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

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.

Keywords: ESE; ESS; RAD51D; VUS; aberrant splicing; breast cancer; clinical interpretation; minigene; ovarian cancer; susceptibility genes.

Figure 1

Structure of the minigene mgR51D_ex2-9 and functional assays of splice-site RAD51D variants. (A) Schematic representation of the RAD51D minigene, with exons 2 to 9. (B) Map of variants. (C) Fluorescent fragment analysis of transcripts generated by the wild type and mutant minigenes. FAM-labeled products (blue peaks) were run with LIZ-1200 (orange peaks) as size standard. FL: full-length transcript.

Figure 2

Analysis of RAD51D exon 3 variants. (A) Map of exon 3 microdeletions and tested variants. Boxes: red, total splicing disruptions; yellow, weak to moderate disruptions. Spliceogenic variants are shown in red. (B) Fluorescent fragment analysis of transcripts generated by selected microdeletions (left) and variants (right). FAM-labeled products (blue peaks) were run with LIZ-1200 (orange peaks) as the size standard.