Phenotyping and genotyping study of thiopurine S-methyltransferase in healthy Chinese children: A comparison of Han and Yao ethnic groups

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1 et al. DOI: /j x British Journal of Clinical Pharmacology Phenotyping and genotyping study of thiopurine S-methyltransferase in healthy Chinese children: A comparison of Han and Yao ethnic groups Jian-ping Zhang, 1 Yong-yuan Guan, 1 Jue-heng Wu, 2 An-Long Xu, 3 Shufeng Zhou 3 & Min Huang, 2,4 1 Departments of Pharmacology, 2 Department of Clinical Pharmacology, 1 Zhongshan Medical College, 2 School of Pharmaceutical Sciences and 3 School of Life Sciences, Sun Yat-sen University, Guangzhou , P.R.China; 3 Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore Correspondence Dr Min Huang, School of Pharmaceutical Sciences, Sun Yat-sen University, 74 Zhongshan Road, section 2, Guangzhou , Peoples Republic of China. Tel: Fax: huangm@gzsums.edu.cn This work was supported by the National Nature Science Fund of China (No ); Elite Grant from Higher Education Office of Guangdong Province, China (No ); Guangdong Nature Science Fund (No ) Keywords thiopurine S-methyltransferase, phenotype, genotype, TPMT*3C Received 6 June 2003 Accepted 29 January 2004 Aims Ethnicity is an important variable influencing drug response. Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurine drugs. Previous population studies have identified ethnic variations in both phenotype and genotype of TPMT, but limited information is available within Chinese population that comprises at least 56 ethnic groups. The current study was conducted to compare both phenotype and genotype of TPMT in healthy Han and Yao Chinese children. Methods TPMT activity was measured in healthy Chinese children by a HPLC assay (n = 213, 87 Han Chinese and 126 Yao Chinese). Allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) were used to determine the frequency of TPMT mutant alleles (TPMT*2, TPMT*3 A, TPMT*3B and TPMT*3C) in these children. Results There was no significant difference in the mean TPMT activity between Han and Yao Chinese children. A unimodal distribution of TPMT activity in Chinese children was found and the mean TPMT activity was ± 3.49 U ml -1 RBC. TPMT activity was not found to differ with gender, but tended to increase with age in Yao Chinese children. TPMT*2, TPMT*3B and TPMT*3A were not detected, and only one TPMT*3C heterozygote (Han child) was identified in 213 Chinese children. Erythrocyte TPMT activity of this TPMT*3C heterozygote was U ml -1 RBC. The frequency of the known mutant TPMT alleles was 0.2% [1/426] in Chinese children. Conclusion The frequency distribution of RBC TPMT activity was unimodal. The frequency of the known mutant TPMT alleles in Chinese Children is low and TPMT*3C appears to be the most prevalent among the tested mutant TPMT alleles in this population Blackwell Publishing Ltd Br J Clin Pharmacol 58:

2 J.-P. Zhang et al. Introduction Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of drugs such as azathioprine, 6- mercaptopurine, and 6-thioguanine, which are widely prescribed for immunosuppressive or cytotoxic applications [1]. TPMT is one of the most well characterized enzymes, with the genetic polymorphism having been well defined in most populations [2]. TPMT deficiency is inherited as an autosomal codominant trait. In most large world populations studied to date, approximately 10% of the population has intermediate activity due to heterozygosity at the TPMT locus, and about 0.33% is TPMT deficient [3, 4]. Patients with very low levels of TPMT activity are at greatly increased risk for thiopurine-induced toxicity such as myelosuppression when treated with standard doses of these drugs [5], while subjects with very high activity may be undertreated and some may be resistant to thiopurine therapy and could be at risk of hepatotoxicity through increased 6- mercaptopurine exposure [6]. Previous studies have demonstrated ethnic difference in mean TPMT activity and observed evidence of polymorphic distribution of TPMT activity in each of the large racial groups studied to date [3, 4, 7]. For example, mean erythrocyte TPMT activity in African subjects was 20% percent lower that in Caucasians from