Analysis of GSTP1-1 polymorphism using real-time polymerase chain reaction

Background: Glutathione transferases (GSTs) play an important role in the protection of cells from the products of oxidative stress as well as from several environmental carcinogens. The GSTP1-1 gene class is significantly overexpressed in many human tumors. Four allelic variants have been described for the GSTP1-1 gene (*A, *B, *C, *D) leading to different amino acid substitutions in position 105 and 114 of the protein sequence. The proteins encoded by the different alleles show different abilities to metabolize carcinogens and anticancer agents, suggesting an association between GSTP1 polymorphism and the risk for a variety of cancers as well as between said polymorphism and varying responses to cancer treatments. Methods: The GSTP1-1 polymorphism was determined using a real-time polymerase chain reaction (PCR) and fluorescence resonance energy transfer with a Light-Cycler Instrument. ARMS was used in the case of *A/*C or *B/*D heterozygosity. We used this method to determine the GSTP1-1 polymorphism in 250 free-living Italian subjects of both sexes. Results: Among 250 subjects representative of an Italian population, we observed the following allelic frequencies: f(A) = 0.710, J(B) = 0.236, f(C) = 0.054 and f(D) = 0. The observed phenotypes are in Hardy-Weinberg equilibrium chi(2) = 0.71, df = 4, P = 0.95). Conclusions: We have extended and improved a method of GSTP1-1 complete genotyping. This method provides the ability to genotype 30 samples in 2 h and it represents a fast, reliable and automated methodology to determine GSTP1-1 polymorphism in order to perform large-scale population studies. (C) 2003 Elsevier Science B.V. All rights reserved.