CYP2C19 and ABCB1gene polymorphisms are differently distributed according to ethnicity in the Brazilian general population

Clopidogrel, a prodrug, is a thienopyridine that inhibits adenosine diphosphate-induced (ADP) platelet aggregation. It has been prescribed mainly in patients with acute coronary syndromes (with or without ST-segment elevation) and patients submitted to percutaneous coronary intervention. It is a pro-drug that requires hepatic metabolization and activation by the cytochrome P450 (CYP) in order to produce its active metabolite. Metabolization of the drug is achieved by a number of different CYP isoenzymes. Two oxidative steps are essential, which culminates in the active metabolite. In particular, the isoenzyme CYP2C19 is involved in the two steps contributing to an estimated 45% of first step and 21% of its change to the active metabolite [1‐3].

Several studies have demonstrated an association between CYP2C19 gene polymorphisms and the enzyme activity [4‐9]. The most common genetic variation, designated CYP2C19*2 (c.G681A), leads to a splicing defect that functionally affects the enzyme. Other alterations have also been reported as loss-of-function: CYP2C19*3 (c.G636A; stop codon), CYP2C19*4 (c.A1G; transition in the initiation codon), and CYP2C19*5 (c.C1297T; amino acid substitution) [7‐9].

CYP2C19*2 allelic variant has been associated with higher levels of ADP-induced platelet aggregation values in clopidogrel-treated patients and, consequently, a higher risk of adverse cardiovascular events, such as the occurrence of stent thrombosis [4‐8]. In contrast, an allelic variant recently reported, CYP2C19*17 (c.C806T; 5'-flanking region of the gene), has been associated with increased enzyme function [10]. Thus, individuals harboring this genetic variant could have an enhanced response to antiplatelet treatment with clopidogrel, improving the prevention of thrombotic events, but, on the other hand, having a putative increased risk of bleeding [7, 10].

On the other hand, it is known that the bioavailability of clopidogrel and of other drugs is also limited by absorption. The multidrug resistance gene (MDR1 or ABCB1) encodes a drug-efflux transporter called P-glycoprotein, which functions as a physiologic intestinal barrier against drug absorption [11]. Some studies evaluating patients receiving clopidogrel have reported that the rate of cardiovascular events and the plasma concentrations of clopidogrel active metabolite were different according to the genotype for the polymorphism c.C3435T in the ABCB1 gene [8, 11].

In addition, it has been observed that the genotypic frequencies of CYP2C19 and ABCB1 polymorphisms present some degree of variation among different ethnic groups [12, 13]. As a result, the prevalence of the predicted metabolic phenotypes associated with these polymorphisms may vary significantly among different world-wide populations, leading the impact of pharmacogenetic testing for these variants extremely dependent on a particular country/population genetic architecture.

The Brazilian population is one of the most heterogeneous in the world, being a mixture of different ethnic groups, composed mainly of European descent, African descent and Amerindians [14]. The main aims of this study were to evaluate the frequencies of CYP2C19 and ABCB1 polymorphisms and to identify the clopidogrel-predicted metabolic phenotypes according to ethnic groups.

Probably the main contribution of the present study was the demonstration, in the Brazilian population of significant differences between the allelic and genotypic frequencies of polymorphisms associated to clopidogrel, according to ethnicity. To our knowledge this is the first study evaluating CYP2C19 polymorphisms in Brazilian Amerindians plus a sample representative of the Brazilian general population. These results could be very useful in the strategic planning of the implementation of pharmacogenetic testing for these variants, aiming at an individual therapeutic approach and adverse drug effect profile prediction both, at the individual and regional country level. To our knowledge, there is no implemented genetic-based prescription program in Brazil; however, the study by Gladding et al concluded that genotyping for the relevant gene polymorphisms may help to individualize and optimize clopidogrel treatment [19].

One potential limitation relates to the self-referred ethnicity. However, this type of classification is the one most probable to be encountered in real-life situations. In addition, even self-referred ethnicity was able to clearly differentiate groups of individuals with rather different allele and genotype frequencies. Finally, different allele and genotype frequencies were also observed when individuals were separated by the country's geographic region. Although a great amount of this effect was probably due to different ethnic distribution according to regions, both variables were independently predictive of genotype distribution in multivariate models adjusted by both ethnic group and country region (data not shown). Probably interaction effects, as well as other confounders, could explain these findings. In fact, one should not dissociate ethnic group self-referral from country region, since cultural aspects may modulate the way people describe themselves regarding ethnicity and this certainly may differ in different parts of the country. This effect should be studied in future works.

