Allele and genotype frequencies of CYP2C9, CYP2C19 and CYP2D6 in an Italian population

Abstract

The polymorphic cytochrome P450 isoenzymes (CYPs) 2C9, 2C19 and 2D6 metabolise many important drugs, as well as other xenobiotics. Their polymorphism gives rise to important interindividual and interethnic variability in the metabolism and disposition of several therapeutic agents and may cause differences in the clinical response to these drugs. In this study, we determined the genotype profile of a random Italian population in order to compare the CYP2C9, CYP2C19 and CYP2D6 allele frequencies among Italians with previous findings in other Caucasian populations. Frequencies for the major CYP2C9, CYP2C19 and CYP2D6 mutated alleles and genotypes have been evaluated in 360 unrelated healthy Italian volunteers (210 males and 150 females, aged 19–52 years). Genotyping has been carried out on peripheral leukocytes DNA by molecular biology techniques (PCR, RFLP, long-PCR). CYP2C9, CYP2C19 and CYP2D6 allele and genotype frequencies resulted in equilibrium with the Hardy–Weinberg equation. One hundred and fourteen subjects (31.7%) carried one and 23 subjects (6.4%) carried two CYP2C9 mutated alleles. Sixty-eight (18.9%) volunteers were found to be heterozygous and six (1.7%) homozygous for the CYP2C19*2, while no CYP2C19*3 was detected in the evaluated population. Volunteers could be divided into four CYP2D6 genotypes groups: 192 subjects (53.3%) with no mutated alleles (homozygous extensive metabolisers, EM), 126 (35.0%) with one mutated allele (heterozygous EM), 12 (3.4%) with two mutated alleles (poor metabolisers, PM) and 30 (8.3%) with extracopies of a functional gene (ultrarapid metabolisers, UM). Frequencies of both CYP2C9 and CYP2C19 allelic variants, as well as CYP2D6 detrimental alleles, in Italian subjects were similar to those of other Caucasian populations. Conversely, the prevalence of CYP2D6 gene duplication among Italians resulted very high, confirming the higher frequency of CYP2D6 UM in the Mediterranean area compared to Northern Europe.

Introduction

Genetic polymorphisms are known to contribute to interindividual variations in the metabolism of numerous drugs in humans [1], [2]. Mutation in a gene coding for a drug metabolising enzyme can cause enzyme variants with high, low or no activity [3]. Pharmacogenetic polymorphisms divide the population into at least two phenotypes, poor (or slow) metabolisers (PM) and extensive (or rapid) metabolisers (EM), and, eventually, ultrarapid metabolisers (UM) [4]. The PM condition can lead to an excessive or prolonged therapeutic effect or drug-related toxicity after a normal dose, conferring a genetic predisposition to drug-induced adverse effects. Moreover, in case of compounds that need to be activated, the PM condition may result associated with decreased response. On the contrary, UMs may not achieve therapeutic levels of the drug given at a standard dose and this might account for lack of therapeutic effect. Furthermore, in cases where the enzyme bioactivates the drug, increased plasma levels of the active metabolite can be expected in UMs. Determination of an individual’s metabolic capacity by use of phenotyping or genotyping tests may become an important tool for a more rational and safe drug administration, especially for agents with a narrow therapeutic index. The major genetic polymorphisms affecting drug metabolising enzymes activity of potential clinical relevance are those related to drug oxidation by cytochrome P450 enzymes (CYP) 2C9, 2C19 and 2D6 [5], [6].

The human CYP2C subfamily consists of at least four isoforms, 2C8, 2C9, 2C18 and 2C19, whose genes are located together on chromosome 10. CYP2C9, the most abundant among human CYP2C isoforms, metabolises a number of therapeutically important drugs, including most nonsteroidal anti-inflammatory drugs, S-warfarin, phenytoin and losartan (Table 1), and is polymorphically expressed [7]. In vitro studies have shown that even relatively conservative changes in the aminoacid sequence may significantly alter both enzymatic activity and substrate specificity [8]. To date, three different allelic variants (CYP2C9*1, CYP2C9*2, CYP2C9*3), coding for enzymes with different catalytic activity, have been well characterised [7]. The frequencies of the detrimental alleles CYP2C9*2 and CYP2C9*3 vary between 8 and 12% and 3 and 8%, respectively, among Caucasians, while they are lower in Orientals and Black Africans [7], [9]. Additional rare defect alleles have been described, but so far their impact on the enzyme activity in vivo is unclear (http://www.imm.ki.se/CYPalleles).

