A deep intronic recurrent CHEK2 variant c.1009-118_1009-87delinsC affects pre-mRNA splicing and contributes to hereditary breast cancer predisposition.

Germline CHEK2 pathogenic variants confer an increased risk of female breast cancer (FBC). Here we describe a recurrent germline intronic variant c.1009-118_1009-87delinsC, which showed a splice acceptor shift in RNA analysis, introducing a premature stop codon (p.Tyr337PhefsTer37). The variant was found in 21/10,204 (0.21%) Czech FBC patients compared to 1/3250 (0.03%) controls (p = 0.04) and in 4/3639 (0.11%) FBC patients from an independent German dataset. In addition, we found this variant in 5/2966 (0.17%) Czech (but none of the 443 German) ovarian cancer patients, three of whom developed early-onset tumors. Based on these observations, we classified this variant as likely pathogenic.

Germline CHEK2 Gene Mutations in Hereditary Breast Cancer Predisposition - Mutation Types and their Biological and Clinical Relevance.

BACKGROUND: Hereditary mutations in the CHEK2 gene (which encodes CHK2 kinase) contribute to a moderately increased risk of breast cancer (BC) and other cancers. Large variations in the frequency of CHEK2 mutations and the occurrence of variants of unknown clinical significance (VUS) complicate estimation of cancer risk in carriers of germline CHEK2 mutations. PATIENTS AND METHODS: We performed mutation analysis of 1,526 high-risk Czech BC patients and 3,360 Czech controls. Functional analysis was performed for identified VUS using a model system based on a human RPE1-CHEK2-KO cell line harboring biallelic inactivation of endogenous CHEK2. RESULTS: The frequency of ten truncating CHEK2 variants differed markedly between BC patients (2.26%) and controls (0.11%; p = 4.1 × 1012). We also found 23 different missense variants in 4.5% patients and in 4.0% of controls. The most common was p.I157T, which was found in patients and controls with the same frequency. Functional analysis identified nine functionally deleterious VUS, another nine functionally neutral VUS, and four intermediate VUS (including p.I157T). We found that carriers of truncating CHEK2 mutations had a high BC risk (OR 8.19; 95% CI 4.11-17.75), and that carriers of functionally deleterious missense variants had a moderate risk (OR 4.06; 95% CI, 1.37-13.39). Carriers of these mutations developed BC at 44.4 and 50.7 years, respectively. Functionally neutral and functionally intermediate missense variants did not increase the BC risk. BC in CHEK2 mutation carriers was frequently ER-positive and of higher grade. Notably, carriers of CHEK2 mutations developed second cancers more frequently than BRCA1/BRCA2/PALB2/p53 or mutation non-carriers. CONCLUSION: Hereditary CHEK2 mutations contribute to the development of hereditary BC. The associated cancer risk in mutation carriers increases with the number of affected individuals in a family. Annual follow-up with breast ultrasound, mammography, or magnetic resonance imaging is recommended for asymptomatic mutation carriers from the age of 40. Surgical prevention and specific follow-up of other tumors should be considered based on family cancer history. The work was supported by grants from the Czech Health Research Council of the Ministry of Health of the Czech Republic NR 15-28830A, 16-29959A, NV19-03-00279, projects of the PROGRES Q28/LF1, GAUK 762216, SVV2019 / 260367, PRIMUS/17/MED/9, UNCE/MED/016, Progress Q26, LQ1604 NPU II and project AVČR Qualitas. The analysis of a set of unselected controls was made possible by the existence and support of the scientific infrastructure of the National Center for Medical Genomics (LM2015091) and its project aimed at creating a reference database of genetic variants of the Czech Republic (CZ.02.1.01/0.0/0.0/16_013/0001634). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 2. 4. 2019 Accepted: 14. 5. 2019.

Contribution of Massive Parallel Sequencing to Diagnosis of Hereditary Ovarian Cancer in the Czech Republic.

