Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

Abstract

Pathogenic protein-truncating variants of RAD51C, which plays an integral role in promoting DNA damage repair, increase the risk of breast and ovarian cancer. A large number of RAD51C missense variants of uncertain significance (VUS) have been identified, but the effects of the majority of these variants on RAD51C function and cancer predisposition have not been established. Here, analysis of 173 missense variants by a homology-directed repair (HDR) assay in reconstituted RAD51C-/- cells identified 30 nonfunctional (deleterious) variants, including 18 in a hotspot within the ATP-binding region. The deleterious variants conferred sensitivity to cisplatin and olaparib and disrupted formation of RAD51C/XRCC3 and RAD51B/RAD51C/RAD51D/XRCC2 complexes. Computational analysis indicated the deleterious variant effects were consistent with structural effects on ATP-binding to RAD51C. A subset of the variants displayed similar effects on RAD51C activity in reconstituted human RAD51C-depleted cancer cells. Case-control association studies of deleterious variants in women with breast and ovarian cancer and noncancer controls showed associations with moderate breast cancer risk [OR, 3.92; 95% confidence interval (95% CI), 2.18-7.59] and high ovarian cancer risk (OR, 14.8; 95% CI, 7.71-30.36), similar to protein-truncating variants. This functional data supports the clinical classification of inactivating RAD51C missense variants as pathogenic or likely pathogenic, which may improve the clinical management of variant carriers.

Significance: Functional analysis of the impact of a large number of missense variants on RAD51C function provides insight into RAD51C activity and information for classification of the cancer relevance of RAD51C variants.

Figure 1.

Evaluation of 173 RAD51C missense variants by HDR assay. A, DR-GFP reporter assay showing the range of HDR activity for 173 missense variants in CL-V4B cells, measured as fold change in GFP-positive cells (normalized to 1–5 scale, WT = 5 and p.Leu138Phe = 1). Neutral (>2.5 scale, gray bars), deleterious (<1.25 scale, red bars), and intermediate effects (>1.25, <2.5 scale, light gray bars). Purple, L138F and C135Y deleterious controls. Amino acid changes in one letter code are labeled on the x-axis in clusters (black, blue, and orange) in order of presentation on the bar chart. Error bars, SEM of three independent experiments. B, Illustration of the location of missense variants within the RAD51C linear sequence identifying a deleterious variant hotspot. Key functional domains of RAD51C are indicated and neutral (blue), deleterious (orange), and intermediate (green) variants are shown at top. C, Circos plot of the RAD51C variants and functional assay (HDR, cisplatin and olaparib sensitivity, binding to XRCC3, RAD51D, and XRCC2) results. RAD51C variants are indicated by residue position in the outer ring. Track 1 shows the final score based on all functional assays. For HDR, variants were classified as neutral (light blue; ≥51.1% relative to WT), intermediate (green; 48.7%–26.8%), or deleterious (orange; ≤22.7%). Cisplatin sensitivity was classified as neutral (≥83.5% relative to WT), intermediate (43%–82%), or deleterious (≤13.1%). Olaparib sensitivity was classified as neutral (≥ 80.4% relative to WT), intermediate (78.7%–51.7%), or deleterious (≤42%). Dark blue, interactions with XRCC3, RAD51D, and XRCC2; yellow, partial interaction; red, no interaction.

Figure 2.

Influence of RAD51C missense variants on response to cisplatin and olaparib treatment. Relative IC₅₀ values (normalized to 1–15 scale, WT = 15 and p.Cys135Tyr = 1) from an MTS assay of CL-V4B cells, transduced with selected RAD51C variant lentivirus, 5 days after treatment with varying doses of cisplatin (A) and olaparib (B). Variants defined as neutral (gray), intermediate (light gray), and deleterious (red) by the HDR assay are shown. Purple, L138F and C135Y deleterious controls. Error bars, SEM of three independent experiments.

Figure 3.

Drug sensitivity of human U2OS landing pad cells. A, Olaparib sensitivity associated with RAD51C variants. Cell survival of U2OS cells expressing RAD51C variants was quantified after 4 days of treatment and calculated relative to mock-treated cells. Mean ± SEM was calculated from three independent experiments, each performed in triplicate. B, RAD51C protein levels in stable U2OS landing pad cell lines. Expression of RAD51C WT and variant proteins in U2OS cells was determined by Western blot analysis after 24 hours of doxycycline induction.

Figure 4.

Characterization of U20S landing pad cells expressing RAD51C variants. A, Colony formation assays of RAD51C variants. B, RAD51 foci quantification and representative microscopy images for each variant. RAD51 foci formation was quantified after exposure of landing pad cells to 5 Gy of γ-irradiation. Each dot represents a geminin (S/G₂ phase)-positive cell and the bars designate the mean number of foci in at least 1,000 cells obtained in at least three independent experiments. The mean change percentage was calculated for each variant relative to the WT.

Figure 5.

Coimmunoprecipitation analysis of RAD51C CX3 and BCDX2 complexes. Western blotting of coimmunoprecipitated Flag-tagged RAD51C variant proteins with XRCC3 and RAD51D, XRCC2 paralogs 48 hours after transfection of HEK293T cells with FLAG-tagged RAD51C variant expression plasmids. IP, immunoprecipitation; TCL, total cell lysate.

Figure 6.

Evaluation of RAD51C deleterious variants in RAD51C 3D structure prediction model. A, Locations of 30 neutral and 30 deleterious variants in the 3D model of two ATP-bound RAD51C monomers (yellow and green). The locations of neutral and deleterious variants are shown with green and red dots, respectively. B, Magnified view of the 11 deleterious variants predicted to disrupt the binding of ATP.