Genetic Variations and Frequencies of the Two Functional Single Nucleotide Polymorphisms of SLCO1B1 in the Thai Population

Aim To investigate the variations and the frequencies of the SLCO1B1 gene in the Thai population. Methods Collected samples were categorized into five regions of Thailand. DNA samples were genotyped for two variants, c.388A>G and c.521T>C of the SLCO1B1, using TaqMan® real-time PCR. Results The minor allele frequencies (MAFs) of two single nucleotide polymorphisms (SNPs) were not significantly different among the five regions. The most frequent haplotype was SLCO1B1*1b (frequency: 0.654), followed by *1a (frequency: 0.217), *15 (frequency: 0.128), and *5 (frequency: 0.001). We observed a similar frequency of OATP1B1 transporter phenotypes compared to other populations. 75.85% of the Thai subjects showed normal OATP1B1 activity, 22.5% showed intermediate OATP1B1 activity, and 1.58% showed low OATP1B1 activity. Conclusion This study reported the frequencies of the SLCO1B1 variants and the subsequent OATP1B1 activity in a large cohort of Thais that can provide important information for the guidance of personalized drug therapy.


INTRODUCTION
The transmembrane protein transporters can be divided into two groups, the solute-linked carrier (SLC) superfamily or known as influx transporters, which uptake the substrate through the cells, and the ATP-binding cassette superfamily (ABC) or efflux transporters which pump the substrates out of the cells (Gong and Kim, 2013). Several groups of the influx transporters uptake a variety of drugs and organic compounds from the blood into the cell, especially, the organic anion transporting polypeptides (OATPs) which are expressed in many organs such as the intestine, liver, and kidneys. OATPs have shown an important role in clinical implications for the pharmacokinetics of many drugs, including drug absorption, distribution, and elimination (Gong and Kim, 2013;Maeda, 2015;Alam et al., 2018). Evidence of changes in OATP transport function has been found to affect the efficacy and safety of many drugs and leading to instability in drug disposition and response (Gong and Kim, 2013;Shitara et al., 2013).
Currently, there is a lack of studies reporting the frequency of the SNPs of SLCO1B1 in the Thai population. The translational decision in the clinical practices has always used the data from the reports in other populations, the Han Chinese population, for instance. The objective of this study was to investigate the regional frequencies of the two functional SNPs of SLCO1B1 in the Thai population. The findings of this study will serve as the Thai pharmacogenetic data source for decision in drug therapy in a specific group of patients, especially, in patients who will be treated with statins or other medications that are affected by these genetic variants.

Samples
In the present study, we enrolled 1,205 samples from the previous cohort of Wongkittichote et al. (2013), which were collected from August 2008 to March 2009 by the Health System Research Institute. The selected samples were then categorized into five regions of Thailand, including Northern, Northeastern, Central, Southern, and Bangkok.

Genotyping Analysis
Genotyping of SLCO1B1 polymorphisms was performed using allele-specific TaqMan ® MGB probe 5' nuclease assay with realtime polymerase chain reaction (PCR) ViiA7 ™ system (Applied Biosystems, Life Technologies). The allele-specific TaqMan ® MGB probe 5' nuclease chain reaction assay was performed with primers of SLCO1B1 c.388A>G (rs2306283; on reference sequence NM_006446.4, assay ID: C:_1901697_20) and c.521T>C (rs4149056; on reference sequence NM_006446.4, assay ID: C:30633906_10). Each 6 ml of PCR mixture contained 2 ml of genomic DNA in a concentration of 5 ng/ml, 2.5 ml of TaqMan ® Genotyping Mastermix, 0.25 ml of allelespecific TaqMan ® MGB probe and sequence-specific primer kit, and 1.25 ml of DNase-free water. The thermal cycler program started with 10 min at 95°C, followed by 50 cycles of 15 s at 92°C and 90 s at 60°C. The allelic discrimination plot was analyzed by ViiA7 ™ software (Applied Biosystems, Life Technologies). Allele 1 was labeled with VIC ® dye fluorescence, and allele 2 was labeled with FAM ™ dye fluorescence.

Statistical Analysis
The frequencies of two SNPs of SLCO1B1, c.388A>G and c.521T>C, were checked for Hardy-Weinberg equilibrium using the R statistic version 3.6.1, the R Foundation for Statistical Computing. Fisher's Exact and Chi-square tests were used to determine the statistical difference between the minor alleles and haplotype frequencies between the geographical regions of Thailand using SPSS version 18.0 for Window, SPSS Inc., Chicago, IL, United States. A p-value of less than 0.05 was considered significant.

