chr3-37008801-A-G

Variant names:

• chr3-37008801-A-G
• rs267607749
• NM_000249.4:c.454-13A>G

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 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 intron
• Clinical Significance: Likely pathogenic reviewed by expert panel P:12
• 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 rank	MANE	Protein	UniProt
MLH1	NM_000249.4	c.454-13A>G		intron_variant	Intron 5 of 18	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.454-13A>G		intron_variant	Intron 5 of 18	1	NM_000249.4	ENSP00000231790.3

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:12

Revision: reviewed by expert panel

Submissions by phenotype

not provided Pathogenic:4

-

Diagnostic Laboratory, Department of Genetics, University Medical Center Groningen

Significance: Pathogenic

Review Status: no assertion criteria provided

Collection Method: clinical testing

- -

-

Joint Genome Diagnostic Labs from Nijmegen and Maastricht, Radboudumc and MUMC+

Significance: Pathogenic

Review Status: no assertion criteria provided

Collection Method: clinical testing

- -

Jun 14, 2023

GeneDx

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

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) -

Sep 11, 2024

ARUP Laboratories, Molecular Genetics and Genomics, ARUP Laboratories

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The MLH1 c.454-13A>G variant (rs267607749) is reported in the literature in several individuals with diagnosed or suspected Lynch syndrome (Lagerstedt Robinson 2007, Nishikubo 2021, Sidenna 2022). This variant is absent from the Genome Aggregation Database (v2.1.1), indicating it is not a common polymorphism. This is an intronic variant in a moderately conserved nucleotide, and computational analyses (Alamut v.2.11) predict that this variant may impact splicing by weakening the nearby canonical acceptor splice site. Consistent with predictions, functional analyses of patient transcripts demonstrate skipping of exon 6, which is predicted to lead to a frameshift (Nishikubo 2021, Thompson 2020). Based on available information, this variant is considered to be likely pathogenic. References: Lagerstedt Robinson K et al. Lynch syndrome (hereditary nonpolyposis colorectal cancer) diagnostics. J Natl Cancer Inst. 2007 Feb 21;99(4):291-9. PMID: 17312306. Nishikubo T et al. Immunohistochemistry and RNA-sequencing have been useful in evaluating the pathological significance of a non-consensus site intronic variant in suspected cases of Lynch syndrome. Int Cancer Conf J. 2021 Mar 6;10(3):186-190. PMID: 34221829. Sidenna M et al. Prevalence and Genotype-Phenotype Correlation of Lynch Syndrome in a Selected High-Risk Cohort from Qatar's Population. Genes (Basel). 2022 Nov 21;13(11):2176. PMID: 36421850. Thompson BA et al. Contribution of mRNA Splicing to Mismatch Repair Gene Sequence Variant Interpretation. Front Genet. 2020 Jul 27;11:798. PMID: 32849802. -

Hereditary cancer-predisposing syndrome Pathogenic:3

Apr 06, 2020

Color Diagnostics, LLC DBA Color Health

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

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. -

Jul 18, 2023

Ambry Genetics

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

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. -

Feb 24, 2025

Molecular Diagnostics Laboratory, Catalan Institute of Oncology

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

PVS1, PM2_Supporting, PP1_Moderate, PP4_Strong MLH1 c.454-13A>G is an intronic variant located close to a canonical splice site in which computational tools predict a deleterious effect of the variant on splicing. An ex vivo splicing analysis using a pCAS splicing reporter mini gene and RNA analysis performed with lymphoblastoid cell lines from affected carrier patients, in the presence/absence of puromycin, showed the skipping out-of-frame of coding exon 6 (r.454_545del, p.Glu153Phefs*8) (PMID: 23729658, 32849802, 34221829) (PVS1). It is not present in the population database gnomAD v2.1.1, non cancer dataset (PM2_supporting). MLH1 c.454-13A>G has been reported in at least 6 CRC tumors with loss of MMR protein expression consistent with the variant location and/or MSI-H and in the absence of MLH1 methylation/BRAFwt (PMID: 32849802, 23729658; LOVD case #00201531 and 2 internal cases from our cohort of patients) (PP4_Strong). Moreover, this variant has also been found to segregate with disease in at least 1 family from India (5 affected carriers, 4 informative meiosis) (Doctoral thesis from Homi Bhabha National Institute: LIFE09201004003) (PP1_Moderate). In addition, it was also identified in the following databases: InSiGHT (Class 4 Likely pathogenic: Splicing aberration shown to cause skipping), ClinVar (5x likely pathogenic, 5x pathogenic), LOVD (6x uncertain significance, 1x likely pathogenic, 3x pathogenic). Based on the currently available information, c.454-13A>G is classified as a pathogenic variant according to ClinGen-MMR Guidelines Draft version v3.1. -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:2

Jan 11, 2023

Baylor Genetics

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Jul 14, 2023

Myriad Genetics, Inc.

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

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]. -

Carcinoma of colon Pathogenic:1

-

Department of Pathology and Laboratory Medicine, Sinai Health System

Significance: Likely pathogenic

Review Status: no assertion criteria provided

Collection Method: clinical testing

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

Oct 18, 2018

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Significance: Likely pathogenic

Review Status: reviewed by expert panel

Collection Method: curation

Splicing abberation shown to cause skipping -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Sep 03, 2024

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

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 altered 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; internal data). It has also been observed to segregate with disease in related individuals. Invitae Evidence Modeling of clinical and family history, age, sex, and reported ancestry of multiple individuals with this MLH1 variant has been performed. This variant is expected to be pathogenic with a positive predictive value of at least 99%. This is a validated machine learning model that incorporates the clinical features of 1,370,736 individuals referred to our laboratory for MLH1 testing. ClinVar contains an entry for this variant (Variation ID: 90230). Studies have shown that this variant results in skipping of exon 6, and produces a non-functional protein and/or introduces a premature termination codon (internal data). 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;