Allele frequencies of single nucleotide polymorphisms of clinically important drug-metabolizing enzymes CYP2C9, CYP2C19, and CYP3A4 in a Thai population

Prior knowledge of allele frequencies of cytochrome P450 polymorphisms in a population is crucial for the revision and optimization of existing medication choices and doses. In the current study, the frequency of the CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP2C19*6, CYP2C19*17, and CYP3A4 (rs4646437) alleles in a Thai population across different regions of Thailand was examined. Tests for polymorphisms of CYP2C9 and CYP3A4 were performed using TaqMan SNP genotyping assay and CYP2C19 was performed using two different methods; TaqMan SNP genotyping assay and Luminex x Tag V3. The blood samples were collected from 1205 unrelated healthy individuals across different regions within Thailand. Polymorphisms of CYP2C9 and CYP2C19 were transformed into phenotypes, which included normal metabolizer (NM), intermediate metabolizer (IM), poor metabolizer (PM), and rapid metabolizers (RM). The CYP2C9 allele frequencies among the Thai population were 0.08% and 5.27% for the CYP2C9*2 and CYP2C9*3 alleles, respectively. The CYP2C19 allele frequencies among the Thai population were 25.60%, 2.50%, 0.10%, and 1.80% for the CYP2C19*2, CYP2C19*3, CYP2C19*6, and CYP2C19*17 alleles, respectively. The allele frequency of the CYP3A4 (rs4646437) variant allele was 28.50% in the Thai population. The frequency of the CYP2C9*3 allele was significantly lower among the Northern Thai population (P < 0.001). The frequency of the CYP2C19*17 allele was significantly higher in the Southern Thai population (P < 0.001). Our results may provide an understanding of the ethnic differences in drug responses and support for the utilization of pharmacogenomics testing in clinical practice.

. Genetic polymorphisms within CYP2C9, CYP2C19, and CYP3A4 significantly affect most clinically used drugs, and the prediction of phenotypes by detecting polymorphisms of these CYP genes is useful in drug therapy. The CYP2C9*2 (R144C) and CYP2C9*3 (I359L) alleles are the clinically relevant defective variants associated with decreased enzyme activity and impaired drug metabolism phenotypes. CYP2C9 metabolizes approximately 25% of clinically-administered drugs including phenytoin, warfarin, glipizide, tolbutamide and non-steroidal anti-inflammatory drugs (NSAIDs) 2,3 . The frequency of CYP2C9*2 is 12.68% in European, 4.60% in South Asian, 2.35% in African, and < 1% in East Asian ancestry. The frequency of CYP2C9*3 is 11.31% in South Asian, 6.88% in European, 3.38% in East Asian, and 1.26% in African ancestry 6 .
Among the CYP2C19 polymorphisms, CYP2C19*2, CYP2C19*3, CYP2C19*6, and CYP2C19*17 are the common variants responsible for interindividual differences in the pharmacokinetics and response to CYP2C19 substrates 7 . CYP2C19 is involved in the metabolism of tricyclic antidepressants, selective serotonin reuptake inhibitors, voriconazole, and clopidogrel 8 . The allelic frequencies of CYP2C19*2 and CYP2C19*3 responsible for the majority of PM phenotypes in the metabolism of CYP2C19 substrate drugs are higher in Asian populations 9 .
CYP3A4 is responsible for the metabolism of ∼50% of drugs used therapeutically such as clarithromycin, erythromycin, diltiazem, itraconazole, ketoconazole, ritonavir. There is considerable interindividual variability in CYP3A4 activity 10 . CYP3A4 rs4646437 polymorphism was related to the risk of hypertension in the Chinese population 11 . Assessment of inter-individual differences in the allele and genotype frequencies in the Thai population is important for the better outcome of pharmacotherapy. In this study, we have analyzed the frequency of specific gene polymorphisms of CYP2C9, CYP2C19, and CYP3A4 (Table 1) in the Thai population. We also compared the the real-time polymerase chain reaction technique (real-time PCR) (ViiA7) and Luminex beadbased multiplex assays for the detection of CYP2C19 variants.

Methods
Subjects. For the allele frequency of CYP2C9, CYP2C19, and CYP3A4 polymorphisms, a total of 1,205 blood specimens of unrelated healthy donors were obtained from the Thai National Health Examination Survey. The samples were selected from five regions of Thailand, including Northern, Northeastern, Central, Southern, and Bangkok. For the comparison between the real-time PCR technique (ViiA7) and Luminex bead-based multiplex assays for the detection CYP2C19 variants, voriconazole-treated patients (N = 180) were recruited between 2012 to 2015 from the Division of Infectious Disease, Department of Medicine, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Thailand. This study was approved by the ethical committee of the Ramathibodi Hospital, Faculty of Medicine, Mahidol University (MURA2013/292/SP 6 ) and conducted in accordance with the Declaration of Helsinki. The study protocol was clearly explained to all participants and/or their legal guardians, and informed consent was obtained before the study.

