Search

The polymorphisms of the important xenobiotic metabolizing enzymes CYP2D6, CYP2C19 and CYP2E1 have been studied extensively in a large number of populations and show significant heterogeneity in the frequency of different alleles/genotypes and in the prevalence of the extensive and poor metabolizer phenotypes. Understanding of inter-ethnic differences in genotypes is important in prediction of either beneficial or adverse effects from therapeutic agents and other xenobiotics. Since no data were available for Australian Aborigines, we investigated the frequencies of alleles and genotypes for CYP2D6, CYP2C19 and CYP2E1 in a population living in the far north of Western Australia. Because of its geographical isolation, this population can serve as a model to study the impact of evolutionary forces on the distribution of different alleles for xenobiotic metabolizing enzymes. Twelve CYP2D6 alleles were analysed. The wild-type allele *1 was the most frequent (85.81%) and the non-functional alleles (*4, * 5, * 16) had an overall frequency of less than 10%. Only one subject (0.4%) was a poor metabolizer for CYP2D6 because of the genotype *5/*5. For CYP2C19, the frequencies of the *1 (wild-type) and the non-functional (*2 and *3) alleles were 50.2%, 35.5% and 14.3%, respectively. The combined CYP2C19 genotypes (*2/*2, *2/*3 or *3/*3) correspond to a predicted frequency of 25.6% for the CYP2C19 poor metabolizer phenotype. For CYP2EI, only one subject had the rare c2 allele giving an overall allele frequency of 0.2%. For CYP2D6 and CYP2C19, allele frequencies and predicted phenotypes differed significantly from those for Caucasians but were similar to those for Orientals indicating a close relationship to East Asian populations. Differences between Aborigines and Orientals in allele frequencies for CYP2D6* 10 and CYP2E1 c2 may have arisen through natural selection, or genetic drift, respectively.

The polymorphic cytochrome P450 CYP2D6 catalyses the biotransformation of at least 40 drugs. The CYP2D6 genetic polymorphism is responsible for pronounced interindividual differences in plasma concentrations and, hence, in drug action and side-effects after administration of the same dose. Provided there is a close relationship between CYP2D6 genotypes and catalytic function, genotyping could be used in the clinical setting for individualization of drug dose. In the present study, we evaluated the relationship between the in-vivo enzyme activity and 35 different genotypes in order to determine whether genotyping can be used to predict a person's metabolic capacity for CYP2D6-catalysed drug oxidation using sparteine as a probe drug. One hundred and ninety-five Caucasian individuals were genotyped for seven nonfunctional (CYP2D6 x 3, x 4, x 5, x 6, x 7, x 8, x 16) and eight functional alleles (CYP2D6 x 1, x 2, x 2 x 2, x 2B, x 2B x 2, x 9, x 10, x 17). The metabolic ratio distribution for sparteine showed trimodality, with 15 poor metabolizers, 21 intermediate metabolizers, and 1.59 extensive and ultrarapid metabolizers. All poor metabolizers were unambiguously identified as carriers of two nonfunctional alleles. In contrast, the most frequent functional genotypes extensively overlapped and, with few exceptions, genotype was not a useful predictor of function. Gene dose effects among homozygotes and heterozygotes of the major functional alleles were not significant and could not explain the wide variations. Only a minor fraction of phenotypical ultrarapid metabolizers, arbitrarily defined as individuals with a metabolic ratio < 0.2, could be identified as carriers of three functional gene copies, including duplicated CYP2D6 x 2 x 2 alleles. Similarly, only a minor fraction of the intermediate metabolizers had predictive genotypes involving alleles coding for enzyme with impaired function. Thus, genotyping correctly identifies poor metabolizers, but quantitative prediction of drug metabolism capacity among extensive metabolizers is not possible.

The CYP2D6 gene on human chromosome 22 encodes a cytochrome P450 responsible for oxidative metabolism of over 30 clinically used drugs. The CYP2D6 gene is highly polymorphic with more than 20 alleles described to date. Some of these harbour loss-of-function mutations which lead to the poor metabolizer phenotype in 5-10% of Caucasians. These individuals are at increased risk of suffering from adverse side effects or to experience therapeutic failure following drug treatment. Phenotype determination requires ingestion of a probe drug and has other inherent problems. Due to the increasing number of alleles known, comprehensive CYP2D6 genotyping using the conventional assays has become cumbersome and time consuming. We have therefore developed a streamlined and more rapid CYP2D6 genotyping procedure. Use of long distance PCR allowed the amplification of a 4666 bp fragment which contains the entire CYP2D6 gene. The 4.7 kb fragment serves as a template for a multiplex allele-specific PCR assay to simultaneously identify the five PM-associated alleles, CYP2D6*3 (A), *4 (B), *6 (T), *7 (E), and *8 (G). Together with the CYP2D6 deletion allele CYP2D6*5 (D), which can be detected in a separate PCR assay, these alleles are responsible for the PM phenotype in approximately 99% of Caucasian individuals. We tested the reliability of the procedure by analysing DNA from more than 80 individuals with known CYP2D6 genotypes. Twelve different genotypes were present among these samples and all of them were correctly identified.

A mutant Rsa I restriction endonuclease site of high frequency has been identified in individuals of German, Greek, Italian, and Turkish origin. The mutation was found within the alpha-globin gene complex and is located 0.7 kb 5' to the alpha 2-globin gene. In individuals of central European origin 34 out of 58 chromosomes exhibited the alpha-gene linkage to the presence of the polymorphic site, and thus a preliminary estimate of the gene frequency for this allele would be 0.59. DNA analysis data of individuals derived from Mediterranean populations indicate a distribution of this polymorphic marker in similar frequencies.