Suchergebnisse

© 2021 by the authors. ; 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 (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. ; P.D., M.P.G.V., D.F.E., M.d.l.H. and E.A.V. have received funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 634935. E.A.V.'s lab is supported by grants from the Spanish Ministry of Science and Innovation, Plan Nacional de I + D + I 2013–2016, ISCIII (PI17/00227 and PI20/00225), co-funded by FEDER from Regional Development European Funds (European Union) and from the Consejería de Educación, Junta de Castilla y León, ref. CSI242P18 (actuación cofinanciada P.O. FEDER 2014–2020 de Castilla y León). M.d.l.H.'s lab is supported by grants from the Spanish Ministry of Science and Innovation, Plan Nacional de I + D + I 2013–2016, ISCIII (PI15/00059 and PI20/00110) and co-funded by FEDER from Regional Development European Funds (European Union). L.S.-M. is supported by a predoctoral fellowship from the AECC-Scientific Foundation, Sede Provincial de Valladolid (2019–2023). A.V.-P. is supported by a predoctoral fellowship from the Consejería de Educación, Junta de Castilla y León (2018–2022). Programa Estratégico Instituto de Biología y Genética Molecular (IBGM), Escalera de Excelencia, Junta de Castilla y León (Ref. CLU-2019-02).

© 2020 by the authors. ; Hereditary breast and/or ovarian cancer is a highly heterogeneous disease with more than 10 known disease-associated genes. In the framework of the BRIDGES project (Breast Cancer Risk after Diagnostic Gene Sequencing), the RAD51C gene has been sequenced in 60,466 breast cancer patients and 53,461 controls. We aimed at functionally characterizing all the identified genetic variants that are predicted to disrupt the splicing process. Forty RAD51C variants of the intron-exon boundaries were bioinformatically analyzed, 20 of which were selected for splicing functional assays. To test them, a splicing reporter minigene with exons 2 to 8 was designed and constructed. This minigene generated a full-length transcript of the expected size (1062 nucleotides), sequence, and structure (Vector exon V1- RAD51C exons_2-8- Vector exon V2). The 20 candidate variants were genetically engineered into the wild type minigene and functionally assayed in MCF-7 cells. Nineteen variants (95%) impaired splicing, while 18 of them produced severe splicing anomalies. At least 35 transcripts were generated by the mutant minigenes: 16 protein-truncating, 6 in-frame, and 13 minor uncharacterized isoforms. According to ACMG/AMP-based standards, 15 variants could be classified as pathogenic or likely pathogenic variants: c.404G > A, c.405-6T > A, c.571 + 4A > G, c.571 + 5G > A, c.572-1G > T, c.705G > T, c.706-2A > C, c.706-2A > G, c.837 + 2T > C, c.905-3C > G, c.905-2A > C, c.905-2_905-1del, c.965 + 5G > A, c.1026 + 5_1026 + 7del, and c.1026 + 5G > T. ; PD: MPGV: DFE: MdlH and EAV have received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 634935. EAV lab is supported by grants from the Spanish Ministry of Science and Innovation, Plan Nacional de I + D+I 2013–2016, ISCIII (PI17/00227) co-funded by FEDER from Regional Development European Funds (European Union) and from the Consejería de Educación, Junta de Castilla y León, ref. CSI242P18 (actuación cofinanciada P.O. FEDER 2014–2020 de Castilla y León). MdlH is supported by grants from the Spanish Ministry of Science and Innovation, Plan Nacional de I + D+I 2013–2016, ISCIII (PI15/00059) co-funded by FEDER from Regional Development European Funds (European Union).