Statistical analysis.
Statistical analyses were performed using the SPSS software package (SPSS version 18.0 for Windows, SPSS Inc., Chicago, IL, https:// www. ibm. com/ produ cts/ spss-stati stics ). The genotype distributions were evaluated for Hardy-Weinberg equilibrium by using Fisher's exact test. Allele frequencies in the Thai population among the different regions of Thailand were compared using the χ2-test. P-value < 0.05 was considered significance. Cohen's Kappa (κ) was used to calculate the agreement between the Luminex xTAG v3 and TaqMan SNP genotyping methods. Table 1. Locations and effects of CYP2C9, CYP2C19, and CYP3A4 polymorphisms (adopted from 12,13 ). *rs number-reference Single Nucleotide Polymorphism (SNP) ID assigned by the SNP database at National Center for Biotechnology Information (dbSNP).

Polymorphism
Nucleotide change rs number* Location, protein effect Enzyme activity

Results
Genotype and allele frequencies of polymorphisms of CYP2C9, CYP2C19, and CYP3A4 in Thai. The genotype and allele frequencies for all the polymorphisms screened are shown in Table 2. All the genotypes were in Hardy-Weinberg equilibrium (P > 0.05). The sample size for the analysis was 1,205. The major allele was defined as the most commonly occurring allele in the population. The population allele frequencies were calculated from genotype numbers. In our sample of Thai unrelated healthy population, two individuals were heterozygous for the CYP2C9*2 variant. CYP2C9*2 variant allele frequency was 0.08%. Carriers of the CYP2C9*3 variant allele were found in 125 individuals; two of these individuals were homozygous for the CYP2C9*3 variant while 123 individuals were heterozygous for the CYP2C9*3 variant. CYP2C9*3 variant allele frequency was 5.27%.
Carriers of the CYP3A4 (rs4646437) variant allele were found in 595 individuals in this study population; 89 of these individuals were homozygous for the rs4646437 variant while 506 individuals were heterozygous for the rs4646437 variant. The allele frequency of the rs4646437 variant allele was 28.5%.  Table 3 and Fig. 1. The frequency of the CYP2C9*3 allele was significantly lower among the Northern Thai population (P < 0.001). The frequency of the CYP2C19*17 allele was significantly higher in the Southern Thai population (P < 0.001). Overall, the prevalence of CYP2C19 alleles was significantly different among the different regions of Thailand (P < 0.001). The frequency of the CYP3A4 rs4646437 allele in different regions of Thailand was found to be comparable.

