2026-04-13T13:18:56Zhttps://recercat.cat/oai/request

oai:recercat.cat:2445/1786422025-12-05T12:33:31Zcom_2072_1057col_2072_478916col_2072_478917

00925njm 22002777a 4500 dc Bueno Martínez, Elena author Sanoguera Miralles, Lara author Valenzuela Palomo, Alberto author Lorca, Víctor author Gómez Sanz, Alicia author Carvalho, Sara author Allen, Jamie author Infante, Mar author Pérez Segura, Pedro author Lázaro García, Conxi author Easton, Douglas F. author Devilee, Peter author Vreeswijk, Maaike P. G. author Hoya, Miguel de la author Velasco, Eladio A. author 2021-06-25T10:46:24Z 2021-06-25T10:46:24Z 2021-06-07 2021-06-25T09:14:44Z 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 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. Càncer de mama Càncer d'ovari Breast cancer Ovarian cancer RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants