Genetic Polymorphisms Analysis of Pharmacogenomic VIP Variants in Miao Ethnic Group of Southwest China

Background

The large variability among individuals in drug efficacy is a major challenge in current clinical practice, drug development, and drug regulation [1]. It has been suggested that genetic background may be responsible for the variation in response to therapy, and mounting evidence demonstrates that an individual’s genetic makeup accounts for an estimated 20~95% of variability in drug disposition and effects [2,3]. Pharmacogenetics and pharmacogenomics elucidated the inherited nature of individual variation in drug response, with the goal of optimizing efficacy and safety through better understanding of human genetic variability and its influence on drug response, leading to personalized medicine [4,5].

The Pharmacogenetics and Pharmacogenomics Knowledge Base (PharmGKB: http://www.pharmgkb.org) is a publicly available Web-based knowledge base created to aid researchers in understanding how genetic variation among individuals contributes to differences in reactions to drugs [6]. This information is presented in the form of Very Important Pharmacogene (VIP) summaries, pathway diagrams, and curated literature [7]. The PharmGKB currently contains information for more than 3000 drugs, 3000 diseases, and 26 000 genes with genotyped variants [8]. In total, it consists of 126 VIP variants that occur in 44 different genes and variously code for cytochrome P450 oxidases, drug targets, drug receptors, and drug transporters. The relationship between these VIP variants and their effect on drug-related toxicity as well as therapeutic benefit have been studied extensively [9].

Pharmacogenomic research in ethnic populations has great significance for the achievement of personalized drug treatment and development of new drugs. However, we have not found any pharmacogenomics information regarding minority groups, such as the Miao ethnic groups in southwest China. The Miao is an ethnic group mainly distributed in the southwest of China; they mostly live in Guizhou, Yunnan, and Sichuan provinces. It is one of China’s largest ethnic groups, with a long history, distinct culture, and fine traditions. According to a 2000 census, the Miao have an approximate population of 9.6 million.

In the present study, we aimed to identify the allele frequencies of VIP variants in the Miao and to determine the difference in allele frequencies between the Miao and 12 other populations. Our goals were to identify differences and determine their extent and provide a theoretical basis for safer drug administration and better therapeutic treatment in the Miao population. The results of our study will extend our understanding of ethnic diversity and pharmacogenomics, and help clinicians triage patients for better individualized treatments.

Material and Methods

STUDY PARTICIPANTS:

We randomly recruited 98 unrelated, healthy Miao subjects from Guizhou province of China. The subjects selected were judged to be of good health and had exclusively Miao ancestry for at least the last 3 generations. We selected 96 unrelated Chinese Han individuals from Lantian county in Xi’an, Shaanxi province as one of our control groups. All subjects were healthy in terms of their medical history and physical examination. An explanation about the purpose and experimental procedures of the study were given to all individuals. Written informed consent was obtained from all subjects prior to sample donation, and the study protocol was performed in accordance with the Declaration of Helsinki and approved by the Clinical Research Ethics of Northwest University for Approval of Research Involving Human Subjects.

VARIANT SELECTION AND GENOTYPING:

We selected genetic variants from published polymorphisms associated with VIP variants from the Pharm GKB database, and excluded loci that could not be designed. We successfully genotyped 66 VIP variants selected from PharmGKB in 194 participants (98 Miao subjects and 96 Chinese Han controls). Genomic DNA was isolated from whole blood using the GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag Ltd. Xi’an, China) according to the manufacturer’s protocol. DNA concentration was measured by NanoDrop 2000C (Thermo Scientific, Waltham, Massachusetts, USA). We used the Sequenom MassARRAY Assay Design 3.0 software (San Diego, CA, USA) to design Multiplexed SNP MassEXTEND assays [10]. Single-nucleotide polymorphism (SNP) genotyping used the standard protocol recommended by the manufacturer with a Sequenom MassARRAY RS1000 (San Diego, California, USA). Sequenom Typer 4.0 Software (San Diego, California, USA) was used to perform data management and analyze the SNP genotyping data, as described in a previous report [11].

