Kleiblova P, et al. - A total of 1,928 high-risk Czech breast/ovarian cancer (BC/OC) patients and 3,360 population-matched controls were examined in this study to analyze germline checkpoint kinase 2 (CHEK2, a multiple cancer predisposing gene) variants and their link with BC and OC. In human non-transformed RPE1-CHEK2-knock-out cells, a complementation assay was developed quantifying CHEK2-specific phosphorylation of endogenous protein KAP1 for functional classification of VUS. A significant increase in BC risk and a marginal increase in OC risk was observed which was caused by functionally deleterious CHEK2 missense variants. Detection of large intragenic rearrangements was shown to be significant for CHEK2 analysis. For risk estimates, careful consideration of ethnicity in both patients and controls was found to be important. Also, newly developed human non-transformed cell line assay was shown to have promising potential for functional CHEK2 VUS classification.