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Keywords:

  • thiopurine S-methyltransferase;
  • phenotype;
  • genotype;
  • TPMT*3C

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

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 13.32 ± 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 12.36 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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

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 of TPMT exon7 and exon10 were amplified from genomic DNA with the primers listed in Table 1.

Table 1.  Primers and product length for PCR
ExonName of primeraSequence of primerProduct length (bp)
  1. W, wild type-specific; M, mutant-specific; C, common primers.

 5P2W5′-GTATGATTTTATGCAGGTTTG-3′254
P2M5′-GTATGATTTTATGCAGGTTTC-3′254
P2C5′-TAAATAGGAACCATCGGACAC-3′ 
 7P460F5′-GGGACGCTGCTCATCTTCT-3′338
P460R