3-37048515-A-G

Position:

• chr3-37048515-A-G

Variant summary

Our verdict is Pathogenic. Variant got 14 ACMG points: 14P and 0B. PVS1_Moderate PP3_Strong PP5_Very_Strong

The NM_000249.4(MLH1):​c.1897-2A>G variant causes a splice acceptor change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.00000891 in 1,459,008 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a pathogenic outcome for this variant. 2/2 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Likely pathogenic (★★★).

• Frequency:
  • Genomes: not found (cov: 31)
  • Exomes 𝑓: 0.0000089 (0 hom.)
• Consequence: MLH1 NM_000249.4 splice_acceptor
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Likely pathogenic reviewed by expert panel P:3 U:8 B:1
• Conservation: PhyloP100: 8.70

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.

• PVS1: Splicing +-2 bp (donor or acceptor) variant, product NOT destroyed by NMD, known LOF gene, truncates exone, which is 0.04051079 fraction of the gene. Cryptic splice site detected, with MaxEntScore 5.5, offset of -3, new splice context is: cttgtcctttttcctgcaAGcgg. Cryptic site results in inframe change. If cryptic site found is not functional and variant results in exon loss, it results in inframe change.
• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: dbscSNV1_ADA, max_spliceai. No scorers claiming Uncertain. No scorers claiming Benign.
• PP5: Variant 3-37048515-A-G is Pathogenic according to our data. Variant chr3-37048515-A-G is described in ClinVar as [Likely_pathogenic]. Clinvar id is 89932.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37048515-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.1897-2A>G		splice_acceptor_variant		ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.1897-2A>G		splice_acceptor_variant		1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 31

GnomAD3 exomes AF: 0.00000398 AC: 1 AN: 251356 Hom.: 0 AF XY: 0.00 AC XY: 0 AN XY: 135838

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.00

Gnomad SAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.00000880

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.00000891 AC: 13 AN: 1459008 Hom.: 0 Cov.: 30 AF XY: 0.00000964 AC XY: 7 AN XY: 726084

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: 0.0000108

Gnomad4 OTH exome AF: 0.0000166

GnomAD4 genome Cov.: 31

Alfa AF: 0.0000987 Hom.: 0

Bravo AF: 0.00000756

ClinVar

Significance: Likely pathogenic

Submissions summary: Pathogenic:3 Uncertain:8 Benign:1

Revision: reviewed by expert panel

Submissions by phenotype

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1 Uncertain:1 Benign:1

Likely pathogenic, criteria provided, single submitter	clinical testing	Counsyl	Sep 29, 2017	- -
Uncertain significance, criteria provided, single submitter	clinical testing	Baylor Genetics	Oct 18, 2023	- -
Likely benign, criteria provided, single submitter	clinical testing	Myriad Genetics, Inc.	Nov 14, 2023	This variant is considered likely benign. This variant is strongly associated with less severe personal and family histories of cancer, typical for individuals without pathogenic variants in this gene [PMID: 27363726]. -

Lynch syndrome Pathogenic:1 Uncertain:1

Uncertain significance, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Jul 10, 2023	This variant causes an A to G nucleotide substitution at the -2 position of intron 16 of the MLH1 gene. Splice site prediction tools suggest that this variant would impact splicing at the intron 16 splice acceptor site. An external laboratory has reported that this variant activates a cryptic, in-frame splice acceptor site, 3 bp upstream from the canonical acceptor site, that results in a majority transcript predicted to produce a protein with an in-frame insertion of one amino acid (p.Glu632_Glu633insArg) (ClinVar SCV000669547.5). To our knowledge functional studies have not been reported for MLH1 p.Glu632_Glu633insArg, and the functional consequence of this change is unknown, although the change does occur in a conserved region of the EXO1/PMS2/MLH3/PMS1-interacting domain. This variant has been reported in two related individuals affected with Lynch syndrome (PMID: 15345113), and two unrelated individuals affected with colorectal cancer, one of whom was affected with early-onset colorectal cancer (age <50) with a mismatch repair proficient tumor (PMID: 27978560, 28135145). However, the variant has also been seen in unaffected individuals and individuals with non-Lynch Syndrome cancers (DOI: 10.1016/j.gim.2022.01.077, external laboratory communication, Color internal data). This variant has also been identified in 1/251356 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Although there is a suspicion that this variant may have a pathogenic role, additional evidence is necessary to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance. -
Likely pathogenic, reviewed by expert panel	curation	International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	Jun 21, 2019	Interrupts canonical donor splice site -

not provided Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	Jun 20, 2023	Canonical splice variant demonstrated to result in a minor splice defect that may not impact protein function (External communication with outside laboratory); Not observed at significant frequency in large population cohorts (gnomAD); Observed in individuals with colon cancer (Rajkumar et al., 2004; Pearlman et al., 2017; Yurgelun et al., 2017); This variant is associated with the following publications: (PMID: 28135145, 27978560, 15345113, 22753075, 20533529, 12799449, 30720243, 30787465, 33087929, 35710434) -
Uncertain significance, criteria provided, single submitter	clinical testing	Revvity Omics, Revvity	Mar 01, 2023	- -

