Bueno Martínez, ElenaSanoguera Miralles, LaraValenzuela Palomo, AlbertoLorca, VíctorGómez Sanz, AliciaCarvalho, SaraAllen, JamieInfante, MarPérez Segura, PedroLázaro García, ConxiEaston, Douglas F.Devilee, PeterVreeswijk, Maaike P. G.Hoya, Miguel de laVelasco, Eladio A.2021-06-252021-06-252021-06-07https://hdl.handle.net/2445/178642RAD51D 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 similar to 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.20 p.application/pdfengcc by (c) Bueno Martínez et al., 2021http://creativecommons.org/licenses/by/3.0/es/Càncer de mamaCàncer d'ovariBreast cancerOvarian cancerinfo:eu-repo/semantics/article2021-06-25info:eu-repo/semantics/openAccess