Abstract
Simple SummaryIn 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.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.
Highlights
RAD51D [MIM#602954] is one of the five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) that play an important role in the repair of DNA double-strand breaks via homologous recombination [1,2]
A RAD51D minigene with exons 2–9 was constructed in splicing vector pSAD
Summarizing, we evaluated a total of 42 candidate exonic splicing enhancer (ESE) variants (26 identified in BRIDGES subjects), 30 of which impaired RAD51D exon 3 splicing
Summary
RAD51D [MIM#602954] is one of the five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) that play an important role in the repair of DNA double-strand breaks via homologous recombination [1,2]. RAD51D loss-of-function variants confer risk of breast and/or ovarian cancer [3,4]. In a cohort of 6690 families, RAD51D pathogenic variants were associated with relative risks of 7.6 and 1.83 for tubo-ovarian cancer and breast cancer, respectively [3]. Two large-scale studies have estimated an overall breast cancer relative risk of 1.8 and 1.72, respectively [5,6]. During RNA splicing, introns from eukaryotic genes are removed from pre-mRNA, and consecutive exons are precisely joined together to form mature mRNA [7]. It has been proposed that ~60% of disease-causing variants disrupt pre-mRNA processing [9], generating aberrant transcripts that can affect protein function and correlate with an increased risk of a given genetic disease. The lack of accurate in silico predictors makes functional assays vital to doing so
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