Exploring Variation in Known Pharmacogenetic ...

Pharm Res DOI 10.1007/s11095-016-1990-5

RESEARCH PAPER

Exploring Variation in Known Pharmacogenetic Variants and its Association with Drug Response in Different Mexican Populations Vanessa Gonzalez-Covarrubias 1 & José Jaime Martínez-Magaña 2 & Regina Coronado-Sosa 1 & Beatriz Villegas-Torres 3 & Alma D. Genis-Mendoza 2 , 4 & Pablo Canales-Herrerias 1 & Humberto Nicolini 2,4 & Xavier Soberón 1

Received: 5 May 2016 / Accepted: 28 June 2016 # Springer Science+Business Media New York 2016

ABSTRACT Purpose Information on genetic variants that affect the pharmacokinetics and pharmacodynamics (PK/PD) of drugs in different populations from Mexico is still an ongoing endeavor. Here, we investigate allele frequencies on pharmacogenetic targets in Mexican Mestizos and Natives from three different States and its association with drug efficacy in individuals receiving either anticoagulants or antipsychotic drugs. Methods Natives from three different states and Mestizo patients receiving acenocoumarol or antipsychotics were genotyped using the DMET microarray (Affymetrix). Results We provide a collection of genetic variants that indicate that there are 3-times more variation than similarities between populations from Mexico and major continental groups. These differences were observed in several relevant targets including ABCB1, SLCO1A1, NAT2, UGTs, TYMS, VKORC1, and NR1I3. Moreover, Mexican Mestizos also showed allele frequency differences when compared to Natives for variants on DPYD, ADH1A, CYP3A4, SLC28A3, and SLC28A1. Significant allele differences also arose among Electronic supplementary material The online version of this article (doi:10.1007/s11095-016-1990-5) contains supplementary material, which is available to authorized users. * Vanessa Gonzalez-Covarrubias [email protected]

1

Laboratorio de Farmacogenómica, Instituto Nacional de Medicina Genómica (INMEGEN), Ciudad de Mexico, Mexico

2

Laboratorio de Genomica de Enfermedades Psiquiatricas y Neurodegenerativas, Instituto Nacional de Medicina Genomica (INMEGEN), Ciudad de Mexico, Mexico

3

Laboratorio de Diagnostico Genomico, Instituto Nacional de Medicina Genómica (INMEGEN), Ciudad de Mexico, Mexico

4

Servicios de Atención Psiquiatrica (SAP) Secretaria de Salud, Ciudad de Mexico, Mexico

the three Native groups here studied, mostly for transporters of the ABC-binding cassette and the solute carrier gene family. Finally, we explored genotype-drug response associations and pinpointed variants on FMOs (coumarins), and GSTM1 (haloperidol). Conclusions These findings confirm previous results and further delve into the pharmacogenetics of Mexican populations including different Native groups.

KEY WORDS antipsychotics . coumarins . Mexican populations . pharmacogenetics

ABBREVIATIONS ADME ADRs FMO GSTM1 INR PD PK

Absorption, distribution, metabolism, and elimination Adverse drug reactions Flavin monooxygenase Glutathione S-Transferase Mu 1 International normalized ratio (INR) is a calculation based on prothrombin time Pharmacodynamics Pharmacokinetics

INTRODUCTION Population variation in genes coding for pharmacokinetic (PK) or pharmacodynamic (PD) targets has long been acknowledged (1). Today, less than 5% of pharmacogenetic studies include samples from Mexican populations (2016, NCBI searches using the terms, Bpharmacogenetics^ pharmacogenomics^, BMexican Natives^, BMexico^, or BHispanics^), the 1000 Genomes project included 26 Mexicans from Los Angeles (2), and databases show that allele frequency on these do not necessarily represent the geographical diversity of Mexico. For example, previous investigations

Gonzalez-Covarrubias et al.

