rs267607749

Positions:

• chr3-37008801-A-G
• chr3-37008801-A-T

Variant summary

Our verdict is Pathogenic. Variant got 12 ACMG points: 12P and 0B. PM2 PP3_Moderate PP5_Very_Strong

The NM_000249.4(MLH1):​c.454-13A>G variant causes a splice polypyrimidine tract, intron change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.000000688 in 1,453,390 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a benign outcome for this variant. Variant has been reported in ClinVar as Likely pathogenic (★★★).

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 6.9e-7 (0 hom.)
• Consequence: MLH1 NM_000249.4 splice_polypyrimidine_tract, intron
• Clinical Significance: Likely pathogenic reviewed by expert panel P:10
• Conservation: PhyloP100: -0.271

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 12 ACMG points.

• PM2: Very rare variant in population databases, with high coverage
• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: max_spliceai. No scorers claiming Uncertain. No scorers claiming Benign.
• PP5: Variant 3-37008801-A-G is Pathogenic according to our data. Variant chr3-37008801-A-G is described in ClinVar as [Likely_pathogenic]. Clinvar id is 90230.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37008801-A-G is described in Lovd as [Pathogenic]. Variant chr3-37008801-A-G is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.454-13A>G		splice_polypyrimidine_tract_variant, intron_variant		ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.454-13A>G		splice_polypyrimidine_tract_variant, intron_variant		1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome AF: 6.88e-7 AC: 1 AN: 1453390 Hom.: 0 Cov.: 29 AF XY: 0.00000138 AC XY: 1 AN XY: 723652

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: 9.05e-7

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 32

ClinVar

Significance: Likely pathogenic

Submissions summary: Pathogenic:10

Revision: reviewed by expert panel

Submissions by phenotype

not provided Pathogenic:3

Pathogenic, no assertion criteria provided	clinical testing	Diagnostic Laboratory, Department of Genetics, University Medical Center Groningen	-	- -
Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Jun 14, 2023	Published functional studies demonstrate a damaging effect: aberrant splicing (Thompson et al., 2020; Nishikubo et al., 2021); Not observed at significant frequency in large population cohorts (gnomAD); This variant is associated with the following publications: (PMID: 17312306, 27601186, 22949379, 22890886, 26177554, 23729658, 32849802, 34221829, 33467402) -
Pathogenic, no assertion criteria provided	clinical testing	Joint Genome Diagnostic Labs from Nijmegen and Maastricht, Radboudumc and MUMC+	-	- -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Myriad Genetics, Inc.	Jul 14, 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]. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Baylor Genetics	Jan 11, 2023	- -

Hereditary cancer-predisposing syndrome Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Apr 06, 2020	This variant causes an A to G nucleotide substitution at the -13 position of intron 5 of the MLH1 gene. This variant has been shown to cause exon 6 skipping in a minigene assay (PMID: 23729658) and is predicted to result in an absent or non-functional protein product. This variant has been reported in several individuals affected with Lynch syndrome (PMID: 17312306, 23729658, 27601186). This variant has been observed to segregate with colon cancer in one family (Color internal data). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Likely Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Jul 18, 2023	The c.454-13A>G intronic pathogenic mutation results from an A to G substitution 13 nucleotides upstream from coding exon 6 in the MLH1 gene. This alteration has been reported in numerous families meeting Amsterdam criteria I or II; results from the Lynch syndrome-associated tumors of each proband also demonstrated high microsatellite instability (MSI-H) and/or loss of MLH1 or MLH1/PMS2 protein expression on immunohistochemistry (IHC; Lagerstedt Robinson K et al. J Natl Cancer Inst, 2007 Feb;99:291-9; Grandval P et al. Database (Oxford), 2013 May;2013:bat036; Nishikubo T et al. Int Cancer Conf J, 2021 Jul;10:186-190). Furthermore, ex vivo splicing analysis using a pCAS splicing reporter mini gene and in vitro RT-PCR analysis revealed that this alteration results in the skipping of coding exon 6 (Grandval P et al. Database (Oxford), 2013 May;2013:bat036). Aberrant splicing has been confirmed in subsequent RNA analyses performed for this variant (Thompson BA et al. Front Genet, 2020 Jul;11:798; Nishikubo T et al. Int Cancer Conf J, 2021 Jul;10:186-190). This variant has been identified in probands whose Lynch syndrome-associated tumor demonstrated MSI-H and/or loss of MLH1/PMS2 expression by IHC (Ambry internal data). In silico splice site analysis predicts that this alteration will weaken the native splice acceptor site. RNA studies have demonstrated that this alteration results in abnormal splicing in the set of samples tested (Ambry internal data). This nucleotide position is not well conserved in available vertebrate species. 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. -

Carcinoma of colon Pathogenic:1

Likely pathogenic, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

The MLH1 c.454-13A>G variant was identified in 3 of 684 proband chromosomes (frequency: 0.004) from individuals or families with Lynch Syndrome (Lagerstedt-Robinson 2007, Davison 2012 PhD Thesis at University of the Witwatersrand, Tzortzatos 2015). The variant was identified in dbSNP (rs267607749) as “with likely pathogenic allele”, ClinVar (classified as likely pathogenic by InSiGHT expert panel in 2018, Invitae, Color, Ambry Genetics, and GeneDx) and UMD-LSDB (observed 1x). 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). In UMD-LSDB, an ex vivo splicing assay and in vitro RT-PCR have been reported to show that this variant results in the skipping of exon 6, citing Grandval 2013, although the data is not available for review. This variant has been identified by our laboratory in a patient with an MLH1- and PMS2-deficient colon tumour. This variant has also been found to segregate with disease in at least two families (Lagerstedt-Robinson 2207; Invitae internal data per ClinVar entry dated November 15, 2018). The variant occurs outside of the splicing consensus sequence and 2 of 3 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE) predict a greater than 10% difference in splicing; this is not very predictive of pathogenicity. In summary, based on the above information, the clinical significance of this variant cannot be determined with certainty at this time although we would lean towards a more pathogenic role for this variant. This variant is classified as likely pathogenic. -

Lynch syndrome Pathogenic:1

Likely pathogenic, reviewed by expert panel

curation

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Oct 18, 2018

Splicing abberation shown to cause skipping -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Invitae

Jan 05, 2024

This sequence change falls in intron 5 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: 17312306, 23729658; Invitae). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 90230). Studies have shown that this variant results in skipping of exon 6 and introduces a premature termination codon (Invitae). The resulting mRNA is expected to undergo nonsense-mediated decay. For these reasons, this variant has been classified as Pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.61
CADD	Benign	0.0030
DANN	Benign	0.81

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.81		Details are displayed if max score is > 0.2
DS_AL_spliceai		0.81		Position offset: 13

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

Publications

LitVar

Other links and lift over

dbSNP: rs267607749; hg19: chr3-37050292;