BACKGROUND: Ovarian cancer is a disease with high mortality. Approximately 1,000 women are diagnosed with ovarian cancer in the Czech Republic annually. Women harboring a mutation in cancer-predisposing genes face an increased risk of tumor development. Mutations in BRCA1, BRCA2, BRIP1, and Lynch syndrome genes (RAD51C, RAD51D, and STK11) are associated with a high risk of ovarian cancer, and mutations in ATM, CHEK2, NBN, PALB2, and BARD1 appear to increase the risk. Our aim was to examine the frequency of mutations in cancer-predisposing genes in the Czech Republic. MATERIALS AND METHODS: We analyzed 1,057 individuals including ovarian cancer patients and 617 non-cancer controls using CZECANCA panel next-generation sequencing on the Illumina platform. Pathogenic mutations in high-risk genes, including CNVs, were detected in 30.6% of patients. The mutation frequency reached 25.0% and 18.2% in subgroups of unselected ovarian cancer patients and patients with a negative family cancer history, respectively. The most frequently mutated genes were BRCA1 and BRCA2. The overall frequency of mutations in non-BRCA genes was comparable to that in BRCA2. The mutation frequency in ovarian cancer patients aged >70 years was three times higher than that in patients diagnosed before the age of 30. CONCLUSION: Ovarian cancer is a heterogeneous disease with a high proportion of hereditary cases. The lack of efficient screening for early diagnosis emphasizes the importance of identifying carriers of mutations in ovarian cancer-predisposing genes; this is because proper follow-up and prevention strategies can reduce overall ovarian cancer-related mortality. This work was supported by grants AZV 15-27695A, SVV2019/260367, PROGRES Q28/LF1. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 7. 3. 2019 Accepted: 24. 4. 2019.

Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian cancer.

Germline mutations in checkpoint kinase 2 (CHEK2), a multiple cancer-predisposing gene, increase breast cancer (BC) risk; however, risk estimates differ substantially in published studies. We analyzed germline CHEK2 variants in 1,928 high-risk Czech breast/ovarian cancer (BC/OC) patients and 3,360 population-matched controls (PMCs). For a functional classification of VUS, we developed a complementation assay in human nontransformed RPE1-CHEK2-knockout cells quantifying CHK2-specific phosphorylation of endogenous protein KAP1. We identified 10 truncations in 46 (2.39%) patients and in 11 (0.33%) PMC (p = 1.1 × 10-14 ). Two types of large intragenic rearrangements (LGR) were found in 20/46 mutation carriers. Truncations significantly increased unilateral BC risk (OR = 7.94; 95%CI 3.90-17.47; p = 1.1 × 10-14 ) and were more frequent in patients with bilateral BC (4/149; 2.68%; p = 0.003), double primary BC/OC (3/79; 3.80%; p = 0.004), male BC (3/48; 6.25%; p = 8.6 × 10-4 ), but not with OC (3/354; 0.85%; p = 0.14). Additionally, we found 26 missense VUS in 88 (4.56%) patients and 131 (3.90%) PMC (p = 0.22). Using our functional assay, 11 variants identified in 15 (0.78%) patients and 6 (0.18%) PMC were scored deleterious (p = 0.002). Frequencies of functionally intermediate and neutral variants did not differ between patients and PMC. Functionally deleterious CHEK2 missense variants significantly increased BC risk (OR = 3.90; 95%CI 1.24-13.35; p = 0.009) and marginally OC risk (OR = 4.77; 95%CI 0.77-22.47; p = 0.047); however, carriers low frequency will require evaluation in larger studies. Our study highlights importance of LGR detection for CHEK2 analysis, careful consideration of ethnicity in both cases and controls for risk estimates, and demonstrates promising potential of newly developed human nontransformed cell line assay for functional CHEK2 VUS classification.

The PALB2 gene is a strong candidate for clinical testing in BRCA1- and BRCA2-negative hereditary breast cancer.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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%). IMPACT: 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.