Ethnic Background and Cyp2d6 Genetic Polymorphisms in Costa Ricans

CYP2D6 differences have already been demonstrated within Latin American populations by the CEIBA.FP Consortium of the Ibero-American Network of Pharmacogenetics (RIBEF, as per the acronym in Spanish). However, within the population of Costa Rica, no research has been conducted until now, even though this population has a trihybrid component ancestry that represents an interesting condition. Thus, the present study was aimed to determine the frequency of Ultra-rapid Metabolizers (UMs) and Poor Metabolizers (PMs) in a Costa Rican population, as well as to determine whether there are differences in the CYP2D6-predicted phenotype frequencies among three Costa Rican groups with different ethnic backgrounds. Additionally, these frequencies of PMs and UMs obtained were compared with Ibero-American populations published data. Finally, we also aimed to describe allele frequencies among different Costa Rican ethnic groups. This research has been undertaken within the framework of the RIBEF CEIBA Consortium studies on Latin American populations. A total of 385 individuals were included in the study: 139 mestizos, 197 Amerindians, and 49 Afro-Caribbeans. and *41 were also determined. For the entire Costa Rican population, the frequency of PMs and UMs was 6% and 6.5%, respectively. The percentage of UMs in the mestizo population was higher than in the Amerindian population. CYP2D6 UMs vary from 3.6% to 10.1% and PMs from 1.4% to 10.2% among three Costa Rican groups. The highest frequencies of UMs (10.1%) and PMs (10.2%) were found in the mestizo and Amerindian populations, respectively. In conclusion, the frequencies of UMs and PMs for CYP2D6 varied widely across the mestizo, Amerindian and Afro-Caribbean Costa Rican populations. Future research in this population should be oriented to identify new CYP2D6 variants through sequencing methods, as well as to determine CYP2D6 phenotype, in order to establish the phenotype-genotype relation. Finally, further studies involving genetic markers of ancestry are needed in the Costa Rican population. CYP2D6 is involved in the metabolism of widely used drugs, such as antidepressants, antipsychotics, antihypertensives, analgesics, and beta-blockers (Ingelman-Sundberg, 2005). The CYP2D6 gene, located on chromosome 22q13.1, is highly polymorphic, with alleles causing absent, reduced, normal and increased catalytic activity (CYP Alleles Nomenclature Database). Inter-ethnic differences in such cyto-chrome P450 polymorphism might be partially responsible for the variations in drug disposition among populations. During the 16 th century , people from the Iberian Peninsula arrived to different places of America leading current Costa Rican, Latin American, and Caribbean populations to have different degrees of admixture (Gaedigk …

Inter-ethnic differences in such cytochrome P450 polymorphism might be partially responsible for the variations in drug disposition among populations.During the 16 th century, people from the Iberian Peninsula arrived to different places of America leading current Costa Rican, Latin American, and Caribbean populations to have different degrees of admixture (Gaedigk et al., 2010;Llerena et al., 2012;Montané-Jaime, Lalla, Steimer, & Gaedigk, 2013).Latin American populations are products of a process of admixture, mainly including groups of Amerindian, European and African ancestry (Sans, 2000).The Costa Rican population has been described as having estimated mean ancestry proportions for European, Amerindian, and African components of 54%, 32%, and 13%, respectively (Segura- Wang, Raventós, Escamilla, & Barrantes, 2010).Therefore, it could be of relevance to determine potential differences across the multi-ethnic Costa Rican population.
A high frequency of Ultra-rapid Metabolizers (UMs) has been previously described in Spanish population (Llerena, Dorado, & Peñas-Lledó, 2009;Peñas-Lledó et al., 2012).Given the Iberian Peninsula's influence on the hybrid population, we hypothesized that a high frequency of UMs would be present in the mestizo population.It was also hypothesized that high frequencies of CYP2D6*17 would be found in the Afro-Caribbean population due to their African ancestry and CYP2D6*10 within the Amerindian population due to their Asian ancestry (Bradford, 2002).
The present study aimed to determine the frequency of UMs and Poor Metabolizers (PMs) in a Costa Rican population, as well as to determine whether there are differences in CYP2D6-predicted phenotype frequencies among three Costa Rican groups with different ethnic backgrounds.Additionally, the frequency of PMs and UMs obtained in this study was compared with published data from Ibero-American populations, and finally, this study also aimed to describe allele frequencies among different Costa Rican ethnic groups.
The inclusion criteria were: • Phenotype features: For the Amerindian population, the criteria were copper-colored skin, straight hair, slanted eyes, and short stature; in the Afro-Caribbean population, dark skin, curly hair, flat nose, and prominent cheekbones were the criteria; the Mestizo population comprised all those subjects not included in any of the aforementioned groups.• Places of residence: For the Amerindian group, the places of residence were Matambu Indian locality (Chorotega), the South and the Pacific area (Guaymi), the Atlantic Talamanca and the Pacific area (Cabecar), the Talamanca area (Bribri), the Quitirrisi and Zapaton Indian localities (Huetar), and the Margarita and Tonjibe Guatuso Indian localities (Guatuso or Maleku).The samples of the Afro-Caribbean population were collected from volunteers living in the Atlantic coastal region of Limon.The mestizo population was selected from people living in the Guanacaste region, and in the Western or Central Valley of Costa Rica (Fig. 1).The inclusion of an individual in a group excluded that individual from being part of any other population.

