CYP2D6 genotype and phenotype determination in a Mexican Mestizo population

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1 Eur J Clin Pharmacol (2005) 61: DOI /s PHARMACOGENETICS Marisol López. Jorge Guerrero. Helgi Jung Cook. María Elisa Alonso CYP2D6 genotype and phenotype determination in a Mexican Mestizo population Received: 7 May 2005 / Accepted: 7 September 2005 / Published online: 26 October 2005 # Springer-Verlag 2005 Abstract Objective: Although CYP2D6 genetic polymorphism plays an important role in interindividual and interethnic variability in drug response, very few pharmacogenetic data are available from Hispanic populations, including Mexicans. For this purpose, this study was undertaken to determine CYP2D6 genotype and phenotype in a healthy Mexican Mestizo population. Methods: Genotyping of five CYP2D6 mutant alleles by polymerase chain reaction-restriction fragment length polymorphism (PCR RFLP), and CYP2D6*5 and duplicated CYP2D6 alleles by long PCR was performed in 243 Mexican Mestizos. Of these, 100 subjects were also phenotyped using dextromethorphan as the probe drug. Results: The frequency of CYP2D6*2, *3, *4, *5, *10, *17 was 19.34, 1.44, 11.21, 2.67, 12.45, and 1.65%, respectively, while duplicated CYP2D6 alleles were found in 12.76% of the 243 genotyped subjects. Among the 100 phenotyped subjects, we identified 10 (10%, 95% confidence interval of ) individuals as poor metabolizers by using the published antimode for Caucasians. The mean log 10 dextromethorphan/dextrorphan ratio of the total sample was The mean (SD) of the log 10 M. López (*) Department of Biological Systems, Metropolitan Autonomous University Xochimilco Campus, Calzada del Hueso 1100, Col. Villa Quietud, Coyoacán, Mexico City, Mexico mlopez@correo.xoc.uam.mx Tel.: Fax: J. Guerrero. M. E. Alonso Department of Genetics and Molecular Biology, National Institute of Neurology and Neurosurgery Manuel Velasco Suárez, Avenida Insurgentes Sur 3877, Col. La Fama, Tlalpan, Mexico City, Mexico H. Jung Cook Department of Neuropsycopharmacology, National Institute of Neurology and Neurosurgery Manuel Velasco Suárez, Avenida Insurgentes Sur 3877, Col. La Fama, Tlalpan, Mexico City, Mexico metabolic ratio (MR) in the CYP2D6 subgroups was ultrametabolizer (UM)= 2.6 (0.86), extensive metabolizer (EM)= 2.09 (0.98), intermediate metabolizer (IM)= 1.71 (1.06), and poor metabolizer (PM)=0.42 (0.625). These data show a trend toward a smaller mean log MR (higher enzyme activity) as the number of active alleles increases. Conclusions: The PM frequency of CYP2D6 in the population studied was 10%, which is very similar to Spanish Caucasians. The observed frequency of the CYP2D6 alleles tested was unique for the Mexican Mestizo sample analyzed, and in accordance with the Caucasian, Asian, and African admixture in this population. Keywords CYP2D6 polymorphism. Dextromethorphan. Mexican Mestizo population Introduction Variability in drug response is at least partly attributable to individual differences in drug metabolism which include environmental factors (e.g., age, dietary intake, lifestyle, concurrent drug therapies) and polymorphisms within genes encoding drug-metabolizing enzymes. In humans, the best studied example of genetic polymorphism in phase I drug metabolism is the debrisoquine 4-hydroxylase, or cytochrome P450 2D6 (CYP2D6), polymorphism. This enzyme catalyzes the oxidative metabolism of about 25% of the clinically important classes of drugs, including many antidepressants, antipsychotics, β-adrenergic blocking agents, antihypertensives, antiarrhythmics, and opioids [1]. The CYP2D6 locus is localized to human chromosome 22q13.1 and normally consists of the active gene and two pseudogenes [2]. The CYP2D6 gene is highly polymorphic, and at least 50 different allelic variants have been identified [3]. These genetic variants have been correlated to at least three classes of phenotypes based on the extent of drug metabolism: ultrarapid (UM), extensive (EM), and poor (PM) metabolizers resulting in low, normal, and high blood levels of parent drugs [4]. Within the last decade,