the same location [4]. Based on the population phenotype-genotype studies performed to date, assays for the molecular diagnosis of TPMT deficiency have focused on TPMT*2 (G238C) [8], TPMT*3A (G460A/A719G) [9], and TPMT*3C (A719G) [10]. These three mutant alleles account for the majority of low activity alleles in human populations studied to date [11]. Ethnicity is an important variable influencing drug response, and recent pharmacogenomic studies in the Asian population have revealed significant interethnic differences in allelic frequencies of polymorphic genes encoding drug metabolizing enzymes, drug transporters and drug targets [12]. Accordingly, the pattern and frequency of mutant TPMT alleles is different among various ethnic populations [2]. The most prevalent TPMT mutant allele in Caucasians is TPMT*3A [13, 14], while that in African and Asian populations is TPMT*3C [15, 16]. Previous studies on phenotype and/or genotype of TPMT have been conducted in Chinese [16 18]. There are 56 racial groups in China, among which Han Chinese are the ethnic majority, while other racial groups like Yao Chinese and Jing Chinese are minorities. Previous studies on TPMT in Chinese focused on Han Chinese [16, 18], yet there are no data on difference in TPMT activity and allele frequency between different racial groups of Chinese. We therefore compared the erythrocyte TPMT activity and allele frequency of TPMT in healthy Chinese Han and Yao children from the same province. Materials and methods Study population Erythrocyte TPMT activity and TPMT genotype were evaluated in a population of Chinese children. Blood samples were collected in January 2002 from 213 healthy elementary school children (n = 213; 92 boys and 121 girls; age range 9 14 years; mean 11.7 years) who needed venipuncture for school health screening laboratory tests. These children were from two racial groups (87 Han Chinese and 126 Yao Chinese) in Liannan County, Guangdong Province, Peoples Republic of China. The children did not regularly use any drugs. Ethnic approval of this study was obtained from the Ethical committee at Sun Yat-Sen University and written informed consent was obtained from the parents of the study subjects. Erythrocytes (RBC) were isolated for analysis of TPMT activity [4] and leucocytes were used for analysis of TPMT genotype as described below. Erythrocyte TPMT activity assay TPMT activity in erythrocytes was determined by a high performance liquid chromatography (HPLC) assay with small modification, as described previously [19]. One unit of TPMT activity represents the formation of 1 nmol of 6-methylmercaptopurine per hour of incubation. TPMT activity was normalized per milliliter of packed erythrocytes. TPMT genotyping Total genomic DNA was extracted from peripheral leucocytes by phenol-chloroform extraction method as previously described [20]. Conventional PCR-based assays were used to detect the major TPMT inactivating mutations G238C (TPMT*2), G460A and A719G (TPMT*3 A), G460A (TPMT*3B) and A719G (TPMT*3C). An allele-specific PCR was used to analyse the G238C mutation in exon 5, as preciously described in detail [11]. PCR amplification and restriction enzyme digestion were used to detect G460A and A719G mutation, respectively. PCR amplification of exon 7 and exon 10 used primers different from previously described, as listed in Table 1. The PCR product of exon 7 was digested with restriction enzyme MwoI (New England Biolabs, Hertfordshire, UK) to detect G460A mutation. The PCR product of exon 10 was digested with restriction enzyme AccI (New England Biolabs) to detect A719G mutation [Figure 1]. Automated sequencing of the PCR fragment confirmed that the expected sequence :2 Br J Clin Pharmacol