Predicted hydroxylation capacity group:
In order to extrapolate genetic data to metabolic phenotype information, an activity score was utilized as previously described (Gaedigk et al., 2008;Llerena et al., 2012).
The differences in CYP2D6 allele frequencies were compared using the X 2 -test and/or Fisher's exact test.P-values <0.05 were regarded as statistically significant.Hardy-Weinberg equilibrium was determined by comparing the genotype frequencies with the expected values using a contingency table X 2 statistic with Yate's correction.Statistical analyses were performed using the STATISTICA 4.3 (Stat-Soft, Tulsa, OK, USA) and GraphPad Prism 3.02 (GraphPad Software, San Diego, CA, USA) software.

RESULTS
CYP2D6 allele frequencies are given in table 1. Multiplications of active genes (wtxN, *2xN) were present in 15 individuals of the mestizo population and in seven of each of the Amerindian and Afro-Caribbean populations (Table 1).
The CYP2D6 frequencies for each activity score group are given in table 1.The entire Costa Rican population frequency of PMs and UMs were 6% and 6.5% respectively.
As expected, the percentage of UMs in the mestizo population (10.1%) was higher than in the Amerindian population (3.6%, p<0.05) (Table 1).However, the frequency of individuals classified as PMs (zero active genes) was higher in Amerindians (10.2%) than in the mestizo population (1.4%, p<0.05).The frequency of UMs (8.2%) and PMs (2%) of the Afro-Caribbean population was not different to any of the Costa Rican populations studied.
The frequencies of CYP2D6 genotypes are listed in table 2. The mestizo population showed more diversity concerning genotypes in comparison with the other Costa Rican populations studied.In all three groups, the most frequently found CYP2D6 genotypes belonged to the classification of two active genes (Table 2).Published data from Latin American populations is used to compare results with the Costa Rican populations (Table 3).

