A Method For Detecting CYP2A6 Gene Copy Number Based On Fluorescence Quantitative PCR And Its Analytical Study In Four Nationalities

Cytochromes P450 (CYPs), refer to a superfamily of heme-containing monooxygenases. CYP2A6 is one of the major hepatic members of this superfamily in human, which catalyzes the biotransformation of xenobiotic compounds and toxins. The CYP2A6*4 allele, characterized as the whole deletion of this gene, is closely associated with nicotine dependence, cancer susceptibility, and drug responsiveness.Given its relation with nicotine metabolism and antineoplastic drugs, the determin ation of the CYP2A6 gene deletion is gaining increasing attention in clinical practice. Several methods have been developed for this purpose, including direct sequencing, ne sted-PCR applications followed by gel electrophoresis, and PCR-RFLP. Although thes e methods have been proven to be feasible and cost-effective, they are time-consuming and technically demanding. Therefore, we designed and validated a sensitive and spec ific assay for genotyping CYP2A6 gene deletion that was based on gene copy number determination by quantitative real-time PCR. The PCR assay specifically amplifies CY P2A6 by designing a specific set of primers and the probe, which effectively prevent the amplification of other alleles. CYP2A6 gene copy numbers were normalized to alb umin(ALB) which was co-amplified simultaneously in a single-tube duplex reaction an d at a setting as the internal reference gene.Although frequency of CYP2A6*4 has been reported in Chinese Han population, it was largely unknown in other Chinese ethnic population. In this study, we investigated the allele frequency of CYP2A6*4 in four main ethnic groups of China including Han, Uighur, Bouyei and Tiben, based on our newly developed quantitative real-time PCR assay. The frequency of the CYP2A6*4 allele were 7.9%,15%,0% and 2% in Han (N=120), Uighur (N=100), Bouyei (N=100) and Tibetan (N=100) (P<0.0001) respectively. This work greatly expanded our understanding of the distribution of CYP2A6*4 in Chinese population and provided more information to different ethnic population’s smoking behavior and also in disease susceptibility and drug response.