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PHARMACOGENOMICS

CYP2D6 Genotype and Phenotype in Amerindians of Tepehuano Origin and Mestizos of Durango, Mexico

Martha Sosa-Macías, MS, Guillermo Elizondo, PhD, Carmen Flores-Pérez, MS, Janet Flores-Pérez, MS, Francisco Bradley-Alvarez, MD, Ruth E. Alanis-Bañuelos, MS and Ismael Lares-Asseff, MD, PhD

From the Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional del IPN Unidad Durango, CIIDIR-IPN, México (Ms Sosa-Macías, Dr Bradley-Alvarez, Ms Alanis-Bañuelos, Dr Lares-Asseff); Sección Externa de Toxicología del Centro de Investigación y de Estudios Avanzados del IPN, México (Dr Elizondo, Ms Sosa-Macías); Departamento de Farmacología, Torre de Investigación "Dr. Joaquín Cravioto" del Instituto Nacional de Pediatría-SSA, México (Ms C. Flores-Pérez, Ms J. Flores-Pérez, Dr Lares-Asseff).

Address for reprints: Ismael Lares-Asseff, MD, PhD, CIIDIR-IPN Unidad Durango. Laboratorio de Farmacogenómica y Biomedicina Molecular. Calle Sigma s/n Fracc. 20 de Noviembre II Durango, Dgo. México C.P. 34220.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although the drug-metabolizing enzyme CYP2D6 has been studied extensively in subjects of differing ethnicities, limited CYP2D6 pharmacogenetic data are available for the Amerindian population and Mestizos of Mexico. Dextromethorphan hydroxylation phenotype was studied in Tepehuano Amerindian (n = 58) and Mestizo (n = 88) subjects, and 195 individuals (85 Tepehuano Amerindians and 110 Mestizos) were genotyped by polymerase chain reaction–restriction fragment length polymorphism methods to identify the frequencies of the CYP2D6*3, *4, *6, and *10 alleles. Tepehuano Amerindian subjects lacked the poor metabolizer (PM) phenotype, whereas in Mestizos the PM phenotype frequency was 6.8%. The CYP2D6*3, *6, and *10 alleles were not found in Tepehuano Amerindians. The CYP2D6*4 allele had a low frequency (0.006) in this Amerindian group. In the Mestizo group, the CYP2D6*3, *4, and *10 alleles had frequencies of 0.009, 0.131, and 0.023, respectively. The CYP2D6*6 allele was not found in Mestizos. The genotype-phenotype association was strongly statistically significant (r2 = .45; P = .005) in Mestizos. The Tepehuano population was found to have a low phenotypic and genotypic CYP2D6 diversity and differed from other Amerindian groups. On the other hand, the frequencies of the CYP2D6 variant alleles in Mestizos were similar to those reported for whites.

Key Words: Allele frequency • CYP2D6 polymorphism • ethnic difference • Tepehuan Amerindians

Ethnicity is known to be a factor in an individual's drug disposition. The oxidative phase I metabolism by cytochrome P450 superfamily (CYP) of enzymes plays a major role in this respect and consists of multiple isoenzymes that are structurally and functionally distinct.1 CYP2D6 represents ~3% of the total CYP expressed in the liver and has been widely studied.2,3 The CYP2D6 gene locus is highly polymorphic; approximately 80 variants have been identified to date (http://www.imm.ki.se/CYPalleles/cyp2d6.htm). The clinical significance of this enzyme's genetic polymorphism has become apparent because it participates in the metabolism of 20% to 30% of the drugs used in clinical practice. Many of these drugs, including antiar-rhythmic, antidepressive, and neuroleptic drugs, have a narrow therapeutic window.4-6

CYP2D6 polymorphism results in variable enzymatic activity among individuals. Each person can be categorized on the basis of his or her CYP2D6 activity as an extensive metabolizer (EM), an intermediate metabolizer (IM), a poor metabolizer (PM), or an ultrarapid metabolizer (UM).7 The PM phenotype has been observed in about 7% of whites from Europe8 and North America,9 0% to 1% of Chinese10 and Japanese11 populations, and 0% to 19% of African populations.12 In general, for Asian and white populations, the association between genotype and phenotype is high. However, for African and African American populations, genotype-phenotype discordance has been observed.13 Thus, there may be CYP2D6 variant alleles that occur only in these groups.14