on CYP2C9 showed that 79% of Mexican Mestizos are extensive CYP2C9 metabolizers, but up-to 92% of Natives would show this phenotype, confirming that genetic variation is apparent among states and does not reflect average frequencies of other populations (3–5). Another relevant example is the case of CYP2D6, for which Natives have an ultrarapid CYP2D6 metabolism in a higher proportion (>12%) that in Mexican Mestizos, due to a duplication of CYP2D6 active alleles (6). Current knowledge about genes and genotypes that affect drug safety and efficacy have been reported for major continental populations thus, the use of validated variants to tailor drug use cannot be freely extrapolated to Mexicans (7,8). Therefore, the collection and description of the genetic diversity in peoples of the Americas is part of an ongoing endeavor to dissect what genes and variants affect drug safety and efficacy (9). In this regard, a recent study assessed genetic variance in PK/PD targets for 45 Zapoteco Natives from Oaxaca and 225 Mexican Mestizos. Bonifaz et. al showed that although levels of genetic differentiation with other populations were low and Native Americans showed the lowest haplotype diversity, there were large frequency differences for several pharmacogenetic relevant genes, (10). Albeit, the classification of genetic variants for major pharmacogenetic markers in Mexican populations are under investigation, correlations among these variants and drug phenotypes are still scarce (11). Here, we investigate allele frequencies of pharmacogenetic variants in three Native groups, Tepehuano, Mixteco, and Maya, and in Mexican Mestizos with the latter receiving drug treatment. Results from this study are in agreement with previous reports including differences between Mexican populations and Africans or Europeans for variants with large genetic divergence such as those on VKORC1 (10), and highlight the relevance of apparent ancestry differences in PK/PD genes including CYP3A4 and DPD. We also list allele frequency differences and similarities of three Native populations and Mexican Mestizos and comparisons to three major continental populations: Northern Europeans from Utah (CEU), Chinese Han from Bejing (CHB), and Africans from Yoruba (YRI). Finally, we explored pharmacogenetic associations for coumarin or antipsychotic drug response in Mestizos.

MATERIALS AND METHODS Samples One-hundred DNA samples from our various research cohorts were available for microarray analysis. Of these, 69 were from Mexican Mestizos under drug treatment, and 31 DNA samples were from Natives: Tepehuanos from the State of Durango, N = 12, Mayas from Campeche, N = 10, and Mixtecos from Oaxaca, N = 9 (Table I). All individuals signed

Table I

Demographic Characteristics of Samples Total (age, yrs)

Natives

31 (18–85y)

Tepehuanos (Durango)

12

Mixtecos (Oaxaca) Mayas (Campeche)

9 10

Mexican Mestizos & diagnosis Mexico City and near surroundings Cardiovascular disease Mexico City and near surroundings Schizophrenia Total

21 (38–85y) 48 (18–57y) 100

an informed consent, and the study protocol was approved by the Ethics Committee of the National Institute of Genomic Medicine (INMEGEN). Of the 69 DNA samples from patients under drug treatment, 21 were from the National Institute of Cardiology in Mexico City, these patients were recruited between 2006 and 2010 and were receiving warfarin or acenocoumarol for a target INR between 2 and 3 for different diagnoses including, stroke, stent use, and thrombosis. A blood sample was taken as part of routine monitoring, and DNA was extracted using the DNAeasy blood and tissue kit (Qiagen, Valencia CA). During sampling, patients were under stable coagulation for at least three consecutive INR measurements. Forty-eight patients were diagnosed with schizophrenia at The Carraci Medical Group also in Mexico City between 2003 and 2005, where they received haloperidol, or an atypical antipsychotic when a lack of response was observed for the former. Genotyping by Allele Discrimination and Microarrays To assess variation on pharmacogenetic targets we genotyped DNA samples using the Affymetrix DMET Plus Array (Affymetrix, Santa Clara, CA, USA), following the manufacturer’s instructions. Variants with more than 5% of missing data and less than 95% of genotype confidence call were eliminated, 1759 variants were considered for statistical analyses. We validated microarray results with previous genotyping results using allele discrimination in 21 samples for variants CYP2C9*2, CYP2C9*3, CYP2C9*4, CYP2C9*5, VKORC1: rs7196161, rs9923231, rs2884737, rs9934438, rs8050894, rs2359612, rs7294, and CYP4F2 rs2108622, complete agreement between allele discrimination and microarray genotypes was observed. Data Analyses Descriptive statistics, allele genotype frequencies, HardyWeinberg equilibrium, Fisher’s exact and chi-square tests