2 750 there has been increasing recognition of an intermediate metabolizer (IM) status associated with heterozygous genotypes composed of one functional and one reduced or diminished functioning gene [5]. Many clear examples of the therapeutic relevance of CYP2D6 variability have been described [6, 7] due to the large number of clinically important drugs that are substrates of CYP2D6 and the narrow therapeutic window of these drugs. In addition to individual variation in drug response, it is well known that there are interethnic differences in CYP2D6 poor metabolizers frequencies. Among Caucasians, 7% lack functional CYP2D6 enzymes [8], whereas the incidence of PMs among Orientals is low (1%) [9]. Studies in Black African populations have shown a prevalence of PMs ranging from 0 to 19% [10]. The functional alleles include CYP2D6*1 (wild-type) and CYP2D6*2, the most common allele encoding slightly reduced enzyme activity ( 80% of the wild-type) in Caucasians, with a frequency of 28.5% [11], which can result in a strong effect on the metabolic phenotype when present in combination with a null allele. The molecular genetic basis for the PM status has been shown to be the occurrence of combinations of any of a number of defective alleles. In Caucasian populations the most common nonfunctional alleles are CYP2D6*4, CYP2D6*5, and CYP2D6*3 at a frequency of 20%, 2 7%, and 1 2%, respectively; but these alleles are rare among most Asian and Black African populations [12]. There are also CYP2D6 allelic variants that result in diminished or altered drug metabolism. Kagimoto et al. [13] identified a C188T change, resulting in a P34S substitution (CYP2D6*10) and the production of a low activity and unstable enzyme [14]. The CYP2D6*10 allele has a high frequency of 50.7% in Chinese [15] and 38.1% in Japanese populations [16], compared to its low 1.5% frequency in Caucasians [8]. The presence of this mutation causes a right shift of the metabolic ratio (MR) in Chinese EMs compared with Swedish EMs [17]. Among most African populations, the basis for the diminished CYP2D6 activity is the presence of the CYP2D6*17 variant, first identified in a Zimbabwean population at an allelic frequency of 34% [18]. The genetic basis of the UM phenotype has been shown to be gene duplication or amplification of functionally active CYP2D6 alleles, resulting in higher levels of enzyme [19]. In Caucasians the percentage of subjects having duplicated/multiduplicated genes (CYP2D6*XN) is 1 2% for Sweden [20], 3.6% in Germany [8], 7 10% in Spain [21, 22], and 10% on Sicily [23]. Black Ethiopians have the highest percentage of duplicated CYP2D6 alleles thus far studied, 29% [24], followed by Egyptians and Saudi Arabians (20%) [25]. The CYP2D6 polymorphism has been extensively studied at both phenotype and genotype levels. Genotyping methods provide direct information on which cytochrome P450 alleles are present but accurate interpretation requires an understanding of the relationship between any given genotype and the resulting phenotype. Despite its importance in drug metabolism as well as its great ethnic variability, CYP2D6 enzymatic activity and genetic polymorphism have rarely been studied in Hispanic populations, including Mexicans, so we tried to bridge this gap by undertaking the present study in a Mexican Mestizo population. The aim of this study was to perform a genetic analysis of CYP2D6 using a polymerase chain reactionrestriction fragment length polymorphism (PCR RFLP) approach, and to evaluate the CYP2D6 phenotype by using dextromethorphan as a probe drug, in order to determine the frequency of the poor metabolizer status in a Mexican Mestizo population. Taking into account the ethnic origin of Mexican Mestizos and the most frequently reported CYP2D6 variants, our analysis comprised the distribution of CYP2D6*2, CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*10, CYP2D6*17, and duplicated CYP2D6 alleles. Materials and methods Subjects After approval by the Ethics Committee of the National Institute of Neurology and Neurosurgery, a total of 243 healthy unrelated individuals of Mexican