ClinVar Genomic variation as it relates to human health

Canonical splice site variant predicted to result in the in-frame skipping of exon 3, which is also a naturally-occurring isoform (Diez et al., 2007; Muller et al., 2011); Not observed at significant frequency in large population cohorts (gnomAD); Has not been previously published as pathogenic or benign to our knowledge; Also known as 296-1G>T; This variant is associated with the following publications: (PMID: 21939546, 17971607, 19609323, 32641407, 33469799, 29937994)

This variant is located in intron 2 of the BRCA2 gene. It is predicted to abolish the intron 2 splice acceptor site at the AG dinucleotide at the 3' terminus of the intron. However, there is an alternative AG dinucleotide at c.72_73, which if used is predicted to cause a 6-basepair deletion (r.68_73del) and an in-frame deletion of 2 amino acids. RNA studies on similar canonical splice acceptor site variants, c.68-1G>A and c.68-2A>G, reported aberrant splicing with the major product being the predicted in-frame deletion of the first six 6 nucleotides in exon 3 (PMID: 32641407, 33469799). This variant has not been reported in individuals affected with hereditary cancer in the literature. This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). The available evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance.

This sequence change affects an acceptor splice site in intron 2 of the BRCA2 gene. RNA analysis indicates that disruption of this splice site induces altered splicing and likely results in the loss of 2 amino acid residue(s), but is expected to preserve the integrity of the reading-frame. This variant is not present in population databases (gnomAD no frequency). This variant has not been reported in the literature in individuals affected with BRCA2-related conditions. ClinVar contains an entry for this variant (Variation ID: 409412). Studies have shown that disruption of this splice site results in the activation of a cryptic splice site in exon 3 (PMID: 32641407, 33469799; 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.

The c.68-1G>T intronic variant results from a G to T substitution one nucleotide upstream from coding exon 2 of the BRCA2 gene. 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 will result in the creation or strengthening of a novel splice acceptor site. RNA studies have demonstrated that close match alterations BRCA2 c.68-2A>G and BRCA2 c.68-1G>A, which have the same predicted RNA effect as this variant, result in substantial expression of multiple abnormal transcripts including a splice variant which is predicted to result in an in-frame loss of two amino acids as well as one that results in skipping of coding exon 2 (also known as exon 3 in the literature (Ambry internal data; Nix P et al. Fam Cancer, 2021 Jan; personal communication). The loss of coding exon 2 of is strongly associated with hereditary breast and ovarian cancer phenotype based on multifactorial analysis (Caputo SM et al. Oncotarget, 2018 Apr;9:17334-17348); however the functional and clinical impact of the small in-frame loss is unknown. Downstream functional studies showed that close-match alteration BRCA2 c.68-2A>G was able to rescue the growth defect in BRCA2-null mouse embryonic stem cells and that these surviving cells maintained partial activity in a homology directed DNA repair assay (personal communication). BRCA2 c.68-2A>G is also identified in patients who collectively have a phenotype that is not consistent with a high risk BRCA2 pathogenic variant (Nix P et al. Fam Cancer, 2021 Jan). Since supporting evidence is conflicting at this time, the clinical significance of this alteration remains unclear. It cannot yet be ruled out that this variant may be hypomorphic and present with reduced risks and/or biallelic phenotype.

The c.68-1G>T variant was not identified in the literature, nor was it identified in the dbSNP, NHLBI Exome Sequencing Project (Exome Variant Server), Exome Aggregation Consortium (ExAC), LOVD, COSMIC, ClinVar, GeneInsight COGR, BIC or UMD. The c.68-1G>T variant is predicted to cause abnormal splicing because the nucleotide substitution occurs in the invariant region of the splice consensus sequence. In addition, 4 of 5 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) predict a greater than 10% difference in splicing. In summary, based on the above information, this variant meets our laboratory’s criteria to be classified as pathogenic.