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.

RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants

“…Many studies, including previous work by our group, support the ability of minigene assays to accurately reproduce splicing alterations as observed in RNA from carriers [ 16 , 24 , 25 , 47 , 48 , 49 ]. Unfortunately, as far as we know, none of the RAD51D variants here assessed have been tested previously in RNA from carriers (the only exception being a sub-optimal study performed in a c.738+1G>A carrier, see footnote to Table 1 ).…”

“…Forty-seven variants from the intron–exon boundaries (3′ splice-site: intron/exon [IVS-10_IVS-1/2nt]; 5′ splice-site: exon/intron [2nt/IVS+1_IVS+10]) [ 16 ] and 34 from RAD51D exon 3 were collected from the BRIDGES sequencing data [ 5 ]. RNA outcomes and predicted protein products were described according to the Human Genome Variation Society guidelines ( , accessed on 1 April 2021) using Ensembl reference transcript ID ENST00000345365.10 (GenBank NM_002878.3).…”

“…To identify potential splicing variants, in silico studies were performed using MaxEntScan (MES, , accessed on 1 April 2021) (cut-off ≥ 3.0) [ 18 ] and NNSplice ( , accessed on 1 April 2021) [ 19 ] when MES was not informative. Potential spliceogenic variants were selected according to the following criteria: (i) MES score changes (>15%) [ 16 , 20 ] and (ii) creation of alternative sites ( Table S2 ).…”

“…MCF-7 cell growth, transfection and inhibition of the nonsense-mediated decay were performed as previously described [ 16 ]. The triple-negative breast cancer (TNBC) cell line MDA-MB-231 was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 1% non-essential amino acids and 1% penicillin/streptomycin solution.…”

Regulation of RAD51 at the Transcriptional and Functional Levels: What Prospects for Cancer Therapy?

“…The importance of the RAD51 paralogs was further underlined by genetic studies showing an increased risk of developing breast and/or ovarian cancer in women bearing RAD51C, RAD51D or XRCC2 mutations [115][116][117]. This association was confirmed in breast and/or ovarian cancer families and cohorts with no known BRCA1 or BRCA2 mutations, leading to the proposition to include RAD51D and RAD51C to the list of genes screened for breast cancer predisposition [118][119][120].…”