Rare sequence variants in "high-risk" disease genes, often referred as unclassified variants (UVs), pose a serious challenge to genetic testing. However, UVs resulting in splicing alterations can be readily assessed by in vitro assays. Unfortunately, analytical and clinical interpretation of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical International Agency for Research on Cancer guidelines), we performed qPCR and/or minigene assays. The latter were performed with a new splicing vector (pSAD) developed by authors of the present manuscript (patent #P201231427 CSIC). We have identified three clinically relevant Class-5 variants (c.682-2A>G, c.7617+1G>A, and c.8954-5A>G), and 27 analytical Class-2 variants (not inducing splicing alterations). In addition, we demonstrate that rs9534262 (c.7806-14T>C) is a BRCA2 splicing quantitative trait locus. Splicing analyses are widely used in the clinical setting to address the significance of rare genetic variants. However, interpretation of results is often challenging. Here we show that incorporating capillary electrophoresis (EP) and direct sequencing to the analysis of RT-PCR splicing assays often produces clear-cut analytical outputs. Indeed, we have produced clear-cut outputs in 29 out of 31 BRCA2 variants investigated, including the identified of clinically relevant splicing aberrations, and one splicing quantitative trait locus (sQTL).
- Capillary electrophoresis