Use of Duplex PCR-CTPP Methods for CYP2E1 RsaI/IL-2 T-330G and IL-1B C-31T/TNF-A T-1031C Polymorphisms

Abstract

Two duplex polymerase chain reaction (PCR) with confronting two-pair primer (PCR-CTPP) methods were designed for cytochrome P450 (CYP) 2E1 RsaI and interleukin (IL-2) T-330G, and for IL-1B C-31T and tumor necrosis factor-alpha (TNF-A) T-1031C. The four polymorphisms are considered to be functional, and the three cytokines reportedly inhibit CYP2E1 expression. Many studies have reported associations between the above polymorphisms and risk of diseases including cancers and inflammatory diseases. The main objective of this study was to examine the applicability of the established PCR conditions to a real situation. Participants were female examinees aged from 35 to 85 years who attended health checks run by a local government in Japan. The allele frequencies among 325 female health check examinees were 0.804 for CYP2E1 c1 allele, 0.668 for IL-2-330T allele, 0.554 for IL-1B-31T allele, and 0.822 for TNF-A-1031T allele. p-Values from a Hardy-Weinberg equilibrium test were 0.658, 0.955, 0.062, and 0.806, respectively. Clear DNA bands observed with electrophoresis allowed us to genotype the four polymorphisms. The genotype frequencies were within the Hardy-Weinberg equilibrium test proportions, though the p-value for IL-1B C-31T was marginal. Both duplex PCR-CTPP methods may be useful tools for studies on the association between these polymorphisms and disease risk.