956]. This variant has been reported in multiple individuals with clinical features of gene-specific disease [PMID: 8571956]. -
Pathogenic, no assertion criteria provided	clinical testing	Diagnostic Laboratory, Department of Genetics, University Medical Center Groningen	-	- -

Lynch syndrome Pathogenic:2

Pathogenic, reviewed by expert panel	research	International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	Sep 05, 2013	Variant causes splicing aberration interrupting functional domain: full inactivation of variant allele -
Pathogenic, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Jan 08, 2024	This synonymous variant does not change the amino acid sequence of the MLH1 protein. However, this variant causes a G to A nucleotide substitution at the last nucleotide of exon 16 of the MLH1 gene. Splice site prediction tools suggest that this variant may have a significant impact on RNA splicing. A functional RNA study has shown that this variant causes skipping of exon 16 in the RNA transcript and results in truncation of the protein product (PMID: 8571956). This variant has been reported in at least 5 individuals from two families that have a total of ten individuals with colon cancer (PMID: 8571956, 12414824). This variant has been reported in additional individuals affected with colorectal cancer and endometrial cancer (PMID: 18625694, 22081473, Color internal data). 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. -

Hereditary cancer-predisposing syndrome Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Nov 01, 2023	This synonymous variant does not change the amino acid sequence of the MLH1 protein. However, this variant causes a G to A nucleotide substitution at the last nucleotide of exon 16 of the MLH1 gene. Splice site prediction tools suggest that this variant may have a significant impact on RNA splicing. A functional RNA study has shown that this variant causes skipping of exon 16 in the RNA transcript and results in truncation of the protein product (PMID: 8571956). This variant has been reported in at least 5 affected individuals from two families that have a total of ten individuals with colon cancer (PMID: 8571956, 12414824). This variant has been reported in additional individuals affected with colorectal cancer and endometrial cancer (PMID: 18625694, 22081473). 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. -
Pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Jul 18, 2022	The c.1896G>A pathogenic mutation (also known as p.E632E), located in coding exon 16 of the MLH1 gene. This variant results from a G to A substitution at nucleotide position 1896. This nucleotide substitution does not change the glutamic acid at codon 632. However, this change occurs in the last base pair of coding exon 16, which makes it likely to have some effect on normal mRNA splicing. This mutation has been identified in multiple, unrelated HNPCC/Lynch syndrome families (Wijnen J et al. Am J Hum Genet. 1996 Feb;58(2):300-7; Wijnen J et al. Am J Hum Genet. 1997 Aug;61(2):329-35; Wagner A et al. J Med Genet. 2002 Nov;39(11):833-7; Ramsoekh D et al. Gut. 2008 Nov;57(11):1539-44; van Lier MG et al. J Pathol. 2012 Apr;226(5):764-74; De Lellis L et al. PLoS One. 2013 Nov 20;8(11):e81194; Ambry internal data). In addition, this variant has been identified in probands whose Lynch syndrome-associated tumor demonstrated high microsatellite instability and loss of MLH1 and PMS2 expression by immunohistochemistry (van Lier MG et al. J Pathol. 2012 Apr;226(5):764-74; Ambry internal data). In one report, RT-PCR and protein truncation studies indicated that this alteration led to aberrant splicing resulting in skipping of exon 16 (Wijnen J et al. Am J Hum Genet. 1996 Feb;58(2):300-7). This nucleotide position is well conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice donor site. Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. -

Hereditary nonpolyposis colon cancer Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

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

Sep 30, 2019

Variant summary: MLH1 c.1896G>A (p.Glu632Glu) alters a conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. Several computational tools predict a significant impact on normal splicing: Two predict the variant abolishes a 5' splicing donor site. One predict the variant weakens a 5' donor site. At least one publication reports experimental evidence that this variant affects mRNA splicing (Wijnen_1996). The variant was absent in 251398 control chromosomes. c.1896G>A has been reported in the literature in multiple individuals affected with Lynch Syndrome and colorectal cancer (Wijnen_1996, Wagner_2002, Ramsoekh_2008, vanLier_2012, DeLellis_2013, tenBroeke_2018). These data indicate that the variant is very likely to be associated with disease. Three clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014. All laboratories classified 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

Sep 18, 2023

For these reasons, this variant has been classified as Pathogenic. Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant is associated with altered splicing resulting in unknown protein product impact (PMID: 8571956; Invitae). ClinVar contains an entry for this variant (Variation ID: 89930). This variant has been observed in individual(s) with Lynch syndrome (PMID: 8571956, 12414824, 18625694). 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 affects codon 632 of the MLH1 mRNA. It is a 'silent' change, meaning that it does not change the encoded amino acid sequence of the MLH1 protein. This variant also falls at the last nucleotide of exon 16, which is part of the consensus splice site for this exon. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.39
Cadd	Benign	19
Dann	Benign	0.90

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	1.0
SpliceAI score (max)		0.65		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.65		Position offset: 0

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

Publications

LitVar

Other links and lift over

dbSNP: rs63751632; hg19: chr3-37089174;