Mestizo ethnic origin participated in the study after written informed consent was obtained. Age ranged from 20 to 50 years (mean±sd=25.3±6.2 years); 138 were women and 97 were men. Of the 243 subjects, all participated in genotyping, and 100 were involved in both genotyping and phenotyping analysis. To assess an ethnic homogeneity, only subjects with both parents and four grandparents, both maternal and paternal, from Mexican Mestizo origin were included. We considered as Mexican Mestizo only those who for two generations, including their own, had been born in Mexico. A Mexican Mestizo is defined as someone born in Mexico who is a descendant of the original autochthonous inhabitants of the region and of individuals, mainly Spaniards of Caucasian and/or Black origin, who came to America during the sixteenth century. Genotyping analysis In all subjects genomic DNA was isolated from peripheral blood by standard techniques. The DNA sample was quantified and checked for purity, and it was stored at 4 C until used. For CYP2D6*2, *3, *4, *10, and *17 allele determination we followed PCR RFLP methods. Genotyping for CYP2D6*2 and CYP2D6*10 was performed as described by Sachse et al. [8], with slight modifications. CYP2D6*3 and CYP2D6*4 genotypes were performed according to Schur et al. [26], except for the use of deoxyuracil triphosphate (dutp). In order to genotype the CYP2D6*17 allele we employed the primers and PCR conditions described by Mendoza et al. [27], except for the primer de-

3 751 scribed as mutant, to obtain an initial 237 bp product. We then introduced the use of Hpy 188 III digestion to detect the C111T mutation (wild type, 237 bp; mutant fragments, 218 bp and 19 bp fragments). The CYP2D6*5 allele was detected by multiplex long PCR as described by Hersberger et al. [28]. The CYP2D6*1 allele was assumed when all CYP2D6 alleles tested were absent, since we did not perform DNA sequencing. Determination of CYP2D6*XN was performed using the Expand Long Template PCR System (Roche Applied Science), and the primers and PCR conditions designed by Lovlie et al. [29]. This primer combination amplified a 5.2 kb PCR fragment from the CYP2D7 CYP2D6 intergenic region, as well as a 3.6 kb PCR fragment from the CYP2D7 CYP2D6 region in subjects with an amplification. Phenotyping analysis Of 243 individuals, only 100 agreed to participate in the phenotypic procedure. None of them had a history of alcohol or drug abuse or dependence, and they did not have any medical condition that required treatment. Additionally, the protocol required not to take any natural remedy or over-the-counter drugs 1 week before the phenotype test. Subjects received a single oral dose of 30 mg of dextromethorphan hydrobromide syrup (300 mg/100 ml, Laboratorios Euromex, S.A. de C.V., Mexico). Urine samples were collected during an 8-h period. The total volume of urine was measured and a 10-ml aliquot was stored at 20 C until analysis. The urinary concentrations of dextromethorphan and its O demethylated metabolite dextrorphan were determined by using the high-performance liquid chromatography (HPLC) method with fluorescence detection described by Duchame et al. [30] with slight modifications. Briefly, samples were extracted by passing through a Bond Elut cartridge, washed with 3 ml of water and 1 ml of acetonitrile. The compounds were eluted with 3 ml of methanol acetonitrile 2% phosphoric acid (80: 30:20 v/v). The chromatographic conditions were: a mobile phase of phosphate buffer (ph 3.5, 0.1 M) acetonitrile (60:40 v/v), a Spherisorb phenyl column (Waters) ( mm ID), at a flow rate of 1.2 ml/min. The fluorescence was determined at a wavelength of 280 nm for excitation and 310 nm for emission. The response was linear between and 25 μg/ml. The dextromethorphan MR was defined as log 10 ratio of dextromethorphan/ dextrorphan. Statistical analysis Allele and genotype frequencies of the CYP2D6 gene were obtained by direct counting. Hardy Weinberg equilibrium was evaluated based on the chi-square test and the likelihood ratio test criterion with the use of the PopGen 32 program, and the Markov exact