3 TPMT in Chinese children of TPMT exon7 and exon10 were amplified from genomic DNA with the primers listed in Table 1. Data analysis Data are presented as mean ± SD. Statistical significance of differences in mean erythrocyte TPMT activity values between gender groups and racial groups were tested with one-way ANOVA. Values of P < 0.05 were considered significant. Deviation from the normal distribution of erythrocyte TPMT activity was examined with the Shapiro-Wilk test. Results Erythrocyte TPMT activity in Chinese Children There was no significant difference in the mean TPMT activity between Han and Yao Chinese children (13.01 ± 2.80 vs ± 2.89 U ml -1 RBC, P = 0.280) [Table 2]. The total mean TPMT activity of both racial groups was ± 3.49 U ml -1 RBC. There was a 5.3-fold interindividual variation in the TPMT activity, Table 1 Primers and product length for PCR ranging from 4.54 to U ml -1 RBC. No subject with TPMT deficiency was found in our study population. The frequency distribution of RBC TPMT activity in Chinese children was unimodal (P = , Shapiro-Wilk test) rather than bimodal or trimodal [Figure 2]. In addition, Chinese girls had slightly higher mean TPMT activity than Chinese boys (13.45 ± 3.54 vs ± 3.43 U ml -1 RBC, Table 2), but the difference was not statistically significant (P = 0.197). TPMT genotype in healthy Chinese children Four alleles of the TPMT gene, TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C, were evaluated in 213 Chi- 500bp 400bp 300bp 200bp WT HET WT HET U CUT HOM U WT HET HOM Exon Name of primer a Sequence of primer Product length (bp) 5 P2W 5 -GTATGATTTTATGCAGGTTTG P2M 5 -GTATGATTTTATGCAGGTTTC P2C 5 -TAAATAGGAACCATCGGACAC-3 7 P460F 5 -GGGACGCTGCTCATCTTCT P460R 5 -GCCTTACACCCAGGTCTCTG-3 10 P719F 5 -AAGTGTTGGGATTACAGGTG P719R 5 -TCCTCAAAAACATGTCAGTGTG-3 W, wild type-specific; M, mutant-specific; C, common primers. Allele: G238C G460A A719G Figure 1 Electrophoresis patterns for TPMT alleles analysed by allele-specific PCR and PCR-RFLP. L, 100 bp DNA ladder; Lane 1 and 3, wild-type specific PCR analysis of nucleotide 238; Lane 2 and lane 4, mutation-specific PCR products of nucleotide 238; Lane 5 7, PCR products of nucleotide 460; lane 8 11, PCR analysis of nucleotide 719. U, uncut; WT, homozygous wild-type; HET, heterozygous mutant; HOM, homozygous mutant. The expected PCR fragments was detected using 2% agarose gel electrophoresis Table 2 TPMT activity (U/ml RBC) in Chinese children according to racial and gender groups Gender Han Chinese Yao Chinese Total n Mean ± s.d. n Mean ± s.d. n Mean ± s.d. Male ± ± ± 3.43 Female ± ± ± 3.54 Total ± ± ± 3.49 Br J Clin Pharmacol 58:2 165

4 J.-P. Zhang et al. Number of subjects TPMT activity (U/ml RBC) Figure 2. Frequency distribution histogram of erythrocyte TPMT activity in 213 Chinese children. The heavy line represents the model predicted activity distribution. RBC, erythrocyte nese children. Only one subject (a Han Chinese child) carrying a mutant TPMT allele was identified, while TPMT*2, TPMT*3A and TPMT*3B alleles were not detected. Thus, the total frequency of mutant alleles in Chinese children was 0.2% [1/426]. This child was heterozygous for TPMT*3C and her TPMT activity was U ml -1 RBC. The result indicated that Han Chinese children had a higher mutant allele frequency [1/ 174] compared with Yao Chinese children [0/252]. Discussion The present study indicated that there was no significant difference in the mean TPMT activity between Han and Yao Chinese children, and in both ethnic groups a unimodal distribution of TPMT activity was found. This is consistent with other studies where a unimodal distribution of erythrocyte TPMT activity in healthy Chinese was reported [17, 18]. However, this is somewhat different from what has been reported in a population sample of Chinese adults from Singapore, which had a bimodal distribution in TPMT activity with a clear antimode (range U ml -1 RBC, mean 31 U ml -1 RBC) [21]. The reason for such a difference in the mode of distribution is unknown, but may be related to the differences in the study populations used. Moreover, the radiochemical assay in Singapore Chinese produced results that were approximately 3 times higher than those obtained from this study and Mayo Clinic. The TPMT activity values measured by HPLC were reported to be in close agreement with those measured by the radiochemical assay [19, 22]. It has been shown that differences in assay conditions can cause significant differences in TPMT activity measured in different laboratories [5]. We used a simple HPLC method to determine TPMP activity and produced results that seemed to be comparable to that of white populations (about 13 U ml -1 RBC) [3, 4] and Korean children (12.4 U ml -1 RBC) measured by the