Quantitative detection of ultraviolet-specific p53 mutations in normal skin from Japanese patients

We have previously developed sensitive methods to detect UV-specific p53 mutations (CC to TT tandem mutations) and have reported that such mutations could be found in the normal skin cell populations of sun-exposed body sites, but not in those of covered sites, in Australian cancer patients. We have now further refined our allele-specific PCR method for detecting CC to TT mutations at codons 247/248 of the p53 gene to allow quantitative measurements. Using DNA containing this mutation from a tumor as a standard for calibration and 5 μg of genomic DNA/PCR reaction, we could detect 1 mutant allele in about 10⁶ wild-type alleles. It is essential to use purified primers and 64°C as the annealing temperature for PCR. Our method has been applied in a study of the correlation of sun exposure and accumulation of CC to TT mutations in normal skin biopsies from Japanese patients. There were more p53 mutations in samples taken from sites that were chronically exposed to the sun than in those from covered sites. A significant trend of increased p53 mutation frequency with increase in age of subjects was found, suggesting the cumulative nature of the mutation. On the other hand, the p53 mutation frequency was higher in patients with premalignant tumors or nonmelanocytic skin cancer than in patients with only benign tumors. These results confirm the utility of PCR-based p53 gene mutation assays for the measurement of exposure to UV as well as for predicting the risk of UV-associated skin cancer.