test with the GenePop software pack. The log 10 MR values between groups of allelic combinations were compared using a one-way analysis of variance (ANOVA) (Sigma Stat program). Significance was defined as P<0.05. Results Genotyping for six common CYP2D6 variants and CYP2D6*x2 alleles was performed in all 243 Mexican Mestizo volunteers. The allelic frequency of CYP2D6*2, *3, *4, *5, *10, and *17 was 19.34, 1.44, 11.21, 2.67, 12.45, and 1.65%, respectively, while duplicated CYP2D6 alleles was observed in 12.76% of the individuals. The CYP2D6 genotype frequencies found are summarized in Table 1. Heterozygosity between the wild-type allele and other CYP2D6 alleles were seen in 35.80% of the individuals, followed by homozygous for the CYP2D6*1 allele (24.28%) and heterozygous for two CYP2D6 mutant alleles (22.63%). In 31 subjects a duplicated CYP2D6 allele was identified. Four predicted phenotypes (UM, EM, IM, and PM) were assumed according to the number of functional (*1 and *2), reduced (*10 and *17), and nonfunctional (*3, *4, and *5) alleles in their genotypes. Table 1 CYP2D6 genotype distribution in 243 Mexican Mestizo subjects Predicted phenotype a Active alleles (n) CYP2D6 genotype n Frequency (%) UM 3 *1/* *1/* *1/* *2 2/* *2/* EM 2 *1/* *1/* *1/* *2/* *2/* *2 2/* *1 2/* *10/* *2/* *10/* *17/* IM 1 *1/* *2/* *1/* *4/* *4/* *2/* *2/* *1/* *3/* PM 0 *3/* *4/* a UM ultrametabolizer, EM extensive metabolizer, IM intermediate metabolizer, PM poor metabolizer

4 752 Fig. 1 Frequency histogram and probit plot of CYP2D6 activity as measured by dextromethorphan/dextrorphan metabolic ratios in 100 Mexican Mestizo subjects Allele and genotype frequencies were in equilibrium with the Hardy Weinberg equation. The results of our study showed that the log 10 dextromethorphan/dextrorphan MR of 100 subjects ranged from 4 to 1.01 with a median of 2.28 and a mean of As illustrated in the frequency histogram showed in Fig. 1, a skewed unimodal distribution of CYP2D6 activity for the dextromethorphan/dextrorphan MR was found. This unimodal population distribution did not allow the identification of Mexican subjects as EMs or PMs, and distinct population subgroups were not apparent with use of either histograms or probit plot analyses (Fig. 1). With the use of the established derived antimode value of 0.3 (log 10, ) described for Caucasians [31], we found that of the 100 phenotyped Mexican subjects, 10 (10%) were determined to be PMs. The median and mean log 10 dextromethorphan/dextrorphan ratios were 0.73 and 0.19 (SD= 0.55) for PMs, and 2.34 and 2.2 (SD=0.749) for EMs, respectively. In order to correlate phenotype genotype data, the log 10 MR of the 100 subjects phenotyped with dextromethorphan were plotted versus the CYP2D6 genotype identified in each individual. We also classified individuals into four major phenotypic groups (UM, EM, IM, and PM) according to the number of active alleles present in their genotypes (Fig. 2). The mean (SD) of the log 10 dextromethorphan MR in these groups was UM= 2.6 (0.86), EM= 2.09 (0.98), IM= 1.71 (1.06), and PM=0.42 (0.625). These data show a trend toward a smaller mean log MR (higher enzyme activity) as the number of active alleles increases. However, as seen in Fig. 2, a substantial scatter exists within the specific genotypes and overlap between genotype groups is apparent. Comparison of the different combinations revealed statistically significant differences (P<0.05) only between PM and all other phenotypic groups: PM/UM (P= ), PM/EM (P=0.0018), and PM/IM (P=0.007). Thus, log 10 MR values did not allow a clear distinction of the IM, EM, and UM phenotype as expected by the determined genotype. This could be due to the small sample size and the large interindividual variability observed in our study. Of the ten phenotypically poor metabolizers who were also genotyped, four individuals, two homozygous for the CYP2D6*4 allele and two with a combination of CYP2D6*3 and CYP2D6*4 alleles, are clearly explainable. Four addi- Fig. 2 Plot of CYP2D6 genotype vs log 10 metabolic ratio (MR) of dextromethorphan in 100 Mexican Mestizo individuals. UM, EM, IM, and PM are expected metabolizer phenotypes according to the number of active alleles in the observed genotype. The mean log 10 MR for each genotype is indicated by a small vertical line. The published antimode of 0.3 (log 10 = 0.52) for Caucasians [31] is shown as a vertical line

