New splicing vector pSAD: Splicing functional analysis of a hybrid "maxi-minigene" with exons 19 to 27 of BRCA2

Abstract

At least 50% of all mutations identified in BRCA genes in breast/ovarian cancer patients are variants of unknown clinical significance (VUS). Several studies have shown the link between disease and splicing disruptions due to mutations, including predicted nonsense or frameshift mutations. We aimed to investigate the role of splicing aberrations of BRCA2 in breast/ovarian cancer. We followed a simple strategy consisting of bioinformatics analysis with NNSplice and Human Splicing Finder of DNA variants and splicing functional assays of hybrid minigenes. Splicing reporter plasmids allow to perform functional analysis without the need of patient RNA. We designed a new splicing vector, pSAD, which enabled us the creation of a “maxi-minigene” that includes nine exons of BRCA2 (19 to 27, MGBR2_19-27), where exon 27 substituted the second vector exon. The wt minigene produced a mRNA of 2,174 nucleotides. We validated the new MGBR2_19-27 with four splicing variants of the exons 19, 20, 23 and 24, which were tested in a previous report. Thirty-six out of 166 variants reported in these 9 exons were bioinformatically selected and introduced by PCR-mutagenesis in the wt MGBR2_19-27. Fifteen variants (42%) of all types (synonymous, nonsense, frameshift or missense) altered splicing by different mechanisms. Altogether, most variants disrupted the canonical splice sites; one affected a splicing silencer and another the polypyrimidine tract. Aberrant splicing represents a relevant pathogenic mechanism in hereditary breast/ovarian cancer. Splicing functional assays with the new vector pSAD are valuable tools to discriminate between benign and pathogenic DNA variants of any human disease genes.