Minigene Splicing Assays Identify 20 Spliceogenic Variants of the Breast/Ovarian Cancer Susceptibility Gene RAD51C

Simple Summary Loss-of-function variants of the RAD51C gene are known to confer a risk of breast and ovarian cancers. In this study, we analyzed the impact of RAD51C variants on splicing, a highly regulated gene expression step by which introns are removed and exons are sequentially joined. Exon recognition is guided by specific sequences, the 3 ' and 5 ' splice sites, which define the exon boundaries. Variants of these sequences of susceptibility genes may lead to aberrant splicing and abnormal transcripts that may trigger a disease. Splicing can be tested using a biotechnological tool called minigenes, which mimic the human gene of interest. Thus, we checked 20 RAD51C splice-site variants using the minigene mgR51C_ex2-8. We found that they all disrupted the splicing mechanism, and 16 variants could be classified as likely pathogenic. Our findings are clinically actionable, and variant carriers may benefit from tailored prevention protocols and therapies. RAD51C loss-of-function variants are associated with an increased risk of breast and ovarian cancers. Likewise, splicing disruptions are a frequent mechanism of gene inactivation. Taking advantage of a previous splicing-reporter minigene with exons 2-8 (mgR51C_ex2-8), we proceeded to check its impact on the splicing of candidate ClinVar variants. A total of 141 RAD51C variants at the intron/exon boundaries were analyzed with MaxEntScan. Twenty variants were selected and genetically engineered into the wild-type minigene. All the variants disrupted splicing, and 18 induced major splicing anomalies without any trace or minimal amounts (<2.4%) of the minigene full-length (FL) transcript. Twenty-seven transcripts (including the wild-type and r.904A FL transcripts) were identified by fluorescent fragment electrophoresis; of these, 14 were predicted to truncate the RAD51C protein, 3 kept the reading frame, and 8 minor isoforms (1.1-4.7% of the overall expression) could not be characterized. Finally, we performed a tentative interpretation of the variants according to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, classifying 16 variants as likely pathogenic. Minigene assays have been proven as valuable tools for the initial characterization of potential spliceogenic variants. Hence, minigene mgR51C_ex2-8 provided useful splicing data for 40 RAD51C variants.

Automated summary

This study analyzed the impact of RAD51C gene variants on splicing and found that they disrupted the splicing mechanism, with 16 variants classified as likely pathogenic. These findings have clinical implications for tailored prevention and treatment protocols.