2026-04-13T13:43:06Zhttps://recercat.cat/oai/request

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

RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants Bueno Martínez, Elena Sanoguera Miralles, Lara Valenzuela Palomo, Alberto Lorca, Víctor Gómez Sanz, Alicia Carvalho, Sara Allen, Jamie Infante, Mar Pérez Segura, Pedro Lázaro García, Conxi Easton, Douglas F. Devilee, Peter Vreeswijk, Maaike P. G. Hoya, Miguel de la Velasco, Eladio A. Càncer de mama Càncer d'ovari Breast cancer Ovarian cancer 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. 2021-06-25T10:46:24Z 2021-06-25T10:46:24Z 2021-06-07 2021-06-25T09:14:44Z info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Reproducció del document publicat a: https://doi.org/10.3390/cancers13112845 Cancers, 2021, vol. 13, num. 11 https://doi.org/10.3390/cancers13112845 info:eu-repo/grantAgreement/EC/H2020/634935/EU//BRIDGES cc by (c) Bueno Martínez et al., 2021 http://creativecommons.org/licenses/by/3.0/es/ info:eu-repo/semantics/openAccess MDPI Articles publicats en revistes (Institut d'lnvestigació Biomèdica de Bellvitge (IDIBELL))