Genetic polymorphism of the human cytochrome CYP2A13 in a French population: implication in lung cancer susceptibility

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Abstract

The human cytochrome CYP2A13, which is mainly expressed in the respiratory tract, has been shown to be highly efficient in vitro in the metabolism of tobacco-smoke carcinogens and procarcinogens such as 4-methylnitroso-1-(3-pyridyl)-1-butanone (NNK). In order to investigate the extent of CYP2A13 genetic polymorphism in a French Caucasian population of 102 individuals, a screening for sequence variations in the 5-untranslated and protein encoding regions of its gene was performed using a polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) strategy. Six polymorphisms in the coding region were identified, including two rare missense mutations (C474G or Asp158Glu, G967T or Val323Leu) and one nonsense mutation (Arg101Stop). This deleterious mutation, the most frequent (5%) in our population, presumably encodes a severely truncated protein. The influence of the nonsense mutation in lung cancer susceptibility was examined by PCR-SSCP using peripheral blood DNA from 204 cases of lung cancer and 201 controls. The CYP2A13*7 allele, which harbours the C301T mutation, was present in 2.0% of controls and 3.4% of cases. However, multivariate analysis showed an elevated risk for small cell lung cancer in subjects heterozygous for the null allele (odds ratio OR=9.9; 95% confidence interval CI=1.9–52.2). This increased risk was not linked to other histological types of lung cancer.

Section snippets

Materials and methods

Genomic DNA samples. A group of 102 unrelated French Caucasian subjects has been involved in the study after Ethical Committee approval and informed consents have been obtained. Among the 102 subjects, 76 were healthy volunteers and 26 were patients with various bowel diseases. Nucleic acid extraction from peripheral blood leucocytes was performed using the Nucleon BACC3 kit (Amersham–Pharmacia Biotech, Saclay, France), according to manufacturer's instructions.

Additionally, genotyping of the

Sequence analysis of CYP2A13 in a French population

To investigate the extent of CYP2A13 gene polymorphism in a large French Caucasian population, a PCR-SSCP strategy was applied to genomic DNAs of 102 unrelated volunteers. One DNA sample homozygote for a wild-type allele of CYP2A13, as confirmed by sequencing, was used as a reference for each SSCP analysis. Single strands were well separated and easily discriminated for each of the CYP2A13 regions studied, as illustrated in Fig. 1 for exons 2, 3, 4, 6, and 9.

For 80 samples, electrophoretic

Discussion

In order to carry out a screening for sequence variations in the 5-flanking and coding regions of the CYP2A13 gene, a PCR-SSCP strategy was applied to 102 unrelated French individuals of Caucasian origin [17]. In the past decade, this method has been successfully used for the detection of mutations in several human cytochrome P450 genes [18]. After optimization of several parameters affecting the single-strand separation, such as temperature for migration or acrylamide concentration, we

Acknowledgements

This study was supported by the Centre Hospitalier Régional et Universitaire de Lille, France, the Fondation pour la Recherche Médicale, France, and by the Génopole de Lille, Région Nord-Pas de Calais (FEDER), France.

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