Persistent p53 mutations in single cells from normal human skin

Epidermal clones of p53-mutated keratinocytes are abundant in chronically s un-exposed skin and may play an important role in early development of skin cancer. Advanced laser capture microdissection enables genetic analysis of targeted cells from tissue sections without contamination from neighboring cells. In this study P53 gene mutations were characterized in single cells from normal, chronically sun-exposed skin. Biopsies were obtained from ski n subjected to daily summer sun and skin totally protected from the sun by blue denim fabric. Using laser capture microdissection, 172 single-cell sam ples were retrieved from four biopsies and analyzed using single-cell polym erase chain reaction and direct DNA sequencing. A total of 14 different mut ations were identified in 26 of 99 keratinocytes from which the P53 gene co uld be amplified. Mutations displayed a typical UV signature and were detec ted in both scattered keratinocytes and in a small cluster of p53-immunorea ctive keratinocytes. This minute epidermal P53 clone had a diameter of 10 t o 15 basal cells. Two missense mutations were found in all layers of epider mis within the P53 clone. The presented data show that p53 mutations are co mmon in normal skin and that a clone of keratinocytes with a mutated p53 ge ne prevailed despite 2 months of total protection from ultraviolet light.