Similarly, interethnic differences were observed in the VKORC1 polymorphism in 4,886 individuals of 11 countries (Asians, African descent and Caucasian descent), and the contribution of VKORC1 toward dose requirements is higher in whites than in nonwhites [20]. Other study reported the worldwide allele frequency distribution of four genetic polymorphisms known to influence warfarin dosing and concluded that understanding the worldwide distribution of markers is important for the future application of pharmacogenomic-based algorithms to different population groups [21].

The ABCB1 gene encodes a P-glycoprotein that exports drugs, including clopidogrel. Simon et al (2009) studied 2,208 patients presenting with an acute myocardial infarction and receiving clopidogrel therapy. They reported that patients carrying one or two variant alleles for the ABCB1 c.C3435T polymorphism presented more than five times the rate of adverse events of patients with the wild-type genotype [8].

The ABCB1 c.C3435T variant allele frequencies reported in studies using samples from African individuals (Ghanian and Kenyan [22]) was of 17.0%. We observed in African descent subjects a higher frequency (32.8%) than in the two cited studies, but this frequency was lower than in other ethnic groups of our study (p < 0.001). In studies of Caucasians subjects (French, 43.0% [23]; Spanish, 48.0% [24]; German, 48.0% [25]; Polish, 49.0% [26]), the variant allele frequency was similar (p > 0.050) to the observed in our sample of Caucasian descent (43.2%) and Amerindian (51.4%) individuals. This result is probably explained by undetected (or unreferred) admixture history of the studied individuals self-referred as African descent from the Brazilian population.

The absence of CYP2C19*3 and CYP2C19*5 variant alleles and the rare frequency of CYP2C19*4 variant allele (0.3%) in our study does not exclude the existence of individuals with PM or IM predicted metabolic phenotypes by presence of these loss-of-function alleles in the Brazilian population. Nevertheless, it clearly shows that these variants are less powerful for the cost-effectiveness design of programs aiming at the identification of individuals harboring predicted metabolic phenotypes compared to the CYP2C19*2 and *17 alleles.

The CYP2C19*2 variant allele frequency found in our study was higher (p < 0.05) in African descent subjects (20.2%) compared with other ethnic groups and with Italian [27] (11.1%); European American [13] (13.0%); and German [28] (15.9%) subjects. However, this frequency was similar (p > 0.05) to the observed in samples of other African descent populations: African American [13] (25.0%), and African Venda [29] (22.0%).

In the general population, the PM predicted metabolic phenotype frequency (4.8%; 49/1029) was lower (p < 0.050) than in Japanese [30] (15.0%). In contrast, it was higher (p < 0.05) than in European American [13] (2.0%) and in Italians [27] (1.7%).

The phenotypic implications of the CYP2C19*2 polymorphism have been established in recent studies using clopidogrel. Mega et al (2009) observed that carriers of a reduced-function CYP2C19*2 allele have significantly lower levels of the active metabolite of clopidogrel, diminished platelet inhibition, and a higher rate of major adverse cardiovascular events, including stent thrombosis [6]. Simon et al (2009) reported that this genetic variant was associated with an increase in the risk of death, myocardial infarction, or stroke, especially among patients undergoing percutaneous coronary intervention [8].

The CYP2C19*17 variant allele, and the UM predicted phenotype, prevalence found was higher in African descent subjects (26.3%; 32.3%) and lower in Amerindians (15.8%; 20.8%) compared with other ethnic groups (p = 0.048). Some studies have reported lower frequencies of this variant allele in Chinese (4.0%) [10], Japanese (1.3%) [31] and Korean (0.3%) [32]; and similar frequencies in Swedish (20.0%) [32]; and Greek (19.6%) [33]. CYP2C19*17 is a recently reported variant causing ultra-rapid metabolism of CYP2C19 substrates. Regarding clopidogrel, Sibbing et al. (2010) observed that patients carrying heterozygous or homozygous genotypes (UM predicted phenotype) had lower ADP-induced platelet aggregation values compared with wild-type. In addition, CYP2C19*17 allele carriage was significantly associated with an increased risk of bleeding [7].

It is interesting that the genetic and functional knowledge of a CYP2C19 enzyme provide enough information to identify patients who will fall outside the therapeutic window of clopidogrel. Thus, it would be possible to justify increases in dosage or a change in therapy. However, it is important to consider that this metabolism has many influences (other genetic markers, concomitant disease processes, medications, foods, age and lifestyle), all of which culminate in variations in the drug action and in the effectiveness of the therapeutic regimen [9, 34].

Our findings indicate the existence of interethnic differences in the ABCB1 and CYP2C19 variant allele and predicted metabolic phenotype frequencies in the Brazilian general population plus Amerindians. Recent studies, have reported the clinical importance of these polymorphisms. The results of the present study will be very useful in the development of effective programs in stratifying individuals regarding clopidogrel-predicted metabolic phenotypes.