Pharmacogenetic Variants and Drug Response in Mexican Populations

to assess differences between populations were computed using Microsoft Excel 2010, R statistical programming system (http://www.r-project.org/), and the toolset PLINK [16]. Continental allele frequencies for three major populations, African (YRI), Asia (CHB), and Caucasians (CEU) were obtained from the HapMap project on the dbSNP of The National Center of Bioinformatics (www.ncbi.nlm.nih.gov), when not available minor allele frequency (MAF) data were collected from the closest geographical region reported by the dbSNP. Statistical significance was defined at P < 0.05 after adjusting for multiple testing using the false discovery rate test (FDR). Genotype-Phenotype Associations Exploratory analyses to identify a potential association between genetic variants and coumarin dose or haloperidol response were defined significant at P-value 0.05; Supplemental Table 4). Differences Among Native Populations Our sample of Mexican Natives was small (N = 31), nevertheless we assessed allele frequency differences between Mestizos and Natives and among the latter. Variants on pharmacogenetic relevant genes such as DPYD, CYP3A4, ALDH1A, and SLC28A1/3 were found to simultaneously differ between Mestizos and all three native populations (Table III). Allele frequency differences were also observed among Natives for 14 variants on ABCB4, SLC25A27, SLC22A1, CYP39A1, and ARNT all of which, simultaneously differed among Native (Supplemental Tables 5 and 9). Pharmacogenetic Variants and Drug Response

Similarities in Allele Frequencies When comparing the 1759 variants between Mexicans and the three major populations we observed allele frequency similarities for 67 variants in 45 genes: 15 variants on 10 CYPs, 16 variants on 11 transferases, 4 variants on 2 reductases and Table III Allele Frequency Differences Between Mexican Mestizos and Native Populations for Selected Variants (P < 0.05)

SNP

DPYD rs17376848 ADH1A rs12512110 CYP3A4 rs2242480 SLC28A3 rs7867504 SLC28A1 rs2242046 1

MAF1

0.052 (G) 0.104 (T) 0.422 (T) 0.436 (T) 0.208 (A)

Information on drug treatment efficacy was available for 21 patients receiving acenocoumarol, and 48 patients receiving haloperidol. Interestingly, seven patients were receiving above-average doses of acenocoumarol (>35 mg/wk) even when accounting for demographic, clinical, and genetic parameters. Preliminary genotyping utilizing allele

Alleles

MestizoMAF

MayaMAF

MixtecoMAF

TepehuanoMAF

PGx relevance

A G

0.94 0.06

0.68 0.32

0.78 0.22

0.79 0.21

PK of 5-FU

G T

0.64 0.36

0.36 0.64

0.33 0.67

0.38 0.63

intron/unknown

C T

0.65 0.35

0.41 0.59

0.33 0.67

0.33 0.67

PK of tacrolimus and carbamazepine

C T

0.65 0.35

0.95 0.05

0.94 0.06

0.88 0.13

PK of gemcitabine

G A

0.68 0.32

0.91 0.09

0.94 0.06

0.96 0.04

Gemcitabine toxicity

Global MAF source: dbSNP or 1000Genomes. P-value < 0.05 after FDR correction for all comparisons

Gonzalez-Covarrubias et al.

discrimination for CYP2C9*2 -*5, CYP4F2 rs2108622 (V433M), and VKORC1 9923231 did not explain the high dose requirements. After microarray genotyping, linear regression analyses pinpointed a significant association between CYP1A1 rs1048943, FMO3 rs2266782, FMO1 rs12954, and FMO4 rs2223477 and coumarin dose (P < 0.05, Table IV and Fig. 1). Similarly, patients diagnosed with schizophrenia (N = 48) receiving haloperidol (N = 23) were classified as responders or non-responders by standard medical criteria including SAPS (Scale for Assessment of Positive Symptoms) and SANS (Scale for Assessment of Negative Symptoms). Regression analyses, showed a significant association between 9 variants on ABCB11, SLC22A14, GSTO1, CYP2C18, CHST3 and drug efficacy, for which the presence of the variant allele was associated to a lack of pharmacological response. In addition, unwanted involuntary movements (ADRs) were associated to CYP4A11 (rs11211402), FMO4 (rs2223477), and SLC19A1 (rs1051266) (P < 0.05; Table IV and Fig. 1). Table IV Variants Associated with Drug Efficacy

Furthermore, analyses of five patients with schizophrenia that did not respond to any treatment (haloperidol, risperidone, clozapine, olanzapine, trifluoperazine, piperazine, and zuclopentixol, or their combination) showed that the frequency of the variant allele of SNPs on AOX1, CYP2C18, CYP2E1, and ABCC6 was significantly higher in resistant patients (P < 0.05, Supplemental Table 10).

