Analysis of p16INK4A gene promoter in male germ-cell tumors: identification of a new point mutation

Human male germ-cell tumors of seminoma type display aberrant expression of INK4-family inhibitors of the cell cycle including CDKN2-encoded p16INK4A. The mechanisms underlying the altered p16INK4A expression are not fully understood. Indeed, neither genetic/epigenetic alterations in CDKN2 coding sequence nor its promoter hypermethylation could explain all anomalies. To assess whether the aberrant p16INK4A expression could be related to the alterations in CDKN2 regulatory sequence, we screened seminoma DNAs from 19 patients for the promoter mutations. Combined polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and automated DNA-sequencing approaches indicated an adenine insertion at the position-1973 (relative to the ATG codon at + 1) of CDKN2 promoter in one particular patient. The immunohistochemical analysis pointed to the correlation between the observed promoter mutation and the loss of p16INK4A protein expression. These data suggest that in addition to previously characterized anomalies, the identified CDKN2 promoter mutation may be relevant for altered p16INK4A protein expressions in at least some seminoma.