Abstract
BackgroundIdentification of CYP2A6 alleles associated with reduced enzyme activity is important in the study of inter-individual differences in drug metabolism. CYP2A6*12 is a hybrid allele that results from unequal crossover between CYP2A6 and CYP2A7 genes. The 5' regulatory region and exons 1–2 are derived from CYP2A7, and exons 3–9 are derived from CYP2A6. Conventional methods for detection of CYP2A6*12 consist of two-step PCR protocols that are laborious and unsuitable for high-throughput genotyping. We developed a rapid and accurate method to detect the CYP2A6*12 allele by Pyrosequencing technology.MethodsA single set of PCR primers was designed to specifically amplify both the CYP2A6*1 wild-type allele and the CYP2A6*12 hybrid allele. An internal Pyrosequencing primer was used to generate allele-specific sequence information, which detected homozygous wild-type, heterozygous hybrid, and homozygous hybrid alleles. We first validated the assay on 104 DNA samples that were also genotyped by conventional two-step PCR and by cycle sequencing. CYP2A6*12 allele frequencies were then determined using the Pyrosequencing assay on 181 multi-ethnic DNA samples from subjects of African American, European Caucasian, Pacific Rim, and Hispanic descent. Finally, we streamlined the Pyrosequencing assay by integrating liquid handling robotics into the workflow.ResultsPyrosequencing results demonstrated 100% concordance with conventional two-step PCR and cycle sequencing methods. Allele frequency data showed slightly higher prevalence of the CYP2A6*12 allele in European Caucasians and Hispanics.ConclusionThis Pyrosequencing assay proved to be a simple, rapid, and accurate alternative to conventional methods, which can be easily adapted to the needs of higher-throughput studies.
Highlights
Identification of CYP2A6 alleles associated with reduced enzyme activity is important in the study of inter-individual differences in drug metabolism
Since the initial informed consent for use of participant blood for research did not include genetics, the Centers for Disease Control and Prevention (CDC)/National Center of Health Statistics (NCHS) Ethics Review Board approved a revised plan in 2001 which allows for the linkage of genetic results to NHANES data through the NCHS Research Data Center to ensure that confidentiality of study participant's identity is maintained [12]
Pyrosequencing assay genotype results were 100% concordant with those obtained by conventional two-step PCR and cycle sequencing for the 90 Polymorphism Discovery Resource (PDR) and 14 Centre d'Etude du Polymorphisme Humain (CEPH) family members
Summary
Identification of CYP2A6 alleles associated with reduced enzyme activity is important in the study of inter-individual differences in drug metabolism. We developed a rapid and accurate method to detect the CYP2A6*12 allele by Pyrosequencing technology. CYP2A6 is a member of this superfamily. It was first identified as the human coumarin 7-hydroxylase enzyme [2]. CYP2A6 is the primary enzyme responsible for the metabolism of nicotine to cotinine. BMC Medical Genetics 2009, 10:80 http://www.biomedcentral.com/1471-2350/10/80 and inter-ethnic differences in enzyme activity are thought to be attributable to genetics as well as to environmental factors [3]. Individuals lacking a functional CYP2A6 gene have impaired nicotine metabolism and are less prone to nicotine addiction and possibly tobacco-related cancers [4]
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