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Supplementary Tables S1–S3 and figures S1 and S2 can be found at: http://carcin.oxfordjournals.org/.-- et al. ; BRCA2-c.2808_2811del (3036delACAA) is one of the most reported germline mutations in non-Ashkenazi breast cancer patients. We investigated its genetic origin in 51 Spanish carrier families that were genotyped with eleven 13q polymorphic markers. Three independent associated haplotypes were clearly distinguished accounting for 23 (WCL), 20 (ECL) and 6 families (SoS). Mutation age was estimated with the DMLE software in a range of 45-68 and 45-71 generations for WCL and ECL haplotypes, respectively. The most prevalent variants, c.2808_2811del and c.2803G>A, were located in a double-hairpin loop structure (c.2794-c.2825) predicted by Quikfold that was proposed as a mutational hotspot. To check this hypothesis, random mutagenesis was performed over a 923-bp fragment of BRCA2 and 86 DNA variants were characterized. Interestingly, three mutations reported in the mutation databases (c.2680G>A, c.2944del and c.2957dup) were replicated and 20 affected the same position with different nucleotide changes. Moreover, five variants were placed in the same hairpin-loop of c.2808_2811del, and one affected the same position (c.2808A>G). In conclusion, our results support that at least three different mutational events occurred to generate c.2808_2811del. Other highly prevalent DNA variants, such as BRCA1-c.68_69delAG and BRCA2-c.5946delT and c.8537delAG, are concentrated in hairpin-loops suggesting that these structures may represent mutational hotspots. ; Instituto de Salud Carlos III (PI10/2910 to E.A.V., ISCIIIRETIC: RD06/0020/1051 and PI10/01422 to L.F.); Junta de Castilla y León, Consejería de Educación (CSI004A10-2 to E.A.V.); Consejería de Sanidad (BIO39/VA27/10 to E.A.V.); Cancer Prevention Program of the Regional Government of Castilla y León; The European Social Fund and Consejería de Educación de la Junta de Castilla y León under the P.O. Castilla y León 2007–13 to A.A.; Spanish Health Research Fund; Carlos III Health Institute; Catalan Health Institute, Government of Catalonia (2009SGR290 to L.F.); Spanish Association Against Cancer (to L.F.). ; Peer Reviewed

© 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).