HAPMAP GENOTYPE DATA:

The genotype data of the 11 populations were downloaded from the International HapMap Project web site (HapMap_release127) at http://hapmap.ncbi.nlm.nih.gov. The 11 populations are as follows: (1) African ancestry in Southwest USA (ASW); (2) Utah, USA residents with Northern and Western European ancestry from the CEPH collection (CEU); (3) Han Chinese in Beijing, China (CHB); (4) Chinese in metropolitan Denver, CO, USA (CHD); (5) Gujarati Indians in Houston, Texas, USA (GIH); (6) Japanese in Tokyo, Japan (JPT); (7) Luhya in Webuye, Kenya (LWK); (8) Mexican ancestry in Los Angeles, California, USA (MEX); (9) Maasai in Kinyawa, Kenya (MKK); (10) Toscani in Italy (TSI); and (11) Yoruba in Ibadan, Nigeria (YRI).

DATA ANALYSIS:

We used Microsoft Excel (Redmond, WA, USA) and SPSS 17.0 statistical packages (SPSS, Chicago, IL, USA) to perform statistical calculations. The validity of the frequency of each VIP variant in the Miao and Chinese Han data was tested by assessing the departure from HWE using an exact test. We calculated and compared the genotype frequencies of the variants in the Miao data with those in the 11 populations separately using the χ2 test [12]. All p values obtained in this study were 2-sided, and Bonferroni adjustment for multiple tests was applied to the level of significance, which was set at p<0.05/(66*12) [13]. Structure (version 2.3.4) software [14] was used to analysis the genetic structure of the 13 populations. We used Arlequin (version 3.1) software to calculate the value of Fst to infer the pairwise distance between populations [15].

Results

We successfully sequenced 66 VIP pharmacogenomic variant genotypes from 98 Miao individuals. The basic information about the selected VIP loci in Miao is listed in Table 1, including gene name and category, chromosome number and position, amino acid translation, and their allele frequencies in Miao.

We used the χ2 test with the Bonferroni correction for multiple hypotheses and multiple comparisons, and we found 5, 7, 12, 13, 14, 15, 15, 16, 16, 19, 19, and 25 different loci in the frequency distributions when the Miao population was compared to the Xi’an Han, CHD, MEX, ASW, JPT, CHB, GIH, MKK, TSI, CEU, LWK, and YRI populations, respectively (p≤6.3×10−5). These VIP variants are mainly distributed in 23 genes; they mainly involve the cytochrome P450 superfamily, nuclear receptor family, G-protein-coupled receptor family, alcohol dehydrogenase family, adrenergic receptors family, ATP-binding cassette (ABC) transporters superfamily, and eag family. Genotype frequencies of MTHFR, VDR, and VKORC1 in the Miao differed widely from those in the other 12 populations. We found that the rs1801133 was the most significantly different locus between the Miao ethnic group and the other populations (Table 2). Additionally, Rs698, Rs1805124, and Rs 1801131 were found to show a significant difference in the 11 HapMap populations.

Pairwise Fst values were calculated for all population comparisons across loci. As shown in Table 3, we found that pairwise Fst values for comparisons of the Miao population with the other 12 populations ranged from 0.01904 to 0.26192. Fst statistics [16] are measures of population differentiation. It is directly related to the variance in allele frequency among populations and to the degree of resemblance among individuals within populations. If Fst is small, it means that the allele frequencies within each population are similar; if it is large, it means that the allele frequencies are different. The value of Fst for the Miao and CHD populations was the smallest. We therefore conclude that the allele frequencies of the Miao and CHD are similar. We speculate that the genetic backgrounds of the Miao and CHD populations are similar.

We used a model-based clustering approach, as implemented in Structure, to infer population structure among the 13 populations. Different values ranging from 2 to 7 were assumed for K in Structure calculations. K=3 was selected, based on the estimated Ln Prob of Data and other recommendations of the Structure software manual. As shown in Figure 1, when the K value was equal to 3, individuals were independently assigned to 3 affinity groups (subpopulations 1: Miao, Xi’an Han, CHB, CHD, JPT; subpopulations 2: ASW, LWK, MKK, YRI; subpopulations 3: CEU, GIH, MEX, TSI) using the relative majority of likelihood to assign individuals to subpopulations. We tested additional values of K and obtained results suggesting that the genetic backgrounds of the Miao and CHD populations are similar.

