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... Show moreRAD51D 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. Show less
Purpose We assessed the associations between population-based polygenic risk scores (PRS) for breast (BC) or epithelial ovarian cancer (EOC) with cancer risks forBRCA1andBRCA2pathogenic variant... Show morePurpose We assessed the associations between population-based polygenic risk scores (PRS) for breast (BC) or epithelial ovarian cancer (EOC) with cancer risks forBRCA1andBRCA2pathogenic variant carriers. Methods Retrospective cohort data on 18,935BRCA1and 12,339BRCA2female pathogenic variant carriers of European ancestry were available. Three versions of a 313 single-nucleotide polymorphism (SNP) BC PRS were evaluated based on whether they predict overall, estrogen receptor (ER)-negative, or ER-positive BC, and two PRS for overall or high-grade serous EOC. Associations were validated in a prospective cohort. Results The ER-negative PRS showed the strongest association with BC risk forBRCA1carriers (hazard ratio [HR] per standard deviation = 1.29 [95% CI 1.25-1.33],P = 3x10(-72)). ForBRCA2, the strongest association was with overall BC PRS (HR = 1.31 [95% CI 1.27-1.36],P = 7x10(-50)). HR estimates decreased significantly with age and there was evidence for differences in associations by predicted variant effects on protein expression. The HR estimates were smaller than general population estimates. The high-grade serous PRS yielded the strongest associations with EOC risk forBRCA1(HR = 1.32 [95% CI 1.25-1.40],P = 3x10(-22)) andBRCA2(HR = 1.44 [95% CI 1.30-1.60],P = 4x10(-12)) carriers. The associations in the prospective cohort were similar. Conclusion Population-based PRS are strongly associated with BC and EOC risks forBRCA1/2carriers and predict substantial absolute risk differences for women at PRS distribution extremes. Show less