Impact of BRCA1 and BRCA2 variants on splicing: clues from an allelic imbalance study

Nearly one-half of BRCA1 and BRCA2 sequence variations are variants of uncertain significance (VUSs) and are candidates for splice alterations for example, by disrupting/creating splice sites. As out-of-frame splicing defects lead to a marked reduction of the level of the mutant mRNA cleared through nonsense-mediated mRNA decay, a cDNA-based test was developed to show the resulting allelic imbalance (AI). Fifty-four VUSs identified in 53 hereditary breast/ovarian cancer (HBOC) patients without BRCA1/2 mutation were included in the study. Two frequent exonic single-nucleotide polymorphisms on both BRCA1 and BRCA2 were investigated by using a semiquantitative single-nucleotide primer extension approach and the cDNA allelic ratios obtained were corrected using genomic DNA ratios from the same sample. A total of five samples showed AI. Subsequent transcript analyses ruled out the implication of VUS on AI and identified a deletion encompassing BRCA2 exons 12 and 13 in one sample. No sequence abnormality was found in the remaining four samples, suggesting implication of cis- or trans-acting factors in allelic expression regulation that might be disease causative in these HBOC patients. Overall, this study showed that AI screening is a simple way to detect deleterious splicing defects and that a major role for VUSs and deep intronic mutations in splicing anomalies is unlikely in BRCA1/2 genes. Methods to analyze gene expression and identify regulatory elements in BRCA1/2 are now needed to complement standard approaches to mutational analysis. European Journal of Human Genetics (2009) 17, 1471-1480; doi:10.1038/ejhg.2009.89; published online 27 May 2009