Interpretation of BRCA2 Splicing Variants: A Case Series of Challenging Variant Interpretations and the Importance of Functional RNA Analysis

Abstract

A substantial proportion of pathogenic variants associated with an increased risk of hereditary cancer are sequence variants affecting RNA splicing. The classification of these variants can be complex when both non-functional and functional transcripts are produced from the variant allele. We present four BRCA2 splice site variants with complex variant interpretations (BRCA2 c.68-3T>G, c.68-2A>G, c.425G>T, c.8331+2T>C). Evidence supporting a pathogenic classification is available for each variant, including in silico models, absence in population databases, and published functional data. However, comprehensive RNA analysis showed that some functional transcript may be produced by each variant. BRCA2 c.68-3T>G results in a partial splice defect. For BRCA2 c.68-2A>G and c.425G>T, aberrant splicing was shown to produce a potentially functional, in-frame transcript. BRCA2 c.8331+2T>C may utilize a functional GC donor in place of the wild-type GT donor. The severity of cancer history for carriers of these variants was also assessed using a history weighting algorithm and was not consistent with pathogenic controls (carriers of known pathogenic variants in BRCA2). Due to the conflicting evidence, our laboratory classifies these BRCA2 variants as variants of uncertain significance. This highlights the importance of evaluating new and existing evidence to ensure accurate variant classification and appropriate patient care.

Keywords: BRCA2; Hereditary cancer syndromes; Pathogenicity; RNA analysis; Splice variants.

Fig. 1

Analysis of BRCA2 c.68-3T>G. (a) Schematic representation BRCA2 regions amplified and digital electrophoresis of control and carrier samples amplified by E1F2-E5R. (b) Fraction of total transcript detected in control and carrier samples determined by quantification of transcripts from isolated traces. (c) Representative isolated sequence traces of observed transcripts from the variant carrier. The splice junction is indicated by a line. The 2 nucleotides inserted in the ▼3p transcript are shaded blue. (d) History weighting algorithm analysis based on 16 observations

Fig. 2

Analysis of BRCA2 c.68-2A>G. (a) Schematic representation BRCA2 regions amplified and digital electrophoresis of control and carrier samples. (b) Fraction of total transcript detected in control and carrier samples determined by quantification of transcripts from isolated traces. (c) Representative isolated sequence traces of observed transcripts from the variant carrier. The splice junction is indicated by a line. The 6 nucleotides missing from Δ3p are underlined in the normal transcript trace. (d) History weighting algorithm analysis based on 11 observations

Fig. 3

Analysis of BRCA2 c.425G>T. (a) Schematic representation BRCA2 regions amplified and digital electrophoresis of control and carrier samples. Some expected products may be obscured by the presence of similarly sized bands and/or may be present at too low a concentration to be visualized by electrophoresis. Not all individual bands are labeled, as the bands were not purified and sequenced separately. (b) Fraction of total transcript detected in control and carrier samples determined by quantification of transcripts from isolated traces. (c) Representative isolated sequence traces of observed transcripts from the variant carrier. The last base of the exon is highlighted blue. (d) History weighting algorithm analysis based on 10 observations

Fig. 4

Analysis of BRCA2 c.8331+2T>C. (a) Schematic representation BRCA2 regions amplified and digital electrophoresis of control and carrier samples amplified by E16F-E20R. (b) Fraction of total transcript detected in control and carrier samples determined by quantification of transcripts from isolated traces. (c) Representative isolated sequence traces of observed transcripts from the variant carrier. The last base of the exon is highlighted blue. (d) History weighting algorithm analysis based on 18 observations