The RAD52 S346X variant reduces risk of developing breast cancer in carriers of pathogenic germline BRCA2 mutations

Abstract

Women who carry pathogenic mutations in BRCA1 and BRCA2 have a lifetime risk of developing breast cancer of up to 80%. However, risk estimates vary in part due to genetic modifiers. We investigated the association of the RAD52 S346X variant as a modifier of the risk of developing breast and ovarian cancers in BRCA1 and BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2. The RAD52 S346X allele was associated with a reduced risk of developing breast cancer in BRCA2 carriers [per-allele hazard ratio (HR) = 0.69, 95% confidence interval (CI) 0.56-0.86; P = 0.0008] and to a lesser extent in BRCA1 carriers (per-allele HR = 0.78, 95% CI 0.64-0.97, P = 0.02). We examined how this variant affected DNA repair. Using a reporter system that measures repair of DNA double-strand breaks (DSBs) by single-strand annealing (SSA), expression of hRAD52 suppressed the loss of this repair in Rad52^-/- mouse embryonic stem cells. When hRAD52 S346X was expressed in these cells, there was a significantly reduced frequency of SSA. Interestingly, expression of hRAD52 S346X also reduced the stimulation of SSA observed upon depletion of BRCA2, demonstrating the reciprocal roles for RAD52 and BRCA2 in the control of DSB repair by SSA. From an immunofluorescence analysis, we observed little nuclear localization of the mutant protein as compared to the wild-type; it is likely that the reduced nuclear levels of RAD52 S346X explain the diminished DSB repair by SSA. Altogether, we identified a genetic modifier that protects against breast cancer in women who carry pathogenic mutations in BRCA2 (P = 0.0008) and to a lesser extent BRCA1 (P = 0.02). This RAD52 mutation causes a reduction in DSB repair by SSA, suggesting that defects in RAD52-dependent DSB repair are linked to reduced tumor risk in BRCA2-mutation carriers.

Keywords: BRCA2; RAD52; breast cancer; double-strand break repair.

Fig. 1

RAD52 S346X is dysfunctional in promoting SSA. (A) The domain map of human RAD52. The N‐terminal domain (1–212) contains DNA binding and self‐association regions. The C‐terminal domain (213–418) contains RPA and RAD51 interacting regions and a nuclear localization signal (NLS). (B) Diagram of the RMD‐GFP SSA reporter. Two tandem repeat (R) sequences are separated by 0.4 Mb and positioned such that SSA generates a Cdkn1a‐GFP fusion gene. SSA is induced by two DSBs between the repeats, with one DSB downstream from the 5′ repeat (5’268) and a second DSB 9.1 kbp upstream of the 3′ repeat. Shown are the single‐guide RNA (sgRNA)/Cas9 targeting sites for each DSB. Not to scale. (C) Flag immunoblot showing expression Flag‐hRAD52 WT and Flag‐hRAD52 S346X in RMD‐GFP Rad52^−/− mESCs. (D) hRAD52 S346X is able to promote SSA but with a > 2‐fold decrease as compared to hRAD52 WT. Rad52^−/− mESCs with RMD‐GFP were transfected with the 268 bp and 9 kbp sgRNA/Cas9 expression vectors along with a control EV, Flag‐hRAD52, or Flag‐hRAD52 S346X complementation vectors. Shown is the percentage of GFP⁺ cells from this experiment, normalized to transfection efficiency. n = 9. Lines represent the mean with SD. The numerical comparisons represent the P‐value determined using multiple t‐test with Holm–Sidak correction. (E) BRCA2 immunoblot showing depletion of BRCA2 in RMD‐GFP Rad52^−/− mESCs, transfected with a pool of four BRCA2 siRNAs (siBRCA2). (∗) Nonspecific band. (F) Depletion of BRCA2 causes an increase in the ability of hRAD52 WT to promote SSA. RMD‐GFP Rad52^−/− mESCs were transfected with the 268 bp and 9 kbp sgRNA/Cas9 expression vectors, either Flag‐hRAD52 WT or Flag‐hRAD52 S346X complementation vectors, along with a nontargeting siRNA (siCTRL) or siBRCA2. Shown is the percentage of GFP⁺ cells from this experiment, normalized to transfection efficiency. n = 9. Lines represent the mean with SD. The numbers shown above each comparison represent the P‐value determined using multiple t‐test with Holm–Sidak correction. (ns) Not significant.

Fig. 2

Subcellular localization of the WT and S346X RAD52 proteins tagged with Flag. RAD52^KO cells transiently transfected with pCAGGS‐RAD52 WT or pCAGGS‐RAD52 S346X expression vectors were examined by immunofluorescence analysis with anti‐Flag antibody (green). Nuclei were stained with DAPI (blue). White scale bars, 10 µm.