Although there have been extensive studies of CYP2D6 polymorphism, scant information is available on the distribution and frequencies of phenotypes and allelic variants in Hispanic populations of Mestizo origin. Recently, a pharmacogenetic study of Mexican Americans from Los Angeles, California, showed a frequency of the PM phenotype of ~3.2%.15 This finding is similar to the 3.6% frequency reported in a study of 137 genetically admixed Nicaraguan subjects, whose frequency was predicted on the basis of inactivating mutations,16 and lower than the 4.5% frequency rate reported in a previous study of 22 Mexican Americans from southern Texas.17 Ngawbe and Embera Amerindian tribes from Panama and Colombia have been reported to have CYP2D6 PM frequencies of 4.4% and 2.2%, respectively. The PM phenotype was not observed among Cuna Amerindians.18-20

Several authors have demonstrated a genetic link between both populations: modern Native American and the aboriginal of Asia21-23; therefore, a similar distribution of allelic frequencies of CYP genes might be expected between Asians and Amerindians. In particular, Muñoz et al24 reported similar allelic frequencies and genotype distributions for the CYP2E1 and CYP2D6 genes between the Mapuche Amerindians of Chile and Asians.

In the preponderance of the literature concerned with polymorphisms, the subjects have been whites, African Americans, and Asians. Far less is known about other ethnic groups. Therefore, there is a need to establish the pharmacogenetic status of populations of different ethnicities. In Mexico, 10 million people, distributed among 56 tribes, are Amerindian. However the distribution patterns of the CYP2D6 genotypes and CYP2D6 enzyme activity phenotypes among the Mexican-Amerindian populations have not been established.

For these studies, we selected a Tepehuan Amerindian group of Durango, Mexico, which is a native population that has inhabited this territory for thousands of years. In Mexico, there are a total of 24 034 Tepehuanos, of which 17 000 live in Durango, making them the most important tribe from the state. On the other hand, Mexican Mestizos are essentially the product of an amalgamation of European, African, and Amerindian groups during the early Spanish colonization of the Americas.25, 26

The aim of the present study was to characterize the phenotypic expression of CYP2D6 in a population of Tepehuano Amerindians and Mexican Mestizos from the State of Durango, Mexico. The drug dextromethorphan (DM) was employed as a probe, and the distributions of 3 CYP2D6 alleles associated with the PM phenotype, CYP2D6*3 (2637A>del), *4 (1934G>A), and *6 (1795T>del), and one linked to IM status, the *10 (188TC>T) allele, were determined. The relationship between CYP2D6 genotype and drug (DM)-metabolizing phenotype was then examined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Two hundred and eleven unrelated healthy Mexicans from Durango State were selected for the study. These subjects included a total of 101 Tepehuano Amerindians between the ages of 15 and 65 years. They were inhabitants of the Guajolota Community (Municipality of Mezquital). Of the 101 Tepehuano Amerindians, 85 participated in genotyping, 58 completed the phenotyping procedure, and 42 were involved in both parts of the study. The 110 Mestizo subjects between the ages of 16 and 71 years were residents of Nuevo Ideal (Municipality of Canatlán). All Mestizos were genotyped, and 88 participated in both the phenotyping and genotyping tests. The ethnicities were determined through self-reporting of subjects and required both parents and all grandparents to be of the same ethnicity as the subject.

When the aim of the study and the possible side effects of drug administration had been explained to the subjects, a written informed consent in their own language was requested and received. The present work was approved by the Ethics and Research Committee of the Durango General Hospital of the Mexican Health Ministry. Each individual was interviewed by written questionnaire, and the subject's clinical and pharmacologic history, nutritional habits, smoking status, use of alcohol and medicinal plant consumption were recorded. Renal, liver, and biochemical tests were performed on each volunteer. Only individuals who had not been exposed to pharmacologic treatment and had not consumed drugs of abuse or alcohol for the 3 weeks before the study were included. Subjects who requested dismissal or who had taken food within 12 hours before the study were eliminated.

