Elsevier

Toxicology

Volume 168, Issue 3, 30 November 2001, Pages 259-268
Toxicology

Comparison of cytochrome P450 2A6 polymorphism frequencies in Caucasians and African-Americans using a new one-step PCR-RFLP genotyping method

https://doi.org/10.1016/S0300-483X(01)00470-XGet rights and content

Abstract

CYP2A6 (cytochrome P450 2A6), which was first identified as the human coumarin 7-hydroxylase, is the most important enzyme in nicotine C-oxidation. The enzyme also metabolically activates the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in vitro. Polymorphisms in the CYP2A6 gene may thus impact on both smoking behavior and lung cancer susceptibility. Several different genotyping methods have been reported with conflicting results in the frequencies of CYP2A6 polymorphic variants. Thus we decided to perform a sequence analysis of the entire CYP2A6 gene. Sequencing confirmed the published CYP2A6 cDNA sequence. However, intron sequences differed considerably from the reported sequence of the CYP2A6*3 (v2) variant. Our analyses revealed that parts of introns shared homologies with the published sequence of CYP2A13. Based on our sequence data we developed a one step protocol for specific amplification of exon 3 of CYP2A6. The resulting PCR product can be used directly for restriction endonuclease digestion with XcmI and DdeI to determine the frequencies of the reported variant alleles CYP2A6*2 and CYP2A6*3. In a population of 305 African-Americans and 145 Caucasians, we found allele frequencies of 0.003 (2/610) for CYP2A6*2 and 0 (0/610) for CYP2A6*3 in African-Americans and allele frequencies of 0.014 (4/290) and 0 (0/290) in Caucasians. We conclude that both alleles are considerably less frequent in populations than previously reported.

Introduction

Members of the cytochrome P450 (CYP) family are involved in the phase I metabolism of a wide range of xenobiotic substances. These bioactivation processes lead to the generation of reactive molecules that are inactivated by phase II enzymes such as glutathion-S-transferases (GST) and can be efficiently excreted due to increased water solubility. However, in the case of procarcinogens it is also possible that mutagenic substances arise through these oxidative mechanisms (Smith et al., 1995). Since many genes encoding xenobiotic metabolizing enzymes are polymorphic in human populations, interindividual differences in metabolic activation or detoxification have become a focus of molecular epidemiologic studies analyzing associations of gene polymorphisms and cancer risk (Rannug et al., 1995). The CYP2A gene cluster is located on the long arm of chromosome 19 and harbors three complete genes (CYP2A6, CYP2A7, CYP2A13) and two truncated pseudogenes (CYP2A7PT and CYP2A7PC) (Fernandez-Salguero et al., 1995). The human cytochrome P450 2A6 (CYP2A6) was first identified as the coumarin-7-hydroxylase (Yamano et al., 1990). It is also responsible for the metabolism of different exogenous compounds including aflatoxin B1, nitrosamines and other xenobiotic substrates (Lewis et al., 1999). In addition, CYP2A6 catalyzes nicotine C-oxidation to cotinine (Nakajima et al., 1996, Messina et al., 1997), which constitutes about 80% of nicotine clearance in most individuals (Benowitz et al., 1994), and the subsequent hydroxylation of cotinine to 3-OH-cotinine (Nakajima et al., 1997). Considerable interindividual differences in CYP2A6 expression levels in human liver were reported (Yun et al., 1991, Nakajima et al., 1996), and several genetic polymorphisms including point mutations and deletions are known (Fernandez-Salguero et al., 1995, Yamano et al., 1990, Oscarson et al., 1999a, Oscarson et al., 1999b, Yokoi and Kamataki, 1998; Nunoya et al., 1998, Nunoya et al., 1999).

Pianezza et al. (1998) analyzed the impact of two polymorphic CYP2A6 variants on human smoking behavior and found that smokers carrying the alleles CYP2A6*2 (v1) or CYP2A6*3 (v2) tend to smoke significantly fewer cigarettes than wild type allele carriers. Additionally, they presented results showing that CYP2A6*2 or CYP2A6*3 allele carriers seem to be protected against becoming smokers.

The genotyping method by Fernandez-Salguero et al. (1995) used in this study is based on a nested polymerase chain reaction (PCR) approach amplifying the entire CYP2A6 gene in the first step and exon 3 in the second step. The restriction endonucleases XcmI and DdeI are used to detect the CYP2A6*2 or CYP2A6*3 polymorphisms located in exon 3 (restriction fragment length polymorphism (RFLP) analysis, Fig. 2a). Since this method has been shown to give unclear results with respect to the coumarin 7-hydroxylation phenotype, an alternative nested allele specific PCR approach for the detection of the CYP2A6*2 variant was developed (Oscarson et al., 1998). Recently, three alternative single-step PCR-RFLP genotyping methods were published (Chen et al., 1999, Kitagawa et al., 1999, Sabol and Hamer, 1999). We cloned and sequenced the CYP2A6 gene to definitively assess the frequencies of the reported polymorphisms. Our sequence analysis revealed considerable discrepancies with respect to intron sequences of the published CYP2A6*3 sequence. Based on these data we developed a novel simple and specific genotyping method for both known polymorphisms in exon 3 (CYP2A6*2 and CYP2A6*3). Allele frequencies were determined in African-Americans and Caucasians.