CYP2C19, an isoform contributing to the clearance of S-mephenytoin, diazepam, omeprazole, proguanil, citalopram, R-warfarin and many antidepressants (Table 1), also exhibits genetic polymorphism [2]. About 3% of Caucasians have been found to be PMs of S-mephenytoin with very little variation noted between the studies [10], [11]. By contrast, several independent studies have shown a much higher incidence of PMs in Orientals, up to 18–23% in Japanese [12], [13]; 15–17% in Chinese [14], [15]; 12–16% in Koreans [16], [17]. In Black Africans, PM frequencies vary between 4 and 7% [18]. The PM condition is inherited as an autosomal recessive trait [11]. The best characterised defect CYP2C19 alleles responsible for the PM phenotype are CYP2C19*2 and CYP2C19*3 [19], [20]. CYP2C19*2 accounts for 75% of the defective alleles in Orientals [19], and 93% in Caucasians [21]. The other well characterised detrimental allele (CYP2C19*3), discovered in Japanese PMs [20], accounts for approximately 25% of all inactive forms in Orientals, being by converse extremely rare in non-Oriental populations [22]. Therefore, the defective forms are still uncharacterised in 15% of the Caucasian PMs. Additional rare defect alleles have been described (http://www.imm.ki.se/CYPalleles), but their impact on the enzyme activity has to be further clarified.

CYP2D6, although expressed at rather low levels compared with other human CYPs, plays an important role in drug metabolism, being partially or to a major extent responsible for the oxidative biotransformation of a variety of psychoactive and cardiovascular drugs (Table 1) [6]. The gene encoding the CYP2D6 enzyme is located on the long arm of chromosome 22. CYP2D6 activity ranges from complete deficiency to ultrarapid metabolism, depending on the existence of a large number of allelic variants of the CYP2D6 gene, causing either absent, decreased or increased enzyme activity in relation to the functional wild type allele [23]. The activity of CYP2D6 is bimodally distributed in Caucasian populations. Individuals with deficient enzyme activity (PM) represent approximately 3–10% of Caucasians, but only 1–2% of Orientals [6]. Among the EMs, 2–10% carry multiple copies of a functional CYP2D6 allele [6]. This genotype has been shown to be associated with extremely high CYP2D6 activity (ultrarapid metabolisers, UM) [24], [25]. The CYP2D6 gene is extremely polymorphic. To date, more than 70 allelic variants have been described (http://www.imm.ki.se/CYPalleles). Four major mutated alleles, CYP2D6*3, CYP2D6*4, CYP2D6*5 and CYP2D6*6, account for 90–95% of the PM alleles in Caucasians. The most common allele associated with the PM phenotype is CYP2D6*4, with an allele frequency of ∼21% in Caucasian populations [26]. The frequency of the CYP2D6*5 allele, with deletion of the entire CYP2D6 gene, is ∼4–6%, very similar in different ethnic populations [26]. Other detrimental alleles frequently (∼2–3%) found among Caucasians are CYP2D6*3, and CYP2D6*6 [6], [26]. Furthermore, alleles with duplication or multiduplication of a functional CYP2D6 gene (*1 or *2), causing increased CYP2D6 activity, have been described. The incidence of CYP2D6 gene duplication is only 1% in Sweden [25], 3% in Germany [23], but as high as 7–10% in Spain [27], [28], 9% in Turkey [29] and 10% in Italy [30]. There is thus a clear north-south gradient of the frequency within Europe. The highest incidence of gene duplication has been found in Ethiopia in Northern Africa with 29% [31].

Considering the pharmacological implications of CYP2C9, CYP2C19 or CYP2D6 genetic polymorphism, the definition of allele distribution pattern might represent a helpful support in the optimisation of pharmacological therapies. Therefore, in this study we first determined the CYP2C9, CYP2C19 and CYP2D6 genotype profile of a random Italian population by screening for the main allelic variants and compared their frequencies with previous findings in other Caucasian, as well African and Oriental populations.