DISCUSSION Endeavors to dissect the pharmacogenetic diversity for several ethnic populations have been the focus of the last few years in countries hosting a wide ethnic diversity (13–15). It is acknowledged that certain populations including those under a BHispanic^ tag, have been underrepresented in research and could be at higher risk of ADRs (3,4,16,17). Currently, about 64 pharmacogenetic studies including Mexican or BHispanic^

Anticoagulants

Allele

Mestizo MAF1

MAF patients on mean dose

MAF patients on high dose

Effect size

P-value

CYP1A1 rs1048943

AG

0.434

0.460

0.286

−8.20

0.041

CT

0.344

0.365

0.150

11.5

0.033

GA

0.287

0.285

0.357

9.40

0.040

AG

0.355

0.381

0.571

11.5

0.043

GA AG GA

0.108 0.108 0.108

Antipsychotics ABCB11 rs3770603 rs4148768 SLC22A14 CYP2C18 rs2281891 GSTO1

Alleles

Mestizo MAF1

0.102 0.102 0.102 MAF Responder

0.150 0.150 0.150 MAF Non-responder

18.3 18.3 18.3 Effect size

0.023 0.023 0.023 P-value

GA CT CT

0.198 0.224 0.455

0.100 0.100 0.370

0.360 0.360 0.710

17.1 17.1 10.1

0.029 0.029 0.041

CT

0.092

0.070

0.500

5.65

0.024

rsrs4925 CHST3 rs3856650 rs4305381 rs6783962 rs1873397

CA

0.165

0.130

0.430

25.5

0.049

GA AC TG GC

0.551 0.480 0.443 0.560

0.250 0.190 0.250 0.250

0.720 0.560 0.610 0.610

5.80 9.44 5.70 5.70

0.035 0.041 0.047 0.047

FMO1 rs12954 FMO3 rs2266782 FMO4 rs2223477 FMO6 1 rs2272797 rs7886938 rs7889839

1

Average MAF in Mexican populations using this and Bonifaz-Pena study [10]; note that for FMO6 listed variants did not comply with Hardy-Weinberg equilibrium (P ≤ 0.004). FMO6 variants MAF were: in Mexican Mestizos of Veracruz (Gulf of Mexico) MAF = 0.033 Mestizos from Guanajuato, Central Mexico M AF = 0.090, Mexico City (this study) MAF = 0.108

Pharmacogenetic Variants and Drug Response in Mexican Populations Fig. 1 Variant size effect for drug response associations.

populations report allele frequencies for a few variants on main pharmacokinetic or pharmacodynamic targets (18). More recently, significant PK/PD genetic variation among Latin American and world populations have been highlighted (10,19). These differences have also been utilized in the scope of continental population substructure (20), and they ought to be expanded to other ethnic groups, validated, and tested for its potential relevance in predicting drug response. Our observations confirmed this trend by providing a list of PK/PD variants that simultaneously differ between Mexicans and three major continental populations. Moreover, allele frequencies in Mexican populations showed significant differences for 203 variants on over 100 genes when compared to YRI, CEU, and CHB, while allele frequency similarities among all these populations were observed for only 67 variants in 45 genes. This shows that there are three times more differences than similarities on these PK/PD targets between major continental groups and Mexican populations. Differences were pinpointed on 112 genes, of which nine are of pharmacogenetic significance to the FDA, including CYP3A4, CYP2B6, CYP2C9, CYP2C19, CYP3A5, VKORC1, and TPMT. Of relevance are certain variants on CYP3A4 and VKORC1 whose MAF is 3.6, 1.3 and 1.2-fold times higher in Mexican populations compared to a global MAF (Table II). These two SNPs make an interesting pharmacogenetic pair since CYP3A4 variants are associated with an increased warfarin clearance, while the presence of the C allele on VKORC1 rs2884737 increases coumarin sensitivity; it is possible that their concomitant presence in Mexican populations may counteract each other’s deleterious effects on coumarin PK/PD. Since, variants on CYP3A4 could decrease drug exposure and variant VKORC1 rs2884747 could increase sensitivity to coumarins hence, these PK/PD variants although affect coumarin pharmacogenetics they may likely render an average phenotype for coumarin PK/PD.^

