A functional assay for the identification of DNA double-strand break repair deficiency in heterozygous carriers of BRCA1/2 and RAD51C mutations.

Abstract

561 Background: Mutations in breast cancer gene 1 and 2(BRCA1/2) account for 50% of the familial aggregation of breast and ovarian cancer. About one third of the mutations are of unknown pathogenetic relevance (unclassified variants, UCV). We recently identified RAD51C as a third high penetrance gene (Meindl et al., 2010). Like BRCA1/2, RAD51C is also involved in homologous recombination repair (HRR) in response to DNA double strand breaks. It has recently been shown that cells with HRR deficiency are sensitive to synthetic lethality mediated by PARP inhibition. Under the assumption that further high risk genes may exist which acts through the HRR pathway we aimed to develop a reliable functional test system which allows the evaluation of HRR deficiency in heterozygous carriers. Methods: We included eight carriers of a pathogenic BRCA2 mutation, two carriers of a pathogenic RAD51C mutation and nine healthy controls as well as seven BRCA2 UCV carriers. Patient and control lymphocytes were γ-irradiated in G2 phase to introduce DNA-DSB. For the assessment of HRR capacity, metaphase chromosomes were stained by multicolour fluorescence in situ hybridisation (M-FISH). Chromosomal translocations and breakages were counted per mitosis and referred to total chromosomal number. The aberration frequency of BRCA2 and RAD51C carriers versus the controls was compared. Results: Lymphocytes from BRCA2 and RAD51C pathogenic mutation carriers versus controls showed a mean aberration frequency of 2.51±0.36 (standard deviation, SD) and 2.7±0.25 (SD), respectively, versus 1.4±0.44 (SD) chromosomal aberrations per metaphase with no overlap (p-value <0.001; Student’s t-test). Seven patients carrying an UCV in BRCA2 could be allocated to either the pathogenic or the control group. Conclusions: Our test system allows the detection of HRR deficiency in heterozygous lymphocytes of BRCA2 and RAD51C gene mutation carriers and is currently evaluated in BRCA1 positive cases. The assay may enable the identification of HRR deficiency irrespective of the underlying gene defect and may also serve as a biomarker for sensitivity to PARP inhibition.