Germline alterations in many genes coding for proteins regulating DNA repair and DNA damage response (DDR) to DNA double-strand breaks (DDSB) have been recognized as pathogenic factors in hereditary cancer predisposition. The ATM-CHEK2-p53 axis has been documented as a backbone for DDR and hypothesized as a barrier against cancer initiation. However, although CHK2 kinase coded by the CHEK2 gene expedites the DDR signal, its function in activation of p53-dependent cell cycle arrest is dispensable. CHEK2 mutations rank among the most frequent germline alterations revealed by germline genetic testing for various hereditary cancer predispositions, but their interpretation is not trivial. From the perspective of interpretation of germline CHEK2 variants, we review the current knowledge related to the structure of the CHEK2 gene, the function of CHK2 kinase, and the clinical significance of CHEK2 germline mutations in patients with hereditary breast, prostate, kidney, thyroid, and colon cancers.

Carriers of mutations in hereditary cancer predisposition genes represent a small but clinically important subgroup of oncology patients. The identification of causal germline mutations determines follow-up management, treatment options and genetic counselling in patients’ families. Targeted next-generation sequencing-based analyses using cancer-specific panels in high-risk individuals have been rapidly adopted by diagnostic laboratories. While the use of diagnosis-specific panels is straightforward in typical cases, individuals with unusual phenotypes from families with overlapping criteria require multiple panel testing. Moreover, narrow gene panels are limited by our currently incomplete knowledge about possible genetic dispositions. We have designed a multi-gene panel called CZECANCA (CZEch CAncer paNel for Clinical Application) for a sequencing analysis of 219 cancer-susceptibility and candidate predisposition genes associated with frequent hereditary cancers. The bioanalytical and bioinformatics pipeline was validated on a set of internal and commercially available DNA controls showing high coverage uniformity, sensitivity, specificity and accuracy. The panel demonstrates a reliable detection of both single nucleotide and copy number variants. Inter-laboratory, intra- and inter-run replicates confirmed the robustness of our approach. The objective of CZECANCA is a nationwide consolidation of cancer-predisposition genetic testing across various clinical indications with savings in costs, human labor and turnaround time. Moreover, the unified diagnostics will enable the integration and analysis of genotypes with associated phenotypes in a national database improving the clinical interpretation of variants.

Several reports indicate that inherited mutations in the PALB2 gene predispose to breast cancer. However, there is little agreement about the clinical relevance and usefulness of mutation screening in this gene. We analyzed the prevalence and spectrum of germline mutations in PALB2 to estimate their contribution to hereditary breast and/or ovarian cancer in the Czech Republic. The entire PALB2 coding region was sequenced in 409 breast/ovarian cancer patients negative for BRCA1 and BRCA2 mutations. Testing for large genomic rearrangements (LGR) was performed by multiplex ligation-dependent probe amplification (MLPA) analysis. We have identified 13 different pathogenic alterations including 10 truncating mutations and three LGRs in 16 of 409 patients (3.9%), whereas one truncating mutation was found in a group of 1,226 controls (0.08%; P = 2.6 × 10(-9)). Three novel LGRs included deletions involving exons 7-8 and 9-10, respectively, and a duplication spanning exons 9-11. Five frameshift and two nonsense mutations were novel, whereas three truncating mutations were described previously. The only recurrent mutation was the c.172_175delTTGT detected in four unrelated breast cancer individuals. Our analyses demonstrated the significant role of the PALB2 gene in breast cancer susceptibility. The highest frequency of PALB2 mutations (comparable with that previously reported for BRCA2) was found in a subgroup of patients with hereditary breast cancer (HBC) (13/235; 5.5%). Our results show that mutation analysis of the PALB2 gene, including the analysis of LGRs, is primarily indicated in patients with HBC in case of their BRCA1 and BRCA2 negativity.

Ovarian cancer (OC) is the deadliest gynecologic malignancy with a substantial proportion of hereditary cases and a frequent association with breast cancer (BC). Genetic testing facilitates treatment and preventive strategies reducing OC mortality in mutation carriers. However, the prevalence of germline mutations varies among populations and many rarely mutated OC predisposition genes remain to be identified. We aimed to analyze 219 genes in 1333 Czech OC patients and 2278 population-matched controls using next-generation sequencing. We revealed germline mutations in 18 OC/BC predisposition genes in 32.0% of patients and in 2.5% of controls. Mutations in BRCA1/BRCA2, RAD51C/RAD51D, BARD1, and mismatch repair genes conferred high OC risk (OR > 5). Mutations in BRIP1 and NBN were associated with moderate risk (both OR = 3.5). BRCA1/2 mutations dominated in almost all clinicopathological subgroups including sporadic borderline tumors of ovary (BTO). Analysis of remaining 201 genes revealed somatic mosaics in PPM1D and germline mutations in SHPRH and NAT1 associating with a high/moderate OC risk significantly; however, further studies are warranted to delineate their contribution to OC development in other populations. Our findings demonstrate the high proportion of patients with hereditary OC in Slavic population justifying genetic testing in all patients with OC, including BTO.

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.