Phenotyping
The studied subjects did not consume food 12 hours before and 3 hours after drug administration. Each individual received a single 30-mg oral dose of DM (Romilar; Roche Laboratories, Nutley, NJ, presentation 15 mg/mL). After 3 hours, a 5-mL blood sample was collected with a vacutainer; the sample was centrifuged, and plasma was separated and stored at –70°C until analysis. The DM metabolic ratio (MR) was defined as the ratio of DM/dextrorphan (DX). Through the use of the established white-derived classification by Schmid et al,27 we identified the EM phenotype (MR < 0.03) and PM phenotype (MR > 0.3), and in accordance with Wennerholm et al,28 the IM phenotype (0.03 ≤ MR ≤ 0.3) was defined.

Analytic Procedure
Before processing, biological samples were incubated for 18 hours at 37°C with 3000 U of beta-glucuronidase (ICN Biomedics Inc, Aurora, Ohio). Plasma concentrations of DM and DX were determined with the high-performance liquid chromatography analytical procedure reported by Yoa-Pu Hu et al29 and modified according to the working conditions at our laboratory. To analyze the samples, an HP Agilent series 1100 chromatographic system consisting of a solvent delivery pump (Model G1311A; Agilent Technologies, Palo Alto, Calif), a florescence detector (Model G132A; Agilent Technologies), a Spherisorb 5-µm phenyl reverse phase column (25 x 0.46 cm inner diameter; Agilent Technologies) and Chem Station for LC3D software, version A.08.03 (847) (Agilent Technologies) were used.

For chromatography, the mobile phase was 10 mM potassium dihydrogen phosphate (pH 4.0)-acetonitrile (50:50 volume/volume [v/v]). After preparation, the mobile phase was passed through a membrane filter under vacuum. The flow rate was constant at 1.0 mL/min–1 at room temperature (25°C). The detector was set at 310 nm excitation and 280 nm emission.

The validation procedure was as follows: Linearity was assessed in the range 1 to 300 ng/mL–1 for DM and for DX. Interday and intraday coefficients of variation were less than 10%. The limits of detection and quantification were 0.003 µg/mL–1 and 0.015 µg/mL–1 for DM and 0.24 µg/mL–1 and 1.0 µg/mL–1 for DX, respectively. Mean recovery values were 94.05% ± 6.8 % for DM and 104.9% ± 7.9% for DX, with coefficient of variation less than 8%.

DNA Extraction
Genomic DNA was isolated from 5 mL whole blood using a sodium perchlorate/chloroform extraction method.30 Briefly, DNA was prepared by combining each blood sample with 35 mL of lysis buffer (320 mM sucrose, 5 mM magnesium chloride [MgCl2], 1% [v/v] Triton X-100, 10 mM tris-hydrochloride [HCl], pH 8). The nuclear pellet was collected by centrifugation at 2000 x g for 10 minutes and resuspended in 2 mL solution B (150 mM sodium chloride, 60 mM EDTA, 1% [weight/volume] sodium dodecyl sulfate, 400 mM tris-HCl, pH 8). The suspension was mixed with 0.5 mL of 5 M sodium perchlorate and then incubated at 65°C for 30 minutes. After the incubation, 2 mL chloroform was added, and the mixture was centrifuged at 1400 x g for 10 minutes. The aqueous DNA-containing upper phase was precipitated by the addition of 2 volumes of 100% ethanol and washed with 70% ethanol. The DNA was then resuspended in 200 µL of 10 mM tris-HCl, 1 mM EDTA, pH 7.4 and quantified by measuring absorbance at 260 nm.

Genotyping Procedure
To detect the CYP2D6*3, *4, *6 and *10 alleles, a 1560 base pair (bp) fragment containing exons 1 and 2 (F1-2) and a 1560 bp containing exons 3 to 6 (F3-6) were amplified by polymerase chain reaction (PCR). Briefly, the F1-2 fragment was amplified using the following primers: 5'-GGTGTATGAGCCTAGCTGG-3' (forward) and 5'-GCCTGTTTCATGTCCACGAC-3' (reverse). The F3-6 fragment was amplified by using the following primers: 5'-GGTGGATGCACAAAGAGTGGG-3' (forward) and 5'-TCCTGGTCAAGCCTGTGCTTGGAG-3' (reverse). The PCR amplification reaction (25 µL) contained 0.2 mM deoxyribonucleoside triphosphates, 1.5 mM MgCl2, 0.5 µM of each primer, ~50 ng genomic DNA, and 2.5 U of Taq DNA polymerase (PE Applied Biosystems, Foster City, Calif). Thermocycling conditions (PE Applied Biosystems 9600) were as follows: initial melting step at 95°C for 2 minutes, 30 cycles of 94°C for 30 seconds, 62°C for 30 seconds, and 70°C for 2 minutes, followed by 70°C for 5 minutes for the final extension.

