No abstract

Cytochrome P450 2A6 is an important human hepatic P450 which activates pre-carcinogens, oxidises some drugs and constitutes the major nicotine C-oxidase. In fact, results have been presented in the literature which suggested a relationship between the distribution of defective CYP2A6 alleles and smoking behaviour as well as cigarette consumption. In the present report, we describe the structure of a novel CYP2A locus where the whole CYP2A6 gene has been deleted, resulting in an abolished cytochrome P450 2A6-dependent metabolism. The origin of this locus is apparently due to an unequal crossover event between the 3P-flanking region of the CYP2A6 and CYP2A7 genes. A rapid PCR-based method for the detection of the CYP2A6del allele was developed and the allele frequency was 15.1% among 96 Chinese subjects, but only 1.0% in Finns (n = 100) and 0.5% in Spaniards (n = 100). In the Chinese population, we did not detect any CYP2A6*2 alleles using an improved genotyping procedure, in contrast to the 11^20% previously reported. It is concluded that genotyping for the CYP2A6del allele is of great importance in studies correlating, for example, smoking behaviour, pre-carcinogen activation or drug metabolism to the CYP2A6 genotype, in particular when oriental populations are investigated.z 1999 Federation of European Biochemical Societies.

The polymorphic human cytochrome P450 2A6 (CYP2A6) metabolises a number of drugs, activates a variety of precarcinogens and constitutes the major nicotine C-oxidase. A relationship between CYP2A6 genotype and smoking habits, as well as incidence of lung cancer, has been proposed. Two defective alleles have hitherto been identified, one of which is very common in Asian populations. Among Caucasians, an additional defective and frequently distributed allele (CYP2A6*3) has been suggested to play a protective role against nicotine addiction and cigarette consumption. Here, we have re-evaluated the genotyping method used for the CYP2A6*3 allele and found that a gene conversion in the 3P P flanking region of 30^40% of CYP2A6*1 alleles results in genotype misclassification. In fact, no true CYP2A6*3 alleles were found among 100 Spaniards and 96 Chinese subjects. In one Spanish poor metaboliser of the CYP2A6 probe drug coumarin, we found two novel defective alleles. One, CYP2A6*5, encoded an unstable enzyme having a G479L substitution and the other was found to carry a novel type of CYP2A6 gene deletion (CYP2A6*4D). The results imply the presence of numerous defective as well as active CYP2A6 alleles as a consequence of CYP2A6/CYP2A7 gene conversion events. We conclude that molecular epidemiological studies concerning CYP2A6 require validated genotyping methods for accurate detection of all known defective CYP2A6 alleles.z 1999 Federation of European Biochemical Societies.

No abstract

The human CYP2A6 enzyme metabolizes certain drugs and pre-carcinogens and appears to be the most important enzyme for nicotine metabolism. At present, more than 10 different allelic variants are known that cause abolished or decreased enzyme activity. Genetic polymorphism in this gene might be of particular importance for an individual's need for nicotine and for susceptibility to lung and/or liver cancer. We have identified a new CYP2A6 allele (CYP2A6*12) which carries an unequal crossover between the CYP2A6 and CYP2A7 genes in intron 2. This results in a hybrid allele where the 5' regulatory region and exons 1-2 are of CYP2A7 origin and exons 3-9 are of CYP2A6 origin, resulting in 10 amino acid substitutions compared to the CYP2A6(*)1 allele. Phenotyping with the CYP2A6 substrate coumarin indicates that it causes reduced CYP2A6 activity in'vivo. Furthermore, when expressed in mammalian COS-1 cells, the enzyme variant catalyzed 7-hydroxylation of coumarin at a rate approximately 60% of that of the wild-type enzyme. The CYP2A6(*)12 allele was present at an allele frequency of 2.2% among Spaniards, but was absent in Chinese.

The mu class glutathione S-transferase gene GSTM1 is polymorphic in humans, with approximately half of the Caucasian population being homozygous deleted for this gene. GSTM1 enzyme deficiency has been suggested to predispose people to lung and bladder cancer. Some people in a Saudi Arabian population, however, have been described previously with ultrarapid GSTM1 enzyme activity. Here we have evaluated the molecular genetic basis for this observation. Genomic DNA from two Saudi Arabian subjects exhibiting ultrarapid enzyme activity and from 13 Swedish subjects having null, one, or two GSTM1 genes were subjected to restriction fragment length polymorphism analysis using the restriction enzymes EcoRI, EcoRV, and HindIII and combinations thereof. Hybridization was carried out using a full-length GSTM1 cDNA or the 5' and 3' parts of the cDNA. The restriction mapping data revealed the presence of a GST mu cluster with two GSTM1 genes in tandem situated between the GSTM2 and GSTM5 genes. A quantitative multiplex polymerase chain reaction method, which simultaneously amplified a fragment of the GSTM1 gene and the beta-globin gene, was developed, and the genomic GSTM1 copy number was determined from the GSTM1/beta-globin ratio. This method clearly separated GSTM1 +/- subjects (ratios between 0.4 and 0.7) from GSTM1 +/+ subjects (ratios between 0.8 and 1.2). The two Saudi Arabians with ultrarapid GSTM1 activities had ratios of approximately 1.5, indicating that they carried three GSTM1 genes. These results demonstrate the existence of a novel mu class GST cluster containing a duplicated active GSTM1 gene causing ultrarapid enzyme activity.

No abstract

No abstract