Purpose: Germline pathogenic variants in CHEK2 confer moderately elevated breast cancer risk (odds ratio, OR ∼ 2.5), qualifying carriers for enhanced breast cancer screening. Besides pathogenic variants, dozens of missense CHEK2 variants of uncertain significance (VUS) have been identified, hampering the clinical utility of germline genetic testing (GGT). Experimental Design: We collected 460 CHEK2 missense VUS identified by the ENIGMA consortium in 15 countries. Their functional characterization was performed using CHEK2-complementation assays quantifying KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1–CHEK2-knockout cells. Concordant results in both functional assays were used to categorize CHEK2 VUS from 12 ENIGMA case–control datasets, including 73,048 female patients with breast cancer and 88,658 ethnicity-matched controls. Results: A total of 430/460 VUS were successfully analyzed, of which 340 (79.1%) were concordant in both functional assays and categorized as functionally impaired (N = 102), functionally intermediate (N = 12), or functionally wild-type (WT)–like (N = 226). We then examined their association with breast cancer risk in the case–control analysis. The OR and 95% CI (confidence intervals) for carriers of functionally impaired, intermediate, and WT-like variants were 2.83 (95% CI, 2.35–3.41), 1.57 (95% CI, 1.41–1.75), and 1.19 (95% CI, 1.08–1.31), respectively. The meta-analysis of population-specific datasets showed similar results. Conclusions: We determined the functional consequences for the majority of CHEK2 missense VUS found in patients with breast cancer (3,660/4,436; 82.5%). Carriers of functionally impaired missense variants accounted for 0.5% of patients with breast cancer and were associated with a moderate risk similar to that of truncating CHEK2 variants. In contrast, 2.2% of all patients with breast cancer carried functionally wild-type/intermediate missense variants with no clinically relevant breast cancer risk in heterozygous carriers.

Polo-like kinases play essential roles in cell cycle control and mitosis. In contrast to other members of this kinase family, PLK3 has been reported to be activated upon cellular stress including DNA damage, hypoxia and osmotic stress. Here we knocked out PLK3 in human non-transformed RPE cells using CRISPR/Cas9-mediated gene editing. Surprisingly, we find that loss of PLK3 does not impair stabilization of HIF1α after hypoxia, phosphorylation of the c-Jun after osmotic stress and dynamics of DNA damage response after exposure to ionizing radiation. Similarly, RNAi-mediated depletion of PLK3 did not impair stress response in human transformed cell lines. Exposure of cells to various forms of stress also did not affect kinase activity of purified EGFP-PLK3. We conclude that PLK3 is largely dispensable for stress response in human cells. Using mass spectrometry, we identify protein phosphatase 6 as a new interacting partner of PLK3. Polo box domain of PLK3 mediates the interaction with the PP6 complex. Finally, we find that PLK3 is phosphorylated at Thr219 in the T-loop and that PP6 constantly dephosphorylates this residue. However, in contrast to PLK1, phosphorylation of Thr219 does not upregulate enzymatic activity of PLK3, suggesting that activation of both kinases is regulated by distinct mechanisms.

PURPOSE: CHEK2 is the frequently detected cancer-predisposing gene in female breast cancer. In addition, the association with the risks of other cancer types has been suggested, and clinical management has also been discussed. Although clinical relevance of germline variants differs across population, there is little evidence of the clinical relevance of CHEK2 germline variants in East Asia. METHODS: Targeted sequencing and functional analyses of missense variants for the coding region of CHEK2 in 111,571 East Asian individuals were performed. Variants classified as pathogenic/likely pathogenic in ClinVar, predicted loss-of-function, or functionally impaired in functional analysis were defined as germline damaging variants (gDVs). We evaluated the association between CHEK2 gDVs and the risk of 23 cancer types. We also compared the clinical characteristics of carriers and noncarriers among patients with CHEK2 -associated cancers. RESULTS: We identified 77 gDVs including 36 functionally impaired missense variants. CHEK2 gDVs were significantly associated exclusively with prostate cancer (odds ratio [OR], 1.8 [95% CI, 1.2 to 2.6]; P = 1.7 × 10 ⁻³ ), in addition to female breast cancer (OR, 1.8 [95% CI, 1.3 to 2.6]; P = 1.2 × 10 ⁻³ ), among 23 cancer types. There were no differences in age at diagnosis, pathologic status, and prognosis between carriers and noncarriers. Besides, there was no association with the risk of cancer types with high incidence rates in East Asian countries. CONCLUSION: CHEK2 gDVs were associated with female breast and prostate cancer risks in East Asia. The necessity of additional systematic clinical management for all CHEK2 gDV carriers should be carefully discussed, and standard cancer screening is recommended unless no other clinical features suggestive of cancer predisposition are noted in East Asia.

Characteristics of 12 case-control datasets from the ENIGMA consortium partners.

Characteristics of 12 case-control datasets from the ENIGMA consortium partners.

List of all analyzed CHEK2 variants with results of KAP1/CHK2 kinase and localization assays and the results from recent previously published functional analyses of the CHEK2 VUS.