Detection of the CYP2D6*3 allele. The F3-6 fragment was used for a subsequent nested PCR. A PCR-based test for detecting the A2637 deletion developed by Sachse et al31 was applied with the use of the following primers: 5'-GCTGGGGCCTGAGACTT-3' (forward) and 5'-GGCTGGGTCCCAGGTCATAC-3' (reverse). The PCR reaction conditions were as follows: 1 minute of denaturation at 95°C, 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 70°C for 20 seconds, followed by 70°C for 5 minutes for final extension. The resulting PCR products were digested with BsaAI. The variant allele generated 2 bands, a 180 bp band and a 20 bp band, whereas the reference allele remained uncut (200 bp). The resultant fragments were electrophoresed in 3% agarose gel containing ethidium bromide. The bands were visualized by UV transillumination.

Detection of the CYP2D6*4 and CYP2D6*6 alleles. The F3-6 fragment was used for nested PCR to detect the *4 and *6 alleles. To identify the G1934 mutation of the *4 allele and the T1795 deletion of *6 allele, a PCR-based test developed by Sachse et al31 was applied with the use of the following primers: 5'-CCTGGGCAAGAAGTCGCTGGACCAG-3' (forward) and 5'-GAGACTCCTCGGTCTCTCG-3' (reverse). The PCR reaction conditions were as follows: an initial denaturation step for 2 minutes at 95°C, 30 cycles of 94°C for 30 seconds, 67°C for 15 seconds, and 70°C for 20 seconds, followed by 70°C for 5 minutes for the final extension. The resulting PCR products were digested with BstNI. The reference allele generated 2 bands of 190 and 163 bp, whereas the CYP2D6*4 allele remained uncut (353 bp). The CYP2D6*6 allele generated 3 bands of 190, 139, and 23 bp. The resultant fragments were electrophoresed in 3% agarose gel containing ethidium bromide and visualized by UV transillumination.

Detection of the CYP2D6*10 allele. The F1-2 fragment was also used for a subsequent nested PCR to detect the *10 allele. To identify the presence of the CYP2D6*10 allele, the following primers were used: 5'-TCAACACAGCAGGTTCA-3' (forward) and 5'-CTGTGGTTTCACCCACC-3' (reverse). The PCR reaction conditions were as follows: 2 minutes at 95°C, 30 cycles of 94°C for 30 seconds, 56°C for 10 seconds, and 70°C for 15 seconds, followed by 70°C for 5 minutes for the final extension. The resulting PCR products were digested with HphI.31 The C188T mutation generated 3 bands of 262, 100, and 71 bp, whereas the reference allele generated 2 bands of 362 and 71 bp. The resultant fragments were electrophoresed in a 3% agarose gel containing ethidium bromide. The bands were visualized by UV transillumination.

Statistical Analysis
The statistical analysis was conducted by SPSS II software (for Windows; SPSS Science Inc, Chicago, Ill). Significant MR differences between Tepehuano Amerindians and Mestizos, as well as differences in allelic frequencies and genotypes between the groups, were detected by the Mann-Whitney U test and Fisher exact test.32 The effects of age, body mass index, gender, and genotypes on the MR were analyzed with a lineal regression. A multiple regression analysis was performed to assess the strength of the phenotype-genotype association. The significance limit used in all analysis was {alpha} = .05. For assessment of deviations of allelic frequencies from Hardy-Weinberg equilibrium, a {chi}2 test33 was used.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phenotyping
A total of 58 Tepehuano Amerindians and 88 Mestizos were phenotyped. All studied subjects were clinically healthy with normal kidney and liver function. Pronounced interethnic differences in the distribution of the DM/DX MR were found. The mean log MR (LMR) for each individual from both populations was calculated, and the results were plotted on frequency histograms and on cumulative frequency distribution graphs (Figures 1A and 1B, respectively). The Tepehuano Amerindian MR histogram revealed a unimodal distribution of CYP2D6 activity for the log DM/DX MR, whereas the Mestizo histogram revealed a bimodal distribution, and a distinct population subgroup was apparent. The median MR values differed between the Tepehuanos and Mestizos (0.0210 vs 0.0171; P < .05). The limits were 0.0123 to 0.0748 (LMR =–1.91 to –1.13) in the Tepehuanos and 0.0068 to 3.21(LMR =–2.16 and 0.51) in Mestizos. By the use of the established white-derived antimode value of 0.3 to define the PM phenotype,27 6 of the 88 Mestizos (6.8%) were classified as PMs. In contrast, no PMs were found in the Tepehuano Amerindian population. In addition, compared with 10.2% of the Mestizo population, only 6.9% of the Tepehuano Amerindian subjects had MR values between 0.03 and 0.3, which was the IM group inclusion criterion.28 Inspection of the histogram indicates that the PMs Mestizos constitute a subpopulation; furthermore, the antimode of the white subjects can be used in the assignment of the phenotypic status in this population.


