Novel pathogenic CHEK2 mutations in African Americans with breast cancer: Results of a Breast Cancer Family Registry study.

A13 CHEK2, encoded on chromosome 22q, is a protein kinase that is involved in cell-cycle checkpoint control by causing arrest in G1 and G2 after DNA damage. The protein-truncating CHEK21100delC mutation abolishes the protein kinase activity of CHEK2 and increases the risk of breast cancer. Germline mutations/variations in CHEK2 were studied in high-risk African Americans from the Northern California site of the Breast Cancer Family Registry (n = 198) and Howard University Hospital (n = 62). 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 or less) breast cancer. BRCA1 and BRCA2 carriers were excluded. The entire CHEK2 coding and intron flanking regions were examined using denaturing high performance liquid chromatography and putative mutations/variations were delineated by DNA sequencing. These experiments revealed two novel mutations: R180C and 1135delTC. Mutation R180C is not located in a functional domain but may affect protein folding and function as the mutation was deemed intolerant by the bioinformatic analysis tool Sort Intolerant From Tolerant (SIFT) that compares homologous sequences in different organisms. Mutation 1135delTC is protein truncating and located in the important kinase domain of CHEK2 in close proximity to the 1100delC mutation; therefore, 1135delTC is expected to be pathogenic. The yeast Rad53 gene is homologous to the human CHEK2 gene. The wild type human CHEK2 gene has been demonstrated to complement the deleted yeast Rad53 gene. Therefore, yeast complementation allows 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, 1135delTC and R180C mutations. Introduction of the human wild type CHEK2 gene into the Rad53 deleted yeast resulted in growth in selective media; whereas introduction of the latter three mutations yielded no growth in selective media and growth in control non-selective media. We conclude that the 1135delTC and R180C mutations are pathogenic because they are detrimental to the function of the protein. These findings are significant because 1135delTC is the only other protein-truncating mutation discovered to date in this gene. Screening for CHEK2 variations in the African-American population should include the pathogenic R180C and 1135delTC mutations.