A group of 96 normal, healthy, non-related Tibetans (including 48 males and 48 females) were recruited between October and December 2009 from Xizang Minzu University in Xianyang. All participants were Tibetan Chinese living in the Tibet Autonomous Region of China, with a minimum of three generations of paternal ancestry within this ethnic group. Subjects with any type of medical illness, organ transplant, drug/alcohol addiction or those who were pregnant were excluded from the study. These exclusion criteria were used to minimize factors that may have influenced genetic variation in the genes of interest.

The present study was approved by The Human Research Committee of the Xizang Minzu University for Approval of Research Involving Human Subjects; all subjects were informed, verbally and in writing, about the experimental procedures, confidentiality and the purpose of the study. All participants gave their written informed consent prior to enrollment in the study.

A blood sample (5 ml) was taken from each subject in an EDTA tube (Jiangsu Kangjie Medical Devices Co., Ltd. Jiangyan, China) and DNA was extracted using the GoldMag-Mini Whole Blood Genomic DNA Purification kit (GoldMag Co., Ltd., Hainan, China), according to the manufacturer's instructions. Primers (presented in Table I; Sangon Biotech, Shanghai, China) were designed to amplify the 5′-flanking regions, all exons, and all introns of the CYP2C19 gene. Polymerase chain reaction (PCR) was performed for all single nucleotide polymorphisms (SNPs) in 10 µl reactions with 1 µl template DNA, 5 µl HotStar Taq Master mix (Qiagen, Germantown, MD, USA), 0.5 µl each primer (5 µM) and 3 µl deionized water. The thermal cycling conditions were as follows: Denaturation step of 15 min at 95°C; followed by 35 cycles of denaturation at 95°C for 30 sec, 55–64°C for 30 sec and 72°C for 1 min; and a final extension step at 72°C for 3 min. PCR products were incubated with 0.5 µl shrimp alkaline phosphatase (Roche Diagnostics, Basel, Switzerland), 8 µl HotStar PCR product, and 1.5 µl deionized water (to a total volume of 10 µl), at 37°C for 30 min, followed by heat inactivation at 80°C for 15 min. Purified PCR products were sequenced directly using the ABI Prism BigDye Terminator Cycle Sequencing kit version 3.1 (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA), using an ABI Prism 3100 sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc.).

Sequencher 4.10.1 (http://www.genecodes.com/) software was used to analyze the sequences, including manual curation, fragment assembly and mutation detection. CYP2C19 variants were designed based on the nucleotide reference sequence NG_008384.2, which is searched from the National Center for Biotechnology Information database (NCBI database, http://www.ncbi.nlm.nih.gov/). The CYP allele nomenclature is quoted from the Human Cytochrome P450 Allele Nomenclature Committee tables (http://www.cypalleles.ki.se/). The χ² test was used to compare allele and genotype frequencies, with descriptive analysis used to compare allele frequencies between the Tibetan and other populations (12). Haploview 4.1 (http://broad.mit.edu/mpg/haploview) was used to assess linkage disequilibrium (LD) and Hardy-Weinberg equilibrium for each genetic variant (13). Haplotypes were constructed from the selected tag SNPs and haplotype frequencies were derived for the Tibetan population.

Non-synonymous SNPs in the CYP2C19 coding regions were analyzed to predict the coded protein function. Two algorithms, Sorting Intolerant From Tolerant (SIFT; http://sift.bii.a-star.edu.sg/) and Polymorphism Phenotyping version 2 (PolyPhen-2; http://genetics.bwh.harvard.edu/pph2/), were used to perform the functional prediction of non-synonymous SNPs (14). Depending on the metabolic activity of CYP2C19, the subjects were divided into three phenotypic groups: Poor metabolizer (PM), extensive metabolizer and ultra-rapid metabolizer, based on CYP allele nomenclature (http://www.cypalleles.ki.se/) (15).

CYP2C19 was sequenced in the group of 96 Tibetan participants (48 males and 48 females) and the results successfully identified a total of 27 CYP2C19 polymorphisms in this population. Three of the polymorphisms had not previously been reported in either the National Center for Biotechnology Information database or the Human Cytochrome P450 Allele Nomenclature Committee tables (Table II), two of the novel polymorphisms were synonymous mutations within exon five and one was a non-synonymous mutation in exon eight. No duplications or deletions within the sequenced CYP genes were observed.

A total of five CYP2C19 alleles were detected in the Tibetan study population (Table III). The CYP2C19^*1A allele had the highest frequency (50%), followed by the CYP2C19^*1B allele (28.13%) and the CYP2C19^*2A allele (15.10%). The other two identified alleles, CYP2C19^*3A and ^*17, were relatively rare with frequencies of 5.21% and 1.56%, respectively. These results indicate that alleles that do not affect the function of CYP2C19 (CYP2C19^*1A and ^*1B), are the most prevalent with a combined frequency of 68.13%, followed by alleles that inactivate enzyme function (CYP2C19^*2A and ^*3A) with a combined frequency of 20.31%. The CYP2C19^*17 allele, which may increase the activity of CYP2C19, had the lowest frequency of 1.56%.

Four CYP2C19 genotypes were identified, with frequencies ranging from 3.13 to 56.25% in the Tibetan population under study (Table III). Of the identified genotypes, one exhibits normal enzymatic activity, two exhibit reduced enzymatic activity and one exhibits increased enzymatic activity. The CYP2C19 allele frequencies were further compared with previously published data from different countries and ethnic groups in Eastern Asia (16–19), Southern Asia (20), Europe (21–25) and Africa (26–28) (Table IV). The results of the present study demonstrated that the frequency of the wild-type allele, CYP2C19^*1, in the Tibetan group was significantly lower than in Caucasian populations (P<0.05), however was highest in Asian populations. Furthermore, the frequencies of CYP2C19^*2 and CYP2C19^*3 were significantly higher (P<0.05) among those of Tibetan descent compared with Caucasian and African populations.

LD analysis was performed using Haploview with confidence intervals to define LD blocks (Fig. 1). The LD was determined for CYP2C19 using those SNPs whose minor allele frequencies were >0.05, as SNPs with low frequency have little power to detect LD. The deviation from the expected (D') was calculated, in addition to the correlation of alleles at two loci (r²) and the LD statistic for all possible pairs of SNPs. Two LD blocks across CYP2C19 were identified. Block 1, the larger of the two blocks, spans a 61 kb region from nucleotide 19154 (rs4244285) in the promoter region to nucleotide 80160 (rs3758580). Block 2 includes five tightly clustered SNPs (rs4917623, rs17886522, 2C19_87331, rs17882572 and rs17885052), each with a D' value equal to 1 (indicating complete LD).

Three novel CYP2C19 variants were identified with only one of these being a non-synonymous mutation. SIFT was used to predict the effect of mutations on CYPC219 function, with the Ala423Lys substitution predicted to affect protein function with a score of 0.01 (results are divided into four levels: Intolerant, 0.00–0.05; potentially intolerant, 0.051–0.10; borderline, 0.101–0.2; tolerant, 0.201–1.00). By contrast, PolyPhen-2 analysis predicted that this mutation is benign with a score of 0.005 (Fig. 2, the results here were divided into five levels: Benign, 0.000–0.999; borderline, 1.000–1.249; potentially damaging, 1.250–1.499; possibly damaging, 1.500–1.999; probably damaging, >2.000; and when the closer the score is to zero, the less damage is predicted).