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.

Genome-wide association studies have revealed a locus at 8p12 that is associated with breast cancer risk. Fine-mapping of this locus identified 16 candidate causal variants (CCVs). However, as these variants are intergenic, their function is unclear. To map chromatin looping from this risk locus to a previously identified candidate target gene, DUSP4, we performed chromatin conformation capture analyses in normal and tumoural breast cell lines. We identified putative regulatory elements, containing CCVs, which looped to the DUSP4 promoter region. Using reporter gene assays, we found that the risk allele of CCV rs7461885 reduced the activity of a DUSP4 enhancer element, consistent with the function of DUSP4 as a tumour suppressor gene. Furthermore, the risk allele of CCV rs12155535, located in another DUSP4 enhancer element, was negatively correlated with looping of this element to the DUSP4 promoter region, suggesting that this allele would be associated with reduced expression. These findings provide the first evidence that CCV risk alleles downregulate DUSP4 expression, suggesting that this gene is a regulatory target of the 8p12 breast cancer risk locus.

High stage and recurrent ovarian clear cell carcinoma (OCC) are associated with poor prognosis and resistance to chemotherapy. A distinguishing histological feature of OCC is abundant cytoplasmic stores of glucose, in the form of glycogen, that can be mobilized for cellular metabolism. Here, we report the effect on preclinical models of OCC of disrupting glycogen utilization using the glucose analogue 2-deoxy-D-glucose (2DG). At concentrations significantly lower than previously reported for other cancers, 2DG markedly improves the efficacy in vitro of carboplatin chemotherapy against chemo-sensitive TOV21G and chemo-resistant OVTOKO OCC cell lines, and this is accompanied by the depletion of glycogen. Of note, 2DG doses-of more than 10-fold lower than previously reported for other cancers-significantly improve the efficacy of carboplatin against cell line and patient-derived xenograft models in mice that mimic the chemo-responsiveness of OCC. These findings are encouraging, in that 2DG doses, which are substantially lower than previously reported to cause adverse events in cancer patients, can safely and significantly improve the efficacy of carboplatin against OCC. Our results thus justify clinical trials to evaluate whether low dose 2DG improves the efficacy of carboplatin in OCC patients.

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 (<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.