Assessing the RNA effect of 26 DNA variants in the BRCA1 and BRCA2 genes.

Comprehensive genetic testing of the breast cancer susceptibility genes BRCA1 and BRCA2 identified approximately 16% of variants of unknown significance (VUS), a significant proportion of which could affect the correct splicing of the genes. Our aim is to establish a workflow for classifying VUS in these complex genes, the first stage of which is splicing analysis. We used a combined approach consisting of five in silico splicing prediction programs and RT-PCR analysis for a set of 26 variants not previously studied at the mRNA level and six variants that had already been studied, four of which were used as positive controls as they were found to affect the splicing of these genes and the other two were used as negative controls.