rs587781518

Positions:

• chr3-36996714-G-A
• chr3-36996714-G-C

Variant summary

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

The NM_000249.4(MLH1):​c.207+5G>A variant causes a splice donor 5th base, intron change involving the alteration of a 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: 32)
• Consequence: MLH1 NM_000249.4 splice_donor_5th_base, intron
• Scores: Splicing: ADA: 0.9964
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:2 U:1
• Conservation: PhyloP100: 9.39

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 3-36996714-G-A is Pathogenic according to our data. Variant chr3-36996714-G-A is described in ClinVar as [Conflicting_classifications_of_pathogenicity]. Clinvar id is 926930.We mark this variant Likely_pathogenic, oryginal submissions are: {Likely_pathogenic=2, Uncertain_significance=1}. Variant chr3-36996714-G-A is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
MLH1	NM_000249.4	c.207+5G>A		splice_donor_5th_base_variant, intron_variant		ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.207+5G>A		splice_donor_5th_base_variant, intron_variant		1	NM_000249.4	ENSP00000231790	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 29

GnomAD4 genome Cov.: 32

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	Dec 02, 2020	This variant causes a G to A nucleotide substitution at the +5 position of intron 2 of the MLH1 gene. A functional RNA study has shown that this variant causes the out-of-frame skipping of exon 2, resulting in premature truncation (LOVD (https://databases.lovd.nl/shared/variants/0000426800#00013676) and external lab communication). This variant has been observed in an individual affected with bowel cancer that exhibited microsatellite instability (LOVD individual#00188772 and external lab communication). A similar variant, c.207+5G>C, has also been reported in individuals affected with colorectal cancer (PMID: 27978560, 32620519) and to cause the same RNA splicing defect (PMID: 27978560). 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 Likely Pathogenic. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Nov 13, 2019	The c.207+5G>A intronic variant results from a G to A substitution 5 nucleotides after coding exon 2 in the MLH1 gene. This nucleotide position is highly conserved in available vertebrate species. This variant has been identified in an individual who was diagnosed with early-onset colon cancer that showed high microsatellite instability (MSI-H) (Fokkema IF et al. Hum. Mutat., 2011 May;32:557-63). This variant was also identified as somatic in a MSI-H colon tumor that displayed loss of both MLH1/PMS2 on immunohistochemistry (IHC) with MLH1 copy-neutral loss of heterozygosity (CN-LOH) and MLH1 promoter hypermethylation was absent (Ambry internal data). Another alteration at the same nucleotide position,c.207+5G>C, has been classified as likely pathogenic based on RNA studies demonstrating abnormal splicing with out-of-frame exon 2 skipping resulting in a premature stop codon, co-segregation with disease, and being identified in a patient meeting clinical diagnostic criteria for Lynch syndrome (Pearlman R et al. JAMA Oncol. 2017;3(4):464-471; Ambry internal data). Using two different splice site prediction tools, this alteration is predicted by ESEfinder to weaken the efficiency of the native splice donor site, but is not predicted to have a deleterious effect on this splice donor site by BDGP. RT-PCR using patient-derived RNA from a c.207+5G>A carrier was also reported to result in out-of-frame exon 2 skipping (Fokkema IF et al. Hum. Mutat., 2011 May;32:557-63). Based on the majority of available evidence to date, this variant is likely to be pathogenic. -

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Dec 15, 2023

This sequence change falls in intron 2 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. This variant is not present in population databases (gnomAD no frequency). This variant has not been reported in the literature in individuals affected with MLH1-related conditions. ClinVar contains an entry for this variant (Variation ID: 926930). Variants that disrupt 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. 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_noAF	Benign	-0.19
CADD	Uncertain	25
DANN	Uncertain	0.98
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.1

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.92
SpliceAI score (max)		0.71		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.71		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: rs587781518; hg19: chr3-37038205;