Discussion

Individuals’ differences in drug reactions can directly influence the efficacy and safety of the drug, and has become a worldwide problem in the treatment of some major diseases. However, it is almost impossible to predict whether a drug will be beneficial, lack efficacy, or cause serious adverse effects [17]. Because genetic variations play an important role in determining the metabolism of and reactions to some specific drugs in individual patients, in this study we genotyped the variants related to drug response (pharmacogenomics) in the Miao ethnic group and compared the genotype frequencies with those in 12 other populations. The χ2 test results show that the allele frequencies of the VDR rs1544410 and VKORC1 (rs9934438) variants in the Miao population are quite different from that in other ethnic groups. We found that genotype frequencies of rs1801133 (MTHFR) in the 13 selected populations are significantly different. Using Fst calculations and analysis of population structure, we also found that the genetic backgrounds of the Miao and CHD population are similar.

Methylenetetrahydrofolate reductase (MTHFR), located on chromosome 1 at 1p36.3, is an important enzyme involved in the folate metabolic pathway. Rs1801133 (677 C>T) is a significant variant of the MTHFR gene. In our present study, rs1801133 was found to be a significant variant that existed in the 13 selected populations. It has been widely reported that the polymorphism of rs1801133 is associated with many diseases, such as breast cancer [18], colorectal cancer [19], and bladder cancer [20]. A previous meta-analysis demonstrated that the 677 C allele was significantly associated with breast cancer risk (OR=0.942, 95%CI = 0.898 to 0.988) when compared with the 677 T allele in the additive model [18]. In our study, the C allele frequency in Miao was somewhat high (28%) in our present study, suggesting that Miao have an intermediate susceptibility to breast cancer. Sohn et al. [21] demonstrated that the MTHFR 677T mutation decreased chemosensitivity of breast cancer cells to methotrexate (MTX), a common cancer chemotherapeutic agent. Cáliz et al. [22] also reported that the C677T polymorphism (rs1801133) was associated with increased MTX toxicity [odds ratio (OR) 1.42, 95% confidence interval (CI) 1.01–1.98, p=0.0428] in a Spanish rheumatoid arthritis population. These findings suggest that the MTHFR C677T polymorphism may be a useful pharmacogenetic determinant for providing rational and effectively tailored therapy for the Miao ethnic group.

Vitamin D receptor (VDR) gene maps to chromosome 12q13.11, whose function has been widely reported. It is an important regulator of the vitamin D pathway and a number of common single-nucleotide polymorphisms (SNP) have been identified in this gene [23]. Clinical evidence suggests that the VDR genotype could modify the efficacy of anti-osteoporotic treatments such as etidronate and alendronate in postmenopausal women [24]. Other studies have demonstrated that the SNP rs1544410 in VDR might modulate the risk of breast, skin, and prostate cancers, as well as other forms [25,26]. One study reported that GA and AA genotypes of rs1544410 were associated with decreased cutaneous malignant melanoma (CMM) risk (odds ratio=0.78 and 0.75, respectively) compared with the GG genotype [26]. We found that the GG genotype frequency of rs1544410 in the Miao is very high, suggesting that the Miao should consider more aggressive screening for CMM.

The VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene encodes the VKORC1 (vitamin K epoxide reductase) protein, which is considered a candidate gene for the variability in warfarin response, mainly including 3 common polymorphisms [27]. The C6484T (rs9934438), or 1173C>T (rs9934438), is a SNP in the first intron of VKORC1, which was the first SNP associated with the low-dose warfarin phenotype [28]. A previous study demonstrated that patients with the 1173T (rs9934438) allele require a lower warfarin dose (mean dose 24–26 mg/week) compared with 35 mg/week for the wild-type carriers [29]. In our study, the frequency of carriers of the allele T of rs9934438 is lower in the Miao population, suggesting that patients in this population will require a lower dose of warfarin.

Our study also demonstrated the correlation between the ethnic groups by Fst calculations and population structure analysis. The Structure plot (Figure 1) showed that the 13 ethnic groups were independently assigned into 3 affinity groups, suggesting they have a homogeneous genetic background. Genetic homogeneity among some populations separated by large geographic distances has been observed in migratory insects [30,31]. Our results are consistent with those findings, which could be explained by the migration theory described by Curry et al. [32].

Despite the current study possessing enough power, some limitations should be considered. First, the sample size of our study was relatively small, which may limit the statistical power. Second, the SNPs tested in our study were not large enough. Therefore, the association between these polymorphisms requires further investigation in a large sample before definitive conclusions can be drawn.

Conclusions

Our results provide the first pharmacogenomics information in the Miao population and illustrate the difference in selected genes between Miao and 12 other populations around the world. These results could be used to create individualized treatment strategies, including appropriate drugs and dosage selections for the Miao ethnic group.