Comprehensive splicing functional analysis of DNA variants of the BRCA2 gene by hybrid minigenes

Abstract

Introduction: The underlying pathogenic mechanism of a large fraction of DNA variants of disease-causing genes is the disruption of the splicing process. We aimed to investigate the effect on splicing of the BRCA2 variants c.8488-1G > A (exon 20) and c.9026_9030del (exon 23), as well as 41 BRCA2 variants reported in the Breast Cancer Information Core (BIC) mutation database.

Methods: DNA variants were analyzed with the splicing prediction programs NNSPLICE and Human Splicing Finder. Functional analyses of candidate variants were performed by lymphocyte RT-PCR and/or hybrid minigene assays. Forty-one BIC variants of exons 19, 20, 23 and 24 were bioinformatically selected and generated by PCR-mutagenesis of the wild type minigenes.

Results: Lymphocyte RT-PCR of c.8488-1G > A showed intron 19 retention and a 12-nucleotide deletion in exon 20, whereas c.9026_9030del did not show any splicing anomaly. Minigene analysis of c.8488-1G > A displayed the aforementioned aberrant isoforms but also exon 20 skipping. We further evaluated the splicing outcomes of 41 variants of four BRCA2 exons by minigene analysis. Eighteen variants presented splicing aberrations. Most variants (78.9%) disrupted the natural splice sites, whereas four altered putative enhancers/silencers and had a weak effect. Fluorescent RT-PCR of minigenes accurately detected 14 RNA isoforms generated by cryptic site usage, exon skipping and intron retention events. Fourteen variants showed total splicing disruptions and were predicted to truncate or eliminate essential domains of BRCA2.

Conclusions: A relevant proportion of BRCA2 variants are correlated with splicing disruptions, indicating that RNA analysis is a valuable tool to assess the pathogenicity of a particular DNA change. The minigene system is a straightforward and robust approach to detect variants with an impact on splicing and contributes to a better knowledge of this gene expression step.

Figure 1

Splicing outcomes of variants of exons 19-20 (A-D) and 23-24 (E-K) of BRCA2. RT-PCRs labelled with FAM (shadowed blue peaks) of mutant minigenes were run in an ABI3130 sequencer with Genescan ROX 500 (red peaks) as size standard. Aberrant isoforms are indicated by arrows. RFU: Relative Fluorescence Units. A-D) Chromatograms of the wt minigene and variants of c.8488-1G > A (intron 19), c.8488-2A > G (intron 19), and c.8486A > T (exon19). E-K) Chromatograms of the wt minigene and variants c.9026_9030del (exon 23), c.8954-1_8955delinsAA (intron 22-exon23), c.8954-3C > G (intron 22), c.9117+1G > A (intron 23), c.9118-2A > G (intron 23), and c.9256+1G > A (intron 24). wt, wild type.

Figure 2

Splicing isoforms generated by DNA variants of exons 19 and 20. A) Fluorescent RT-PCRs (blue peaks) of mutant minigenes were run in an ABI3130 DNA sequencer with Genescan ROX 500 (red peaks) as size standard. Chromatograms of different variants were overlaid to generate this picture. B) Diagrams of the splicing outcomes caused by variants of exons 19 and 20.

Figure 3

Splicing isoforms generated by DNA variants of exons 23 and 24. A) Fluorescent RT-PCRs (blue peaks) of mutant minigenes were run in an ABI3130 DNA sequencer with Genescan ROX 500 (red peaks) as size standard. Chromatograms of different variants were overlaid to generate this picture. B) Diagrams of the splicing outcomes caused by variants of exons 23 and 24.