Allele and Haplotype Frequencies of SLCO1B1 in Thai Population
The allele frequencies of the non-synonymous polymorphic variants in the coding region c.388A>G (N130D) and c.521T>C (V174A) of SLCO1B1 gene and haplotype frequencies of SLCO1B1*1a, *1b, *5, and *15 in 1,205 healthy Thai samples distributed over five regions of Thailand are shown in Table 1. All detected variations were in Hardy-Weinberg equilibrium (p>0.05). The allele frequencies of c.388A>G were similar among five regions. At the same time, the SNP c.521T>C showed the most frequency in Bangkok and the least in the Southern region, however, there were no significant differences in minor allele frequencies of these two SNPs among the five regions of Thailand. The most frequent haplotype was SLCO1B1*1b (frequency: 0.654), followed by *1a (frequency: 0.217), *15 (frequency: 0.128), and *5 (frequency: 0.001). We did not observe significant differences in haplotype frequencies among the five regions ( Table 1).

The Phenotypes of OATP1B1 Transporter Based on SLCO1B1 Diplotypes in Thai Population
The phenotypes of the OATP1B1 transporter have been assigned based on the diplotype at c.388A>G and c.521T>C of the SLCO1B1 gene. The phenotype frequencies distributed over the five regions of Thailand and worldwide are shown in Table 2.

DISCUSSION
Numerous data have reported the genetic variations of the SLCO1B1 gene for the determination of clinical drug response.  The allele frequencies of the polymorphic variations and haplotype frequencies of the SLCO1B1 gene, which are SLCO1B1*1a, SLCO1B1*1b, SLCO1B1*5, and SLCO1B1*15, have been studied in the various population groups. Among the published reports, SLCO1B1 c.388A>G (N130D) and SLCO1B1 c.521T>C (V174A) are the most commonly investigated SNPs in the various ethnic groups. This present study investigated the frequencies of these two common polymorphic variations in the Thai population distributed across five regions of Thailand. We found that allele frequencies of the c.388A>G and c.521T>C variants were close to the frequencies reported in other Asian populations, including Han Chinese, Japanese, Korean, and Vietnamese (Kim et al., 2008;Namgoong et al., 2015). When compared separately, the frequencies of c.388A>G were similar in Asian ancestry but showed differently in Asian Indians and Caucasians, while the frequencies of c.521T>C showed similarity in all ethnicity except Asian Indian and African ancestry ( Table 3 and Figure 1). The most remarkable haplotypes of the SLCO1B1 gene, SLCO1B1*5 and SLCO1B1*15, have been reported to reduce the number and function of the OATP1B1 transporter. These haplotypes not only alter the disposition of many therapeutic drugs, including various statins, but also affect the adverse events, especially statin-induced myotoxicity (0.3% in Thai population). Many reports have confirmed that the SLCO1B1*5 allele is associated with statin-induced myotoxicity in several populations (Hamann et al., 2013;Sakamoto and Kimura, 2013;Ramsey et al., 2014). Nevertheless, the frequency of SLCO1B1*5 haplotype in Asian population was very low when compared with other geographical groups (Figure 1). Published research on haplotype frequencies of the SLCO1B1*5 have shown the prevalence of 1.2% in a Chinese population, 0.7% in Japanese, and absent in Korean, and Vietnamese. We observed the SLCO1B1*5 haplotype having a frequency of 0.08% in the Thai population. SLCO1B1*1b is the most abundant haplotype in Asian populations (approximately 55% to 70%). The frequency of SLCO1B1*15 in our study showed a similar range compared to the Asian populations but lower when compared with South American populations (Nozawa et al., 2002;Kim et al., 2008).
We observed similar diplotype frequencies of the OATP1B1 transporter in the Thai population compared to previous reports in Asian populations, including Han Chinese, Japanese, Korean,   and Vietnamese (Nozawa et al., 2002;Kim et al., 2008). The SLCO1B1 phenotypes based on diplotypes in the Thai population were in agreement with the comprehensive data of the function of the OATP1B1 transporter (Table 3).
In conclusion, frequencies of the SLCO1B1 variants and the subsequent OATP1B1 activity in a large cohort of Thais can provide important information for the guidance of personalized drug therapy.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Committee on Human Rights Related to Reseach Involving Human Subjects, Faculty of Medicine Ramathibodi Hospital, Mahidol University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

AUTHOR CONTRIBUTIONS
CN and CS were responsible for analysis, interpretation of data, and final approval of the manuscript. JW and CD were responsible for concept and design. PS was responsible for the analysis of data. SW and CS were responsible for supervising the overall conduct of the study.