standard radiochemical assay [23]. The 5.3-fold range in TPMT activity among Chinese Children in this study is similar to the 4.0-fold range observed in Korean Children [23]. In addition, this study observed minor and nonsignificantly higher TPMT activity in girls than boys. This was different from others studies where males had slightly higher activity than females [24, 25]. Genotype was determined in all subjects and only a single TPMT*3C heterozygote was found in this study. Several previous studies demonstrated that TPMT*3C was the major mutant allele in Chinese [16, 26]. However, the total frequency of mutant TPMT alleles in our study population [0.2%] is lower than that in Chinese [ %] reported by other groups [16, 26] and white or black Americans [15], but compatible with that in Koreans [0.6%] [27] and Japanese [0.8%] [28]. Low frequency of mutant TPMT alleles in Chinese children conformed to the normal distribution of TPMT activity in this population. The large interindividual variation of the TPMT activity in Chinese children suggests that additional molecular genetic mechanisms might be involved in the regulation of the level of TPMT activity. In this study, only four TPMT mutant alleles, TPMT*2, *3A, *3B and *3C were genotyped in Chinese children. We inferred that the samples in which these mutant alleles were not detected had wild-type allele, TPMT*1. However, it remains a possibility that the presence of other mutant allele were not detected in this study. Additional rare TPMT mutant alleles (TPMT*3D, *4, *5, *6, *7, *8, and *10) recently have been identified [27, 29 31]. Their allele contribution to total variation in Chinese has not been defined yet. Mutations in the TPMT promoter region would be an alternative explanation [32, 33]. However, a recent study demonstrated that the variable number tandem repeats (VNTR*3 to VNTR*9) in the promoter region of the TPMT gene had no significant impact on enzyme activity in British Asians and Caucasians [34]. Further studies are needed to sequence the open reading frame and promoter region of TPMT gene for novel mutations in Chinese, especially in patients with an enzyme activity of 8 U ml -1 RBC. Both TPMT activity measurement and genotyping methods can be used to diagnose TPMT deficiency [35]. A simple activity assay by HPLC or radiochemical methods would allow the identification of rapid or slow metabolisers. Proper dose adjustment is needed :2 Br J Clin Pharmacol

5 TPMT in Chinese children for rapid metabolisers and they should be treated with an alternative therapeutic agent if drug resistance is highly possible [6, 36], whereas dose reduction is certainly necessary for avoiding toxicity in slow or deficient metabolisers who are intolerant to thiopurine therapy. However, the standard activity assay is associated with a number of significant limitations. For example, this method can t be used on patients who have received a blood transfusion because the donor erythrocytes may affect the result [37]. On the other hand, genotyping methods can reliably detect the major and rare mutant allele at human TPMT locus, in particular when genetic polymorphism is highly likely to provide an explanation for TPMT deficiency in individuals [35]. To date, it has become possible to detect TPMT inactivating mutations with more than 95% concordance between genotype and phenotype [37]. In the current study, TPMT activity of the only subject carrying TPMT*3C allele is U ml -1 RBC, while none of the four known mutant alleles were revealed in other subjects with relatively low TPMT activity (<10.0 U ml -1 RBC). This suggests that the difference in TPMT activity between heterozygotes and patients with no mutations is less clear-cut with no clear antimode. In conclusion, this study did not observe significant difference in the mean TPMT activity between Han and Yao Chinese children, while only one Han Chinese child carried a mutant TPMT allele. The frequency distribution of erythrocyte TPMT activity in Chinese children was normal. The frequency of the known mutant TPMT alleles in Chinese Children is low and TPMT*3C appears to be the most prevalent among the known mutant TPMT allele in this population. 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