RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants

SIMPLE SUMMARY: In the BRIDGES project, the breast/ovarian cancer gene RAD51D has been sequenced in >113,000 women. In the present study, we focused on the impact that 11 pre-selected RAD51D variants at the intron/exon boundaries had on the splicing process (intron removal). For this purpose, we developed a splicing reporter minigene, containing RAD51D-exons 2–9 wherein any variant could be introduced and functionally assayed for splicing alterations. All variants impaired splicing, 10 of which caused complete splicing aberrations. Moreover, we developed a minigene-based strategy to search for non-canonical, spliceogenic variants that disrupted splicing enhancers/silencers in the non-constitutive exon 3. Twenty-six BRIDGES and 16 artificial exon 3 variants were also tested. Thirty variants impaired splicing by producing variable amounts of the FL transcript. In total, up to 9 variants were classified as Likely Pathogenic, and therefore were clinically actionable. Carriers may benefit from tailored prevention protocols and therapies. ABSTRACT: RAD51D loss-of-function variants increase lifetime risk of breast and ovarian cancer. Splicing disruption is a frequent pathogenic mechanism associated with variants in susceptibility genes. Herein, we have assessed the splicing and clinical impact of splice-site and exonic splicing enhancer (ESE) variants identified through the study of ~113,000 women of the BRIDGES cohort. A RAD51D minigene with exons 2–9 was constructed in splicing vector pSAD. Eleven BRIDGES splice-site variants (selected by MaxEntScan) were introduced into the minigene by site-directed mutagenesis and tested in MCF-7 cells. The 11 variants disrupted splicing, collectively generating 25 different aberrant transcripts. All variants but one produced negligible levels (<3.4%) of the full-length (FL) transcript. In addition, ESE elements of the alternative exon 3 were mapped by testing four overlapping exonic microdeletions (≥30-bp), revealing an ESE-rich interval (c.202_235del) with critical sequences for exon 3 recognition that might have been affected by germline variants. Next, 26 BRIDGES variants and 16 artificial exon 3 single-nucleotide substitutions were also assayed. Thirty variants impaired splicing with variable amounts (0–65.1%) of the FL transcript, although only c.202G>A demonstrated a complete aberrant splicing pattern without the FL transcript. On the other hand, c.214T>C increased efficiency of exon 3 recognition, so only the FL transcript was detected (100%). In conclusion, 41 RAD51D spliceogenic variants (28 of which were from the BRIDGES cohort) were identified by minigene assays. We show that minigene-based mapping of ESEs is a powerful approach for identifying ESE hotspots and ESE-disrupting variants. Finally, we have classified nine variants as likely pathogenic according to ACMG/AMP-based guidelines, highlighting the complex relationship between splicing alterations and variant interpretation.