Mutations in CHEK2 associated with prostate cancer risk

Abstract

The DNA-damage-signaling pathway has been implicated in all human cancers. However, the genetic defects and the mechanisms of this pathway in prostate carcinogenesis remain poorly understood. In this study, we analyzed CHEK2, the upstream regulator of p53 in the DNA-damage-signaling pathway, in several groups of patients with prostate cancer. A total of 28 (4.8%) germline CHEK2 mutations (16 of which were unique) were found among 578 patients. Additional screening for CHEK2 mutations in 149 families with familial prostate cancer revealed 11 mutations (5 unique) in nine families. These mutations included two frameshift and three missense mutations. Importantly, 16 of 18 unique CHEK2 mutations identified in both sporadic and familial cases were not detected among 423 unaffected men, suggesting a pathological effect of CHEK2 mutations in prostate cancer development. Analyses of the two frameshift mutations in Epstein Barr virus-transformed cell lines, using reverse-transcriptase polymerase chain reaction and western blot analysis, revealed abnormal splicing for one mutation and dramatic reduction of CHEK2 protein levels in both cases. Overall, our data suggest that mutations in CHEK2 may contribute to prostate cancer risk and that the DNA-damage-signaling pathway may play an important role in the development of prostate cancer.

Figure 1

CHEK2 germline mutations in prostate cancers. A, Mutations found in the CHEK2 gene. “T” indicates the clinic or sporadic prostate tumor samples (numbers as shown in table 1), and “F” indicates families with prostate cancer in which CHEK2 mutations were identified (numbers as indicated in fig. 2). B, Sequence analysis shows the five CHEK2 germline mutations identified in families with familial prostate cancer. DNA sequence analyses were performed on either genomic DNA (first four pairs of panels) or cDNA (right-most panels). Sequences are presented in the 5′→3′ direction, and arrows mark the location of each mutation. The upper panels depict the regions from wild-type alleles and the lower panels show the respective sequences with the mutations. All mutations were detected with genomic DNA and were confirmed with cDNA.

Figure 2

Segregation of CHEK2 mutations in nine families with prostate cancer (families 1–9). Where known, the individual’s age is indicated to the right side of each cancer. A dot (●) is present at the lower right corner of the symbol if a blood sample was available and was analyzed. An asterisk (*) to the right of the symbol indicates the presence of the CHEK2 mutation carriers in each family. The individual indicated with a dot but without an asterisk has tested negative for CHEK2 mutation. Arrows (↗) indicate probands. Squares denote males; circles denote females; completely blackened symbols denote patients with prostate cancer for whom pathology records were available; 3/4 blackened symbols denote patients with prostate cancer for whom records were unavailable; 1/2 blackened symbols denote patients with other types of cancer; all symbols with a diagonal denote deceased individuals. The cancer type for each individual is shown underneath each symbol.

Figure 3

Abnormal splicing and abnormal protein syntheses of the two CHEK2 frameshift mutations. A, Schematic representation of the abnormal splice for the IVS2+1G→A mutant. A 4-bp insertion is created in the mutant transcript because of the usage of the new splice donor site (underlined). B, Sequences of the wild-type (left) and mutant (right) CHEK2 transcripts (between exons 2 and 3) from the cell line established from the affected men carrying the IVS2+1G→A germline mutation. C, Western blot analyses showing the reduction of CHEK2 in the cell lines carrying the frameshift mutations, compared with the normal lymphocyte cells and the cells carrying CHEK2 missense mutations.