rs267607845

Positions:

• chr3-37042267-G-A
• chr3-37042267-G-C
• chr3-37042267-G-T

Variant summary

Our verdict is Pathogenic. Variant got 18 ACMG points: 18P and 0B. PVS1 PM2 PP5_Very_Strong

The NM_000249.4(MLH1):​c.1668-1G>A variant causes a splice acceptor change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.00000207 in 1,448,254 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 Likely pathogenic (★★★).

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 0.0000021 (0 hom.)
• Consequence: MLH1 NM_000249.4 splice_acceptor
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Likely pathogenic reviewed by expert panel P:8 U:1
• Conservation: PhyloP100: 8.93

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 18 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
• PP5: Variant 3-37042267-G-A is Pathogenic according to our data. Variant chr3-37042267-G-A is described in ClinVar as [Likely_pathogenic]. Clinvar id is 89826.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37042267-G-A is described in Lovd as [Likely_pathogenic]. Variant chr3-37042267-G-A is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
MLH1	NM_000249.4	c.1668-1G>A		splice_acceptor_variant		ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.1668-1G>A		splice_acceptor_variant		1	NM_000249.4	ENSP00000231790	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome AF: 0.00000207 AC: 3 AN: 1448254 Hom.: 0 Cov.: 29 AF XY: 0.00 AC XY: 0 AN XY: 721404

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: 0.00

Gnomad4 NFE exome AF: 0.00000273

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 32

ClinVar

Significance: Likely pathogenic

Submissions summary: Pathogenic:8 Uncertain:1

Revision: reviewed by expert panel

Submissions by phenotype

Lynch syndrome Pathogenic:3

Likely pathogenic, reviewed by expert panel	curation	International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	Jun 21, 2019	Interrupts canonical donor splice site -
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.1668-1G>A was observed in one family with several cases of early-onset colon and endometrial cancer (Shirts et al 2016, PMID 26845104). It was also seen as an apparent passenger mutation in a tumor with other somatic mutations apparently responsible for the observed MSI status, reducing the likelihood of pathogenicity (Shirts et al 2018, PMID 29887214). This has been reported as likely pathogenic by the InSiGHT consortium and as pathogenic by several laboratories. We currently consider this variant to be likely pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	Apr 19, 2019	The c.1668-1G>A variant in MLH1 has been reported in at least 4 individuals with Lynch Syndrome (Lamberti 2006, Arnold 2009, Pérez-Carbonell 2012, Shirts 2016) and was found to segregate with disease in 2 additional affected individuals in one family (Arnold 2009). This variant was absent from large population studies. This variant occurs in the invariant region (+/- 1,2) of the splice consensus sequence and has been observed to cause altered splicing leading to an abnormal or absent protein (Arnold 2009, Pérez-Carbonell 2012). Heterozygous loss of function of the MLH1 gene is an established disease mechanism in Lynch Syndrome. Additionally, this variant has been classified as Likely pathogenic on Sep 5, 2013 by the ClinGen-approved InSiGHT panel (ClinVar SCV000106296.2). In summary, this variant meets criteria to be classified as pathogenic for Lynch Syndrome in an autosomal dominant manner. ACMG/AMP criteria applied: PM2, PS4_Supporting, PVS1. -

Hereditary cancer-predisposing syndrome Pathogenic:3

Pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Jan 01, 2022	This variant causes a G to A nucleotide substitution at the -1 position of intron 14 of the MLH1 gene. Functional RNA studies have shown that this variant causes out-of-frame deletion of exon 15 (PMID: 2707453). This variant is expected to result in an absent or non-functional protein product. This variant has been reported in individuals affected with Lynch syndrome or colorectal cancer with tumors showing microsatellite instability and loss of MLH1 protein expression (PMID: 2707453, 15855432, 17095871, 19267393, 21868491). 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. -
Likely pathogenic, criteria provided, single submitter	research	Academic Department of Medical Genetics, University of Cambridge	Jan 26, 2018	Application of AMCG guidelines 2015. Used other ClinVar submission evidence where relevant. Loss of heterozygosity in tumours or immunohistochemistry abnormalities considered functional evidence of pathogenicity. -
Pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	May 19, 2021	The c.1668-1G>A intronic pathogenic mutation results from a G to A substitution one nucleotide upstream from coding exon 15 of the MLH1 gene. This alteration has been previously reported in several individuals with colorectal cancers demonstrating microsatellite instability and absent MLH1 and/or PMS2 IHC staining, including individuals meeting Amsterdam criteria (Taylor CF, et al. Hum. Mutat. 2003 Dec; 22(6):428-33; Piñol V, et al. JAMA 2005 Apr; 293(16):1986-94; Lamberti C, et al. Digestion 2006 ; 74(1):58-67; Pérez-Carbonell L, et al. Gut 2012 Jun; 61(6):865-72; Walker M et al. Br J Surg, 2007 Dec;94:1567-71). One splicing assay using cell lines from mutation carriers found that this alteration results in out-of-frame skipping of exon 15 (Arnold S, et al. Hum. Mutat. 2009 May; 30(5):757-70). In silico splice site analysis predicts that this alteration will weaken the native splice acceptor 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. -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1

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. 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]. -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Mar 14, 2023

ClinVar contains an entry for this variant (Variation ID: 89826). Disruption of this splice site has been observed in individual(s) with Lynch syndrome (PMID: 14635101, 15855432, 17095871, 19267393, 21868491). This variant is not present in population databases (gnomAD no frequency). This sequence change affects an acceptor splice site in intron 14 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. Studies have shown that disruption of this splice site results in exon skipping and introduces a premature termination codon (PMID: 19267393). The resulting mRNA is expected to undergo nonsense-mediated decay. For these reasons, this variant has been classified as Pathogenic. -

not provided Uncertain:1

Uncertain significance, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

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Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.63	D
BayesDel_noAF	Pathogenic	0.42
CADD	Pathogenic	34
DANN	Uncertain	1.0
Eigen	Pathogenic	1.2
Eigen_PC	Pathogenic	1.1
FATHMM_MKL	Pathogenic	0.99	D
MutationTaster	Benign	1.0	D;D;D;D;D;D
ClinPred		0.99	D
GERP RS		5.4

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.93
SpliceAI score (max)		0.71		Details are displayed if max score is > 0.2
DS_AL_spliceai		0.71		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: rs267607845; hg19: chr3-37083758;