Another interesting set of variants are those on UGT2B7 and UGT1A6 (Table II), the MAF in Mexicans is 3, and 1.3fold higher compared to a global MAF, which may translate in altered metabolism of efavirenz, zidovudine, opioids, antipsychotics, valproic acid, and aspirin resistance (21,22). Also, variant rs11265572 on the nuclear receptor factor NR1I3 (CAR), which mediates the expression of CYPs and UGT1A, was 5 times more frequent in Mexican populations. This may be of relevance, since this variant has been reported to decrease tacrolimus metabolism via CYP3A5*3 expression (23). Given its high frequency, it is possible that NR1I3 rs11265572 affects the PK/PD of several other drugs more frequently in Mexicans than in other world populations, contributing to the high phenotypic variation among individuals (24). There are several other variants with a reported clinical impact with a higher allele frequency in Mexican populations that may influence drug response, a full list is presented in Supplemental Table 3. Allele frequency differences between Natives and Mexican Mestizos were also pinpointed, for example, DPYD rs17376848, associated with 5-FU toxicity and used in the clinic to individualized therapy (25,26), was significantly more frequent in Natives compared to Mestizos (MAF >0.22 vs. 0.06, P < 0.05) (27), CYP3A4 rs2242480, related to a higher tacrolimus elimination in organ transplanted patients, was also present in Natives at a significant higher frequency than in Mestizos (MAF 0.59 vs. 0.35, P < 0.05). It is apparent, that many other variants have allele frequency differences between Mestizos and Native groups; here, we only provided variants that differed simultaneously between Mestizos and these three Natives groups (Table III and Supplemental Table 9). Finally, in patients receiving coumarins or antipsychotics, a high dose or lack of response was associated with a higher frequency of the minor allele for certain variants except for FMO1 rs12954, for which the reference variant was prevalent in coumarin-resistant patients (Table IV). Hence, we may

Gonzalez-Covarrubias et al.

speculate that the accumulation of certain variants on oxidases for coumarins and phase II enzymes for haloperidol may be increasing drug clearance conveying the absence of drug efficacy. Variant CYP1A1 rs1048943 has already been correlated to coumarin response (28), but an association between coumarin dose and FMO6 variants seemed unexpected given its unreported role in pharmacogenetics. Interestingly, we observed two non-linked variants on CYP2C18 one in haloperidol non-responders and another in schizophrenic patients resistant to all medications. This may underscore the relevance of CYP2C18 in the metabolism of antipsychotic drugs (29). It is important to highlight that pharmacogenetic variation for the Native individuals here studied may not draw definitive conclusions given its small sample size; it does however, provide some insight into possible differences in allele frequency among Natives and Mestizos from Mexico.

MD. Jose Luis Lopez for their clinical assistance during patient treatment and sample management. Funding was provided by INMEGEN to the Pharmacogenomics Laboratory, project No.14/2014/I.

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CONCLUSIONS It has become common to investigate relevant PK/PD targets, in individuals from different geographical backgrounds, a strategy well documented for coumarin response (3,4), but not for all pharmacogenes, nor for all ethnic groups. Some polymorphisms can be used across different populations to aid in the implementation of genotype-guided dosing (30), but pharmacogenetic differences, and phenotypic associations among the peoples of Mexico are still under investigation (9,31–33). Pharmacological treatment is not always completely effective, it may vary depending on the therapeutic class or population studied (34), a lack of control over drug safety and efficacy motivates research to pinpoint markers to predict them. The use of genetic polymorphisms represents an approach, although incomplete, to address these predictions, since the expression of PK and PD genes may vary several hundred times among individuals, compared to other genes including Bhousekeeping^ genes (34). Investigations on the functional impact and correlation between genotypes and the expression of PK/PD genes relevant to Mexican populations ought to be the scope of future endeavors.

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ACKNOWLEDGMENTS AND DISCLOSURES The technical expertise of M.Sc. Fabiola Morales Mandujano and M.Sc. Raul Mojica Espinosa during microarray experiments is gratefully acknowledged. We are also grateful to Drs. Alejandra V. Contreras and Alfredo Hidalgo-Miranda from INMEGEN for they kindly procured DNA and microarrays for the analyses in Mexican Natives. We would also like to thank the staff at the National Institute of Cardiology, Marisol Serna Galarza, Sandra J. Rodriguez Duarte, and

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