rs267607878

chr3-37048614-G-Achr3-37048614-G-Cchr3-37048614-G-T

Variant summary

Our verdict is Uncertain significance. Variant got 4 ACMG points: 4P and 0B. PM2 PP3 PP5

The NM_000249.4(MLH1):c.1989+5G>A variant causes a splice donor 5th base, intron change involving the alteration of a non-conserved nucleotide. 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 Conflicting classifications of pathogenicity (no stars).

• Frequency: Genomes: not found (cov: 31)
• Consequence: MLH1 NM_000249.4 splice_donor_5th_base, intron
• Scores: Splicing: ADA: 0.9982
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:2 U:1
• Conservation: PhyloP100: 2.41

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 Uncertain_significance. Variant got 4 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 3-37048614-G-A is Pathogenic according to our data. Variant chr3-37048614-G-A is described in ClinVar as [Conflicting_classifications_of_pathogenicity]. Clinvar id is 581631.We mark this variant Likely_pathogenic, oryginal submissions are: {Pathogenic=1, Uncertain_significance=1, Likely_pathogenic=1}.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.1989+5G>A		splice_donor_5th_base_variant, intron_variant		ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.1989+5G>A		splice_donor_5th_base_variant, intron_variant		1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 31

GnomAD4 exome Cov.: 29

GnomAD4 genome Cov.: 31

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:2 Uncertain:1

Revision: criteria provided, conflicting classifications

Submissions by phenotype

Hereditary cancer-predisposing syndrome Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Feb 28, 2022	This variant causes a G to A nucleotide substitution at the +5 position of intron 17 of the MLH1 gene. Functional RNA studies have shown that this variant results in exon 17 skipping and an in-frame deletion of 93 nucleotides (DOI: 10.1093/gastro/goab030). This deletion potentially impacts the EXO1 and PMS2 binding domains (PMID: 11292842, 11427529). This variant has been reported in individuals affected with Lynch syndrome or colorectal cancer (DOI: 10.1093/gastro/goab030). It has been shown that this variant segregates with disease, with multiple affected carriers in a single family (DOI: 10.1093/gastro/goab030). 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	Nov 17, 2021	The c.1989+5G>A intronic pathogenic mutation results from a G to A substitution 5 nucleotides after coding exon 17 in the MLH1 gene. This alteration has been identified in a proband that also had somatic MLH1 copy neutral loss of heterozygosity (CN-LOH) in a MSI-H colon tumor with loss of MLH1 and PMS2 expression by immunohistochemistry (IHC) where MLH1 promotor hypermethylation was negative (Ambry internal data). In addition, this alteration has been identified as somatic in conjunction with a somatic pathogenic MLH1 variant in a MSI-H uterine tumor with loss of MLH1 and PMS2 expression by IHC where MLH1 promotor hypermethylation was negative (Ambry internal data). In silico splice site analysis predicts that this alteration may weaken the native splice donor site and will result in the creation or strengthening of a novel splice donor site. RNA studies have demonstrated that this alteration results in abnormal splicing in the set of samples tested (Ambry internal data). The resulting transcript is in-frame and is not expected to trigger nonsense-mediated mRNAdecay. The exact functional effect of the altered amino acid sequence is unknown; however, the impacted region is critical for protein function (Ambry internal data). 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. -

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Invitae

Oct 04, 2021

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. This variant disrupts the c.1989+5G nucleotide in the MLH1 gene. Other variant(s) that disrupt this nucleotide have been determined to be pathogenic (PMID: 8993976, 14970868, 17473388). This suggests that this nucleotide is clinically significant, and that variants that disrupt this position are likely to be disease-causing. Nucleotide substitutions within the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. This variant has not been reported in the literature in individuals affected with MLH1-related conditions. This variant is not present in population databases (ExAC no frequency). This sequence change falls in intron 17 of the MLH1 gene. It does not directly change the encoded amino acid sequence of the MLH1 protein. It affects a nucleotide within the consensus splice site of the intron. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.78
Cadd	Pathogenic	32
Dann	Benign	0.72

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.91
SpliceAI score (max)		0.86		Details are displayed if max score is > 0.2
DS_DG_spliceai		0.86		Position offset: 26
DS_DL_spliceai		0.33		Position offset: -5

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

Publications

LitVar

Other links and lift over

dbSNP: rs267607878; hg19: chr3-37090105;