Discussion
The interethnic variability in the drug metabolism capacity and pharmacokinetics between populations is the impact of the differences in the allele distribution of pharmacogenes 14 . Drug metabolizing enzymes CYP2C9, CYP2C19, and CYP3A4 are polymorphic and cause substantial interpersonal differences in drug and metabolite exposure 15 . To date, numerous studies have analyzed the frequencies of the polymorphisms in CYP2C9, CYP2C19, and CYP3A4 in the Thai population; yet the available frequency data have been derived from a small sample of the Thai population. In this study, we report the frequency of specific gene polymorphisms of CYP2C9, CYP2C19, and CYP3A4 in 1,205 Thai subjects. Furthermore, this works are able to represent the majority allele frequencies of Thai people. Since the samples were recruited to cover all the regions in Thailand.
Regarding CYP2C9, the frequencies of the allelic variants CYP2C9*2 and CYP2C9*3 in the present study were consistent with the figures reported for East Asians in PharmGKB (https:// www. pharm gkb. org/ varia nt/ PA166 153972, accessed September 06, 2020). The frequency of CYP2C9*3 was slightly higher in our study compared to what has been reported in PharmGKB for East Asians (5.27% vs 3.37%). About differences in the frequency of alleles across different regions of Thailand, the frequency of the CYP2C9*3 allele was significantly lower among the Northern Thai population ( Table 3). The CYP2C9*2 allele was observed with a frequency of 0.08% in our study. By contrast, the CYP2C9*2 allele frequency was zero in previous reports that were examined in Thais 16,17 . Pratt et al. reported that CYP2C9*2 and CYP2C9*3 have decreased function and are the most well-characterized variant CYP2C9 alleles compared with other alleles 18 . Both CYP2C9*2 and CYP2C9*3 alleles have been reported at higher frequencies among the Caucasian population 19 . Concerning the metabolic predicted phenotypes, the frequencies of CYP2C9 IM and PM were 10.37% and 0.17%, respectively. Interestingly, these frequencies were more consistent with the Han Chinese population (8.23% IM and 0.16% PM) 20 . The Clinical Pharmacogenetics Implementation Consortium (CPIC) has published recommendations for pharmacogenetic testing on the CYP2C9 gene for dosing of phenytoin, (NSAIDs), and warfarin [21][22][23] .
In our research, we determined and compared the frequency of four pharmacologically relevant CYP2C19 variants among Thai populations. The frequencies of CYP2C19*2, CYP2C19*3, CYP2C19*6, and CYP2C19*17 were 25.6%, 2.5%, 0.1%, and 1.8% respectively. We observed the significant variability in the distribution of CYP2C19 variants across different regions of Thailand. The CYP2C19*2, CYP2C19*3, and CYP2C19*6 alleles are the no-function alleles and characterized as PM, while the CYP2C19*17 allele is associated with high CYP2C19 activity and characterized as UM [24][25][26][27] . The frequency of the CYP2C19*2 allele in our study is less than the frequency in Asian populations (~ 30%) but higher than the allele frequency of 18% in Africans and Europeans 26 . The prevalence of the CYP2C19*3 allele presented in our study is lower than that of Han Chinese (7%), Koreans (12%), Japanese (11%), and Vietnamese (14%) 28 . The distribution of CYP2C19*2 and CYP2C19*3 alleles in Northeastern Thailand are consistent with the prior study of Tassaneeyakul et al. conducted in 774 Thais 29 . We observed a very low frequency of the CYP2C19*6 allele in our study. The prevalence of the CYP2C19*6 allele is very low, and sometimes absent in general populations 30 . The CYP2C19*17 allele, associated with accelerated metabolism, is prevalent at 18.2% in African-American, 6.2% in Asian, 15.8% in Caucasian, 15.2% in Hispanic, and 19.8% in Ashkenazi Jewish populations 30 . The frequency of the NM, IM, PM, and UM phenotypes was 48.22%, 42.98%, 6.64%, and 2.16% respectively. Notably, a similar trend was observed for the IM among the Thais and Han Chinese in previous studies 20, 31 . The CYP2C19 metabolic phenotypes are uniformly distributed Table 3. Prevalence of CYP2C9, CYP2C19, and CYP3A4 alleles in different regions of Thailand (n = 1205). *Allele frequencies were compared in different regions of Thailand using χ2 test, P-value < 0.05. BKK Bangkok, NE Northeastern. www.nature.com/scientificreports/ across African-American, Asian, Caucasian, Hispanic, and Ashkenazi Jewish populations 30 . Currently, there are dosing guidelines from the CPIC for CYP2C19 genotyping and the personalization of clopidogrel, tricyclic antidepressants, selective serotonin reuptake inhibitors, voriconazole, and proton pump inhibitor therapy [32][33][34][35][36] . The CYP3A4 enzyme is the most abundant metabolic enzyme in the human liver, encoded by the CYP3A4 gene 37 . A lot of studies have reported the effects of CYP3A4 rs4646437 on drug metabolism and outcomes of drug therapy [38][39][40][41] . The frequency of CYP3A4 rs4646437 in our study was 28.5% in the Thai population and 29.9% in the northeastern Thai population, which is slightly higher from the findings in another study conducted in the northeastern Thai population 42 . According to the 1000 Genomes project, the CYP3A4 rs4646437 is highly prevalent among African, East Asian, and South Asian populations, but not so frequent among the Europeans (https:// www. pharm gkb. org/ varia nt/ PA166 157391).
In addition to the allele, genotype, and phenotype frequencies in our study subjects, we also compared the Luminex xTAG v3 and TaqMan SNP genotyping methods for the detection of CYP2C19 variants (Supplementary

Conclusions
In conclusion, our findings confirm the interregional differences in the CYP2C9, CYP2C19, and CYP3A4 allele and genotype frequencies among Thais. Our results further support the need to identify individuals who have altered pharmacokinetics for CYP2C9, CYP2C19, or CYP3A4 substrates so that prescribers could personalize appropriate dosages for optimal drug responses. TaqMan SNP genotyping assay is better than the Luminex xTAG v3 system in the genotyping of CYP2C19 variants in the Thai population.