Hereditary cancer-predisposing syndrome Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	Ambry Genetics	May 09, 2024	The c.1897-2A>G intronic variant results from an A to G substitution two nucleotides upstream from coding exon 17 in the MLH1 gene. This alteration has been identified in two probands with early-onset colorectal cancer, one of whom had a MSI-high tumor and met Bethesda criteria for Lynch syndrome; however, the colon tumor of the second proband was reported to be mismatch repair (MMR) proficient demonstrating microsatellite stability and/or normal immunohistochemistry (Rajkumar T et al. Genet. Test. 2004 ;8(2):157-62; Pearlman R et al. JAMA Oncol. 2017 Apr;3(4):464-471). This variant was also reported in an individual from a cohort of unselected colorectal cancer patients undergoing multigene panel testing (Yurgelun MB et al. J. Clin. Oncol. 2017 Apr;35:1086-1095). This nucleotide position is highly conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice acceptor site and may result in the creation or strengthening of a novel splice acceptor site. RNA studies have demonstrated that this alteration results in a transcript predicted to lead to a protein with an in-frame insertion of one amino acid that is structurally deleterious (Ambry internal data). Of note, this alteration is also designated as IVS16-2A>G in the published literature. Based on the available evidence, the clinical significance of this variant remains unclear. -
Uncertain significance, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	May 12, 2023	This variant causes an A to G nucleotide substitution at the -2 position of intron 16 of the MLH1 gene. Splice site prediction tools suggest that this variant would impact splicing at the intron 16 splice acceptor site. An external laboratory has reported that this variant activates a cryptic, in-frame splice acceptor site, 3 bp upstream from the canonical acceptor site, that results in a majority transcript predicted to produce a protein with an in-frame insertion of one amino acid (p.Glu632_Glu633insArg) (ClinVar SCV000669547.5). To our knowledge functional studies have not been reported for MLH1 p.Glu632_Glu633insArg, and the functional consequence of this change is unknown, although the change does occur in a conserved region of the EXO1/PMS2/MLH3/PMS1-interacting domain. This variant has been reported in two related individuals affected with Lynch syndrome (PMID: 15345113), and two unrelated individuals affected with colorectal cancer, one of whom was affected with early-onset colorectal cancer (age <50) with a mismatch repair proficient tumor (PMID: 27978560, 28135145). However, the variant has also been seen in unaffected individuals and individuals with non-Lynch Syndrome cancers (DOI: 10.1016/j.gim.2022.01.077, external laboratory communication, Color internal data). This variant has been identified in 1/251356 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Although there is a suspicion that this variant may have a pathogenic role, additional studies are necessary to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance. -

Muir-Torré syndrome;C1333991:Colorectal cancer, hereditary nonpolyposis, type 2;C5399763:Mismatch repair cancer syndrome 1 Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Fulgent Genetics, Fulgent Genetics

Oct 31, 2018

- -

not specified Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

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

Feb 27, 2022

Variant summary: MLH1 c.1897-2A>G alters a conserved nucleotide located in a canonical splice-site and is predicted to affect mRNA splicing resulting in a significantly altered protein due to either exon skipping, shortening, or inclusion of intronic material. Several computational tools predict a significant impact on normal splicing: Three predict the variant abolishes the canonical 3' splice acceptor site. However, these predictions have yet to be confirmed by functional studies. The variant allele was found at a frequency of 4e-06 in 251356 control chromosomes. c.1897-2A>G has been reported in the literature as a VUS in individuals affected with colorectal cancer (Pearlman 2017, Yurgelun 2017), although in one of these cases the associated tumor was described as MMR proficient (based on microsatellite instability and/or immunohistochemistry) (Pearlman 2017). The variant appeared to segregate with the disease in a family that fulfilled the Bethesda guidelines for Lynch Syndrome (Rajkumar 2004), and though the tumor status here was described as MMR deficient (assessed by high microsatellite instability), in these patients only MLH1 and MSH2 was studied. These data indicate that the variant may be associated with disease. To our knowledge, no experimental evidence demonstrating an impact on protein function has been reported. Six clinical diagnostic laboratories and an expert panel (International Society for Gastrointestinal Hereditary Tumours, InSiGHT) have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation (Likely Pathogenic, n=6 including the expert panel, VUS, n=1). One submitter reports a VUS classification having re-classified it from Likely Pathogenic as an outcome of RNA analysis demonstrating no impact on splicing at their laboratory. Based on the evidence outlined above, the variant was re-classified as VUS possibly pathogenic. -

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Dec 20, 2023

This sequence change affects an acceptor splice site in intron 16 of the MLH1 gene. RNA analysis indicates that disruption of this splice site induces altered splicing and likely results in the gain of 1 amino acid residue(s), but is expected to preserve the integrity of the reading-frame. This variant is present in population databases (rs267607871, gnomAD 0.0009%). Disruption of this splice site has been observed in individual(s) with colorectal cancer (PMID: 15345113, 27978560, 28135145). This variant is also known as IVS16-2A>G. ClinVar contains an entry for this variant (Variation ID: 89932). Studies have shown that disruption of this splice site results in the activation of a cryptic splice site in intron 16 (Invitae). In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.63	D
BayesDel_noAF	Pathogenic	0.26
CADD	Pathogenic	32
DANN	Uncertain	0.99
Eigen	Pathogenic	1.1
Eigen_PC	Pathogenic	0.99
FATHMM_MKL	Pathogenic	0.99	D
MutationTaster	Benign	1.0	D;D;D;D;D;D
GERP RS		5.3

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
SpliceAI score (max)		1.0		Details are displayed if max score is > 0.2
DS_AG_spliceai		0.33		Position offset: 6
DS_AL_spliceai		1.0		Position offset: 2

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

Publications

LitVar

Other links and lift over

dbSNP: rs267607871; hg19: chr3-37090006;