Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene

Abstract

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.

Keywords: RAD51C; VUS; aberrant splicing; breast cancer; clinical interpretation; functional assay; genetic variants; minigene; ovarian cancer; splicing; susceptibility genes.

Figure 1

Structure of the minigene mgR51C_ex2-8 and functional validation. (A) Schematic representation of the RAD51C minigene with exons 2 to 8. Exons are indicated by boxes, broken arrows indicate the expected splicing reactions in eukaryotic cells and black arrows locate specific vector RT-PCR primers. (B) Functional assay of the wild type minigene mgR51C_ex2-8. cDNAs were amplified with primers SD6-PSPL3_RTFW and RTpSAD-RV (full-length transcript V1-RAD51C ex2-8-V2 = 1062 nt). The RT-PCR product was run by agarose gel electrophoresis (left) and fluorescent capillary electrophoresis (right), where the full-length transcript is shown as a blue peak and the LIZ1200 size standard as orange/faint peaks. (C) Agarose gel (left) and fluorescent capillary electrophoresis (right) of transcripts produced by MCF-7 cells (above) and human breast RNA (below). cDNAs were amplified with primers RTR51C_ex1-FW and RTR51C_ex9-RV (full-length transcript = 957nt). FAM-labelled products (blue peaks) were run with LIZ1200 (orange peaks) as the size standard. FL, full-length transcript.

Figure 2

Splicing functional assays of selected RAD51C variants. (A) Map of tested variants. (B) fluorescent fragment analysis of transcripts generated by the wild type and mutant minigenes. cDNAs were amplified with primers SD6-PSPL3_RTFW and RTpSAD-RV (full-length transcript V1-RAD51C ex2-8-V2 = 1062 nt). FAM-labelled products (blue peaks) were run with LIZ1200 (orange peaks) as the size standard. For transcript descriptions see Table S2; FL, full-length transcript.

Figure 3

Workflow of the minigene protocol. The basic assay includes the following steps: (1) minigene construction; (2) site-directed mutagenesis; (3) transfection of the wild type and mutant minigenes; (3) inhibition of nonsense-mediated decay and RNA purification; (4) transcript sequencing and fragment analysis by fluorescent capillary electrophoresis; (5) data interpretation.