Figure 1
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  		Figure 1. (A) histogram of log metabolic ratio (Log MR); (B) cumulative frequency distribution of Tepehuano Amerindian and Mestizo volunteers' MR.

 
Genotyping
The frequencies of the CYP2D6*3, CYP2D6*4, CYP2D6*6, and CYP2D6*10 alleles were determined among the 110 Mestizo and 85 Tepehuano Amerindian subjects. The genotype distribution differed significantly (Fisher test, P < .001) between Mestizos and Tepehuanos Amerindians. After the *1/*1 genotype (0.736), the most frequent genotypes in Mestizos were *1/*4 (0.173) and *4/*4 (0.036). The *1/*4 genotype was observed in only one Tepehuano Amerindian individual (0.012). The remaining Tepehuan Amerindians were *1/*1.

The CYP2D6 allelic frequencies are shown in Table I. Among the variant alleles tested in the Mestizo group, the most common was the *4 (0.131), followed by *10 (0.023) and *3 (0.009). In the Tepehuano Amerindian group, the *4 allele was the only non *1 allele identified; its frequency was 0.006. The *6 allele was not found in either group. The distribution of genotypes in Mestizos was in equilibrium, according to the Hardy-Weinberg principle. Because the frequency of the *4 allele in Tepehuano Amerindians was less than 1%, a Hardy-Weinberg equilibrium test was not performed.


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  		Table I Comparison of CYP2D6 Allelic Frequencies in 85 Tepehuanos and 110 Mestizos

 

Relationship Between Genotyping and Phenotyping
A total of 42 Tepehuano Amerindians and 88 Mestizo subjects were genotyped and phenotyped. From the Tepehuano Amerindian subgroup, all had the *1/*1 genotype. Forty individuals were identified as EMs (MR < 0.03), and only 2 individuals were identified as IMs. In this group, the genotypic and phenotypic data were in agreement. Of the 6 PMs identified in the Mestizo group, 4 were homozygous for the *4 allele, 1 was heterozygous for *1 and *4, and another who manifested the highest rate of DM metabolism of all subjects tested in this study had the *1/*1 genotype (Table II). Despite the discrepancy between the phenotype and genotype in these last 2 cases, the association remains highly significant (r2 = .45; P = .005).


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  		Table II Relationship Between Phenotyping and Genotyping in 88 Mestizos

 

Some individuals exhibited phenotype/genotype dissociation. One of the EM Mestizo subjects had a MR of 0.023 and *4/*10 genotype, which is much closer to the transition point between extensive and intermediate metabolism (0.03). On the other hand, 6 of the 9 IM subjects had the *1/*1 genotype (Table II). These discrepancies could be attributed to the presence of other alleles with intermediate function (eg, *9, *17, or *41) that were not tested for in this study.

We completed a lineal and multiple regression analysis to examine the effects of age, body mass index, and gender on the MR, in both groups. None of these factors were associated significantly with CYP2D6 activity. A limited association between gender and genotypes was observed in the Mestizo group. A multiple regression analysis that considered only the *4 allele revealed that this allele predicts CYP2D6 enzymatic activity, and the presence or absence of this allele was sufficient to explain 45% of variability of phenotype after adjusting for gender (Table III). No differences were found in phenotype between men and women, only that gender was associated scarcely with the MR, which we show in Table IV. Our data are consistent with those of other groups of investigators34,35; however, this study does not preclude the possibility that probes for CYP2D6 other than DM may identify effects of gender in CYP2D6 activity.