5 753 tional subjects were heterozygous between CYP2D6*1 and mutant alleles (*4 and *10). Since we were not able to sequence the CYP2D6 gene, we cannot rule out that these individuals carry additional mutations not identified in this study. The PM status of the remaining two subjects (*2/*4 and *2/*10) is probably due to nongenetic factors, such as diet, as other individuals with the same genotype exhibited an EM phenotype. Discussion and conclusions To the best of our knowledge, this study represents the first analysis of CYP2D6 phenotype and genotype in a Mexican Mestizo population to date. The frequency of poor metabolizers (10%) in the Mexican Mestizo population studied was very similar to those documented for Spanish Caucasians (6.6 10%) [32, 33], but higher than those reported in other Latin American cohorts. Mendoza et al. [27] found 3.2% of poor metabolizers in Mexican Americans, while Isaza et al. [34] found 6.6% in a Colombian population, and Jorge et al. [35] determined 4.4 and 2.2% as poor metabolizers in Ngawbe and Embera Amerindians of Panama and Colombia, respectively. Among the CYP2D6 variant alleles analyzed, the most common were the *2,*10, and *4, followed by *5,*17, and *3. The allelic frequency of CYP2D6*2 found (19.34%) was very similar to Mexican Americans (22.8%) [27] and Spaniards (22.0%) [21]. The frequencies found for deficient alleles CYP2D6*3 and CYP2D6*4 (1.44 and 11.21%, respectively) were higher than those reported for Orientals (0 and <1%) [12], but very similar to Spaniards (1.2 and 11.6%) [36]. The frequency of CYP2D6*4 in Caucasians has been shown to be 20%, while that for CYP2D6*3 is 1 2% [12]. The frequency of CYP2D6*5 nonfunctional allele identified in our sample was 2.67%, which is within the range reported for Caucasians (2 7%) [12]. CYP2D6*10 and CYP2D6*17 are alleles associated with a decreased enzymatic activity, and the magnitude of their effect varies between substrates. CYP2D6*10 is prevalent in East Asians (33 50%), while CYP2D6*17 is prevalent in Africans and African Americans (27.7%) [12]. We determined an allelic frequency of 12.45% for CYP2D6*10, which is higher than Mexican Americans (7.4%) [27], but closely related to Ngawbe Amerindians of Colombia (17.5%) [35]. The CYP2D6*17 allele frequency in our sample was 1.65%, which is higher than in Mexican Americans (<1.0%) [27], but lower than that observed in Africans (27.7%) [37]. We found CYP2D6 gene duplications in 12.76% of the population analyzed, which is slightly higher than the range reported for Spaniards (7.0 10%) [21, 22]. Among European Caucasians, Spaniards distinguish themselves in having both the lowest frequency of CYP26*4 and the highest percentage of duplicated alleles [21, 22, 32]. In this regard, our data for CYP2D6*4 and CYP2D6*2XN in Mexican Mestizos followed the same behavior. Taken together, the CYP2D6 allelic frequencies found in this study showed a unique distribution in the Mexican Mestizo sample analyzed. These results are in accordance to the trihybrid model, based on blood groups and other DNA markers, which proposes that cosmopolitan Mexican Mestizo populations are derived from three primary sources: Europeans (Spanish), Amerindians, and Africans [38]. Our results showed that log 10 MR values varied among individuals that share the same genotype, but comparison of the mean log 10 MR value among UM, EM, IM, and PM groups suggest a gene dose effect. Further studies, involving a larger sample size and genotyping for additional CYP2D6 mutant alleles, are still needed to clarify the relationship between genotype and phenotype in our population. The overall allelic frequencies of CYP2D6*2, *3, *4, *10, *17, and the percentage of CYP2D6*XN found are unique to the Mexican Mestizo population analyzed. 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