DISCUSSION
To the best of our knowledge, this is the first study in a Costa Rican population that   (Albuquerque et al., 2013), the Mexican-American (Casner, 2005), and the Colombian mestizo (Isaza, Henao, López, & Cacabelos, 2000) populations.Likewise, the frequency of UMs for the Costa Rican population is similar to those reported for the Spanish population (6.1%) (Peñas-Lledó et al., 2012).
Considering the ethnicity of the analyzed populations, the frequency of UMs in the mestizo group (10.1%) is similar to those reported in a Spanish population (6.1%) (Peñas-Lledó et al., 2012), those of a Mexican admixed population (9.1%) (López, Guerrero, Jung-Cook, & Alonso, 2005), and it is lightly greater than the percentage of UMs determined with debrisoquine in a Spanish population (5.2%, p=0.053) (Llerena et al., 2009).Moreover, the high frequency of PMs in the Costa Rican Amerindian population (10.2%) is similar to that reported in an Amerindian population from Argentina and Paraguay (12.8%) (Bailliet et al., 2007).Despite the small number of individuals in the Afro-Caribbean group, the frequencies of PMs and UMs are comparable to those of Brazilian populations with African ancestry (Kohlrausch et al., 2009;Silveira, Canalle, Scrideli, Queiroz, & Tone, 2009).Individuals carrying two inactive alleles will produce a non-functional protein.Therefore, all of them will likewise be determined as PMs in phenotyping studies.However, discordance between the identification of UMs by molecular methods and phenotype has been reported (Llerena et al., 2012;Løvlie, Daly, Molven, Idle, & Steen, 1996), so the predicted phenotype estimation needs to be confirmed with phenotyping studies.
Variability of CYP2D6 alleles was found within this Costa Rican population, in accordance with the finding that the Costa Rican population is genetically heterogeneous (Morera et al., 2003;Morera & Barrantes, 2004).The high frequency of PMs in the Amerindian group can mainly be accounted for by the presence of the null allele CYP2D6*4 in this population (22.6%) at a frequency similar to those found in Amerindian populations of Argentina-Paraguay (17.8%) (Bailliet et al., 2007).However, it is higher than those reported in Panamanian Embera (14%) and Ngwabe (17.1%) populations (p<0.05)(Jorge, Eichelbaum, Griese, Inaba, & Arias, 1999).
A limitation of this study was that the number of individuals in the Afro-Caribbean population was low (n=49).Moreover, the inclusion criteria did not include ancestry informative markers analysis (AIMs).In this sense, further studies involving genetic markers of ancestry are needed in the Costa Rican population.It is necessary to remark that even though this study reports allele frequencies of CYP2D6 for Costa Ricans, they might not be representative of the population and might have been influenced by random effects.
Furthermore, the study of populations with complex ancestry such as Latin American populations comprises the fact that novel or rare variants (Fohner et al., 2013;Gaedigk et al., 2010) might appear, leading to poor metabolism or reduced function (Montané-Jaime et al., 2013).In the future, sequencing the CYP2D6 gene in these populations might be suitable to detect relevant genetic variants.
Given the percentage of UMs found in the mestizo and Afro-Caribbean population and of PMs in the Amerindian group, it might be appropriate to follow available guidelines that provide information relating to the interpretation of CYP2D6 genotype test results to guide the dosing of different drugs (Crews et al., 2012;Hicks et al., 2013).Likewise, the information provided by this study supports that it might be appropriate to consider the development of drug treatment guidelines taking into account population ethnic background, meaning specific alleles of the population tested, to improve drug safety and efficacy in Costa Rican and Latin American populations.
In conclusion, we report here for the first time the frequency of PMs (6%) and UMs (6.5%) in a Costa Rican population.Secondly, we found a difference between the frequency of predicted UM and PM phenotype across ethnicity in Costa Ricans.

TABLE 2 (
Continued) N=2 in one individual, N=3 in one individual.+ N=2 in three individuals from the mestizo group, three from the Amerindian group, and in one individual from the Afro-Caribbean group; N=3 in two individuals from the mestizo group and one from the Amerindian group.^N=2 in three individuals from the mestizo group, three from the Amerindian group and in two individuals from the Afro-Caribbean group; N=3 in one individual from the mestizo group; N=4 in one individual from the mestizo group. #

TABLE 3
Percentages (%) of UMs and PMs for CYP2D6 predicted phenotypes and phenotypes in the Costa Rican population (n=385) and other Latino, Amerindian and Iberian Peninsula populations previously studied CCG was supported by a fellowship of the University of Costa Rica in the PhD program of the University of Extremadura.The study is part of the Research Program entitled "Genética, Ecología y Salud en los Amerindios de Costa Rica" (N˚742-93-903) and the project N˚ 742-90-416 of the University of Costa Rica.