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  		Table III Multiple Regression Analysis of the Relationship Between Metabolic Ratio and Variant Allele in Mestizos

 

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  		Table IV Simple Regression Results Modeling the Metabolic Ratio by Age, Body Mass Index, and Gender in Tepehuanos and Mestizos

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study is the first investigation of drug-metabolizing phenotype and CYP2D6 genotype completed in Mexican Amerindians and Mestizos in the State of Durango. The MR data indicate that the Tepehuanos Amerindian population studied is characterized by the absence of PMs. This finding is evidenced by the absence of an antimode (Figure 1A), as well as by the nature of the cumulative frequency distribution (Figure 1B). The highest value found for MR among Tepehuano subjects was 0.0748 (LMR =–1.13), which about one fourth of the antimode value reported for persons of European descent in whom the value of 0.3 (LMR =–0.5) has been assigned.27 These results are similar to those observed in Cuna Amerindians, who also had an absence of PMs (0/210).20 It is interesting that this population is characterized by a low racial admixture (ie, less than 1%).20 In contrast, in other Amerindian populations such as the Ngawbe and Embera from Panama and Colombia, the PM frequencies were higher, 4.4%, and 2.2%, respectively.18,19 The Ngawbe constitute the largest extant and intact Amerindian population with little or no racial admixture.36,37 These differences have been explained by Arias et al,20 who hypothesized that among the common ancestors of the Amerinidans there was a defined proportion of PMs, and after their divergence, the selective pressures acted in a different manner or intensity, resulting in a decrease in the number of subjects with deficient metabolic activity. Arias and colleagues20 have speculated that the environment acts most strongly through food intake, which was qualitatively different in some way and favored certain differential changes in this metabolic pathway. XbaI restriction fragment length polymorphisms haplotypes were very homogeneous in Amerindians, because the only fragment that hybridized with the CYP2D6 cDNA probe was the 29 kb (not 42/44 kb or 11.5/13 kb).19 This finding indicated no gene cluster recombinations that generate insertions or deletions.19 This characteristic is found preferentially in whites, which present a significant number of PMs.

In contrast to the Tepehuano Amerindian population, 6.8% of Mestizos were characterized as PMs, which is similar to the widely quoted range of 5% to 10% for whites. However, the frequency obtained in this study is higher than both the 4.5% rate cited in a preliminary report for a group of 22 Mexican Americans in southern Texas17 and the 3.2% rate found in a group of Mexican American subjects from Los Angeles County, California.15

In nonwhite populations, the PM and UM phenotypes of CYP2D6 substrates occur with differing prevalences. Among Asians, there are very few PMs and UMs (less than 1%), but the IM phenotype frequency is very high (50%).38,39 By contrast, 20% to 30% of Africans may be considered CYP2D6 IMs.14,40 In our group of Mestizos, the IM frequency was 10.2%, which is similar to that reported for whites (10%-15%).

We found that the CYP2D6 allele frequencies differed between Tepehuanos and Mestizos (P = .05). Comparisons of our findings to those of other studies of Hispanic populations and Amerindian tribes can be seen in Table V. In this study, the only allelic variant identified in Tepehuanos was the inactive allele *4, with a frequency of 0.006, which is similar to the frequency reported for Asians (less than 1%).39,41,42 The *4 frequency is also low among the Mapuche Amerindians of Chile (0.036).24 This rate is lower than the frequencies observed for Ngawbe and Embera Amerindians (0.171 and 0.14, respectively).19 All PMs in these groups possessed either *4 or *6 alleles, and there were no disagreements between genotypic and phenotypic data. The differences between allelic frequencies among Amerindians are likely to have arisen with their genetic divergence over time. According to a report by Jorge et al,43 the presence of the *4 allele in Amerindians suggests that this allele had a far more ancient evolutionary history than previously thought.


