Characterization of germline CHEK2 mutations in African-American women at high risk for hereditary breast cancer

CHEK2 is a protein kinase involved in cell-cycle checkpoint control. The proteintruncating CHEK21100delC mutation abolishes the protein kinase activity of CHEK2 and increases the risk of breast cancer. Germline mutations/variations in CHEK2 were studied in African Americans at high risk for breast cancer (n = 262). High risk criteria for hereditary breast cancer included a family history of breast and/or ovarian cancer, bilateral breast cancer, or early-onset (age ≤ 40 years) breast cancer with BRCA1 and BRCA2 carriers excluded. For the first time, the entire CHEK2 coding and intron flanking regions were examined in high risk African Americans using denaturing high performance liquid chromatography and DNA sequencing. Two novel silent mutations coding for the same amino acid were detected: 660C→T (Tyr220), and 1497G→C (Leu499). Exonic splicing enhancer analysis revealed that the Leu499 mutation may negatively affect splicing; further studies are needed to support this possibility. Two previously described mutations that do not affect function, 254C→T (Pro85Leu) and 252A→G (Glu84Glu), were also detected as well as two novel intronic mutations. Multiplex ligation-dependent probe amplification did not identify any large genomic rearrangements in CHEK2. Most significantly, these experiments detected two pathogenic mutations: Arg180Cys and 1139delTC. Mutation Arg180Cys is not located in a functional domain but may affect protein folding and function as the mutation was deemed intolerant by bioinformatic analysis that compares homologous sequences in different organisms. The 1139delTC variation, located within the important kinase domain, has not previously been reported and may constitute a founder CHEK2 mutation in African Americans. The wild type human CHEK2 gene has been demonstrated to complement the deleted yeast Rad53 gene, a homologue to human CHEK2, and allowed evaluation of the effect of potentially pathogenic mutations. Site-directed mutagenesis of the human wild type gene was used to generate plasmids with the 1100delC, 1139delTC and Arg180Cys mutations. Functional analysis by yeast complementation revealed that the 1139delTC and Arg180Cys mutations are pathogenic because they are detrimental to the function of the protein. Screening for CHEK2 Arg180Cys and 1139delTC in the high risk African-American population may prove useful in the clinical management of breast cancer.