The Genetic and Molecular Analyses of RAD51C and RAD51D Identifies Rare Variants Implicated in Hereditary Ovarian Cancer from a Genetically Unique Population

Abstract

To identify candidate variants in RAD51C and RAD51D ovarian cancer (OC) predisposing genes by investigating French Canadians (FC) exhibiting unique genetic architecture. Candidates were identified by whole exome sequencing analysis of 17 OC families and 53 early-onset OC cases. Carrier frequencies were determined by the genetic analysis of 100 OC or HBOC families, 438 sporadic OC cases and 1025 controls. Variants of unknown function were assayed for their biological impact and/or cellular sensitivity to olaparib. RAD51C c.414G>C;p.Leu138Phe and c.705G>T;p.Lys235Asn and RAD51D c.137C>G;p.Ser46Cys, c.620C>T;p.Ser207Leu and c.694C>T;p.Arg232Ter were identified in 17.6% of families and 11.3% of early-onset cases. The highest carrier frequency was observed in OC families (1/44, 2.3%) and sporadic cases (15/438, 3.4%) harbouring RAD51D c.620C>T versus controls (1/1025, 0.1%). Carriers of c.620C>T (n = 7), c.705G>T (n = 2) and c.137C>G (n = 1) were identified in another 538 FC OC cases. RAD51C c.705G>T affected splicing by skipping exon four, while RAD51D p.Ser46Cys affected protein stability and conferred olaparib sensitivity. Genetic and functional assays implicate RAD51C c.705G>T and RAD51D c.137C>G as likely pathogenic variants in OC. The high carrier frequency of RAD51D c.620C>T in FC OC cases validates previous findings. Our findings further support the role of RAD51C and RAD51D in hereditary OC.

Keywords: French Canadian; RAD51C; RAD51D; genetic drift; ovarian cancer predisposing genes.

Figure 1

Study design and criteria for identifying candidate variants in RAD51C and RAD51D. (A) Schematic diagram summarizing phase I of the study for identifying candidate variants in ovarian cancer (OC) cases, phase II of the study for determining the carrier frequency of the candidate variants in familial and sporadic OC cases, Hereditary Breast and Ovarian Cancer (HBOC) syndrome families and controls from the French Canadian (FC) population of Quebec, and phase III of the study for identifying additional carriers; teal ribbon signifies women with OC; and diagrams contain provincial flag of Quebec denoting geographic ascertainment of cases and controls; and (B) Schematic diagram presenting the filtering and prioritizing criteria applied to identify candidate variants (see Table S5). Variants were prioritized using different in silico tools for conservation: Genomic Evolutionary Rate Profiling v1.0 (GERP++ v1.0 (score ≥ 2.0)); Phylogenetic p value v4.0 of 100 vertebrates (PhyloP 100 way v4.0 (score ≥ 0.2)) and PHAST Conservation v4.0 of 100 vertebrates (PhastCons 100 way v4.0 (score ≥ 0.4)); for predicting damaging effects based on their best predictive performance: Rare Exome Variant Ensemble Learner v4.0 (REVEL v4.0 (score ≥ 0.4)); Meta-analytic Logistic Regression v4.0 (MetaLR v4.0 (score ≥ 0.4)); Meta-analytic support Vector Machine v4.0 (MetaSVM v4.0 (score ≥ 0.0)); Combined Annotation Dependent Depletion v1.3 (CADD v1.4 (Phred score ≥ 20)); Protein Variation Effect Analyzer v4.0 (PROVEAN v4.0 (score ≥ −2.5)); and CONsensus DELeteriousness v2.0 (CONDEL v2.0 (score ≥ 0.4)); and for affecting alternative splicing: Maximum Entropy Estimates of Splice junction strengths v2.0 (MaxEntScan v2.0); Human Splicing Finder v3.1 (HSF v3.1); and two different Database Splicing Consensus Single Nucleotide Variant (dbscSNV) in silico tools: AdaBoost v4.0 (ADA v4.0 (score ≥ 0.4)) and Random Forest v4.0 (RF v4.0 (score ≥ 0.4)) (see Materials and Methods Section 2.2.).

Figure 2

Loss of heterozygosity (LOH) analysis of a RAD51D c.620C>T carrier. Sanger sequencing chromatograms showing evidence of a complete loss of the wild-type variant in genomic DNA from formalin-fixed paraffin embedded (FFPE) tumour tissues from both ovaries.

Figure 3

RAD51C c.705G>T effect on transcript splicing in carrier- and non-carrier-derived lymphoblastoid cell lines (LCLs) (see Figure S3). (A) Agarose gel of cDNA analysis showing different sized bands from carrier- compared to non-carrier-derived LCLs; (B) Sanger sequencing chromatograms of genomic and cDNA from carrier-derived LCLs, confirming the variant status as indicated with * in genomic DNA and showing skipping of exon four in cDNA; and (C) The upper panel depicting the wild-type RAD51C at the genomic, mRNA and protein level whereas the lower panel depicting the predicted effect of RAD51C c.705G>T at mRNA and protein level, resulting in skipping of exon four (44 amino acids) annotated according to genomic (NC_000017.10), mRNA (NM_058216.1) and protein (NP_478123.1) NCBI Reference Sequence (RefSeq) Database (ncbi.nlm.nih.gov/refseq/, accessed on 1 October 2021).

Figure 4

The RAD51D p.Ser46Cys variant impairs protein stability and function in U2OS RAD51D knock-out (KO) cells. (A) Scheme representing the AAVS1 genomic integration system used to complement the RAD51D KO U2OS cell line. (B) U2OS RAD51D KO cells complemented with the AAVS1 system were confirmed by Sanger sequencing. (C) Western blots of U2OS RAD51D KO cells stably complemented with wild-type (WT) or the p.Ser46Cys variant (see Figure S7); CTL was used as non-edited control and Vinculin was used as a loading control. (D,E) Survival curves of U2OS RAD51D KO cells stably complemented with the WT RAD51D, the RAD51D p.Ser46Cys variant or empty vector (EV) and plated in triplicate in a 96 well plate. Cell viability was monitored following (D) olaparib or (E) talazoparib treatments for 96 h and was assessed by counting remaining nuclei. Experiments were performed in three biological replicates.