Section snippets

Study populations and DNA extraction

African-Americans presenting to a community-based primary care clinic in Durham, North Carolina, USA were assessed for their smoking status. Individuals who had smoked 100 or more cigarettes in their lifetime were classified as smokers. Active smokers were approached and asked to donate peripheral venous blood after informed consent was obtained (N=305). Active smokers were those that answered to the question “Do you currently smoke” with ‘Yes’. The blood was immediately stored at 4 °C, and DNA

Results

The CYP2A6 gene was amplified by PCR with the primer pair F4SalI/R4SalI according to the first amplification protocol of the genotyping method published by Fernandez-Salguero et al. (1995). The resulting PCR product was about 7 kB in size. To avoid sequencing of related genes, we cloned the 5′-fragment of the PCR product and sequenced the resulting plasmid pTP37 (Fig. 1a).

With the exception of one nucleotide (silent point mutation in exon 1) sequences of exons 1–3 were identical with the

Discussion

The CYP2A6 gene is crucially involved in the metabolic inactivation of nicotine and was shown in in vitro experiments to activate the procarcinogen NNK. Polymorphisms that affect its enzymatic activity have been described in exon 3. As a result, this gene has been the focus of several molecular epidemiologic investigations of associations between these polymorphisms and smoking behavior as well as lung cancer risk.

When comparing the results of various genotyping studies, it is noticeable that

Acknowledgements

This work was partially supported by grants from the National Cancer Institute (P01-CA-72099 and R01-CA-70317) and from the Roswell Park Alliance Foundation.

References (40)

  • G.-F. Chen et al.

    Low frequency of CYP2A6 gene polymorphism as revealed by a one-step polymerase chain reaction method

    Pharmacogenetics

    (1999)
  • P. Fernandez-Salguero et al.

    A genetic polymorphism in coumarin 7-hydroxylation: sequence of the human CYP2A genes and identification of variant CYP2A6 alleles

    Am. J. Hum. Genet.

    (1995)
  • D.F. Gu et al.

    The use of long PCR to confirm three common alleles at the CYP2A6 locus and the relationship between genotype and smoking habit

    Ann. Hum. Genet.

    (2000)
  • Gullstén, H., Oscarson, M., McLellan, R.A., Ingelman-Sundberg, M., Anttila, S., Rautio, A., Pelkonen, O., Raunio, H.,...
  • K. Inoue et al.

    CYP2A6 genetic polymorphisms and liver microsomal coumarin and nicotine oxidation activities in Japanese and Caucasians

    Arch. Toxicol.

    (2000)
  • M.-A. Loriot et al.

    Genetic polymorphisms of cytochrome P450 2A6 in a case control study on lung cancer in a French population

    Pharmacogenetics

    (2001)
  • E.S. Messina et al.

    A major role for CYP2A6 in nicotine C-oxidation by human liver microsomes

    J. Pharmacol. Exp. Ther.

    (1997)
  • N. Nakajima et al.

    Role of human cytochrome P4502A6 in C-oxidation of nicotine

    Drug Metab. Dispos.

    (1996)
  • N. Nakajima et al.

    Characterization of CYP2A6 involved in 3′-hydroxylation of cotinine in human liver microsomes

    J. Pharmacol. Exp. Ther.

    (1997)
  • M. Nakajima et al.

    Deficient cotinine formation from nicotine is attributed to the whole deletion of the CYP2A6 gene in humans

    Clin. Pharmacol. Ther.

    (2000)
  • Cited by (30)

    • Polymorphism of selected enzymes involved in detoxification and biotransformation in relation to lung cancer

      2007, Lung Cancer
      Citation Excerpt :

      Recently, several additional variant alleles have been found, including CYP2A6*4A and CYP2A6*4D deletion alleles and CYP2A6*5 allele with a Gly479Val substitution [75–77] Several additional variant alleles have been found, for a shortened list of which, refer to Table 3. Nowadays, it seems evident, that variant alleles are much more frequent in Asian populations than in Caucasian or African populations, although some discrepancies due to diversity of the genetic methods used by various research still persist [78,79]. However, some variant alleles might still be found in one population but not in the other while for the others the trend might be completely different [80–82].

    • A safety assessment of coumarin taking into account species-specificity of toxicokinetics

      2006, Food and Chemical Toxicology
      Citation Excerpt :

      The most common polymorphisms are the CYP2A6*2 variant that encodes an inactive enzyme, and the CYP2A6*4A variant that represents a gene deletion (Fernandez-Salguero et al., 1995; Hadidi et al., 1997, 1998; Oscarson, 2001; Raunio et al., 2001; Yamano et al., 1990). In Caucasians, variant allelic frequencies represent less than 2% of the population (Chen et al., 1999; Paschke et al., 2001), whereas 15–20% of the Asian population has the CYP2A6*4 gene deletion (Oscarson et al., 1999). Burian et al. (2003) determined the CYP2A6 genotype of 231 patients who were dosed with 90 mg of coumarin/day in order to determine whether susceptibility to coumarin-mediated hepatotoxicity is due to a polymorphism.

    • CYP2A6 genetic variation and potential consequences

      2002, Advanced Drug Delivery Reviews
    View all citing articles on Scopus
    View full text