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  		Table V CYP2D6 Allele Frequencies in Hispanic Populations and Amerindian Tribes

 

The PM phenotype in Mestizos could be accounted for by the presence of the inactive *4 allele, which had a frequency of 0.14, similar to the 0.156 frequency reported in a study of Nicaraguans, and the 0.17 frequency cited for Spaniards.44 However, the frequency of this allelic variant in the Mestizos in this study was higher than that reported in a study of Mexican Americans (0.103). The high white genetic influence in our sample of Mestizos is consistent with the genetic admixture reported previously among Mexicans and Mexican Americans. Cosmopolitan populations from large cities of Mexico (Nuevo León, Jalisco, and the Distrito Federal) consist of gene pools derived from these 3 primary sources: mostly Spanish-European (50%-60%) and Amerindian (37%-49%) contributions, as well as a minor African (1%-3%) contribution.26,33,45 Hanis et al46 reported a similar heritage for Mexican Americans in the United States, who appear to be 31% Native American derived, 61% Spanish derived, and 8% African derived. Likewise in Costa Rica, the proportions of genes from European, Amerindian, and African ancestry were 61%, 30%, and 9%, respectively.47 The general pattern described above is a prominent European ancestry, followed by Indian and African ancestry. However, in many other Latin American countries, the general pattern has a higher African ancestry. For example, the admixture rates for native Puerto Ricans and Cubans are reported to be 46% African, 4% Amerindian, and 50% Spanish.48 Puerto Ricans and Cubans in the United States presented African inheritance rates of 37% and 20%, respectively, with an 18% Amerindian inheritance.46 Nevertheless, although Latin American countries are truly trihybrid, there are fundamental differences among them in the proportion of the gene flow from the ancestral populations.

Another CYP2D6 allele associated with decreased enzyme activity is 2D6*10. In whites, the CYP2D6*10 allele has a frequency of 2.8%49 and is present in 10% to 20% of individuals with IM phenotype.50 In Asiatic populations, the frequency of CYP2D6*10 exceeds 50% and is responsible for a shift in the median MR of debrisoquine/sparteine to higher values and for the lower average metabolic clearance of CYP2D6 drugs in Asians compared to whites.41,51 The *10 allele was not identified in our population of Tepehuano Amerindians, and its frequency was likewise reported to be very low (0.018) in the Mapuche Amerindians. In Ngawbe and Embera Amerindians, however, the *10 allele frequency was higher (0.175 and 0.069, respectively). In our study, the *10 allele was present in the Mestizo group, with a frequency of 0.02, close to that found in whites. However, none of the Mestizo identified as IMs had this allele, which indicates that in our population, other alleles with intermediate function were responsible for this phenotype.

In conclusion, we observed genetic differences between Tepehuano Amerindians and the Mestizos of Durango, Mexico, that were consistent with previous observations. The oxidative metabolism of Tepehuano Amerindians had a narrower distribution, which indicates less variability, than the Mestizos. We found no PM individuals in the Tepehuano group and 6.8% PMs in the Mestizo group. Differing allele distributions (P < .001) and genotype frequencies (P < .001) were also observed between these populations. We observed that Tepehuano Amerindians differed markedly from Asians in *10 allele frequency. That is, *10 is frequent in Asians but not present in Tepehuanos. Furthermore, the allelic frequencies in Tepehuanos were found to differ from those of other Amerindian groups. Nebert52 has suggested that such notable differences in allelic frequencies may be the result of 2 possible selective pressures, the first being differences in diet that have evolved over thousands of years and the second being the evolution of balanced polymorphisms, such as in alleles that influence resistance to bacterial or viral infections.53,54

Finally, knowing the genotype of subjects involved in a clinical study of drugs that are CYP2D6 substrates can confer important advantages in clinical safety. Moreover, this information could have a positive impact on the outcome of therapeutic decisions of dosage and determinations of the therapeutic window that are better suited to individual patients.

The results of the study are of great relevance because they contribute, for the first time, to the knowledge of genetic polymorphism of CYP2D6 of 1 of the 56 Amerindian tribes from northern Mexico that had not been reported. This work also offers the opportunity to make studies in the future that include genetic polymorphism in the main ethnic groups of Mexico. These studies will be able to demonstrate the possible differences determined by ethnographic factors or the different lifestyles of the studied ethnic groups that have health implications from the anthropological point of view.

The implications of the results of the drug use study that were metabolized by CYP2D6 had direct benefits for the studied subjects because of the result of his or her genotype and phenotype. This result should be considered by his or her physician when the physician is required to use a drug that is eliminated by this metabolic pathway regulated by the CYP2D6 gene. Also, the results will be useful for the best decision making in public health about the rational use of drugs in the studied populations and for each individual.

The authors gratefully acknowledge the CONACYT-Mexico Project G34049 [GenBank] -M for financial support. The experiments comply with the current laws of Mexico, where they were performed, inclusive of ethics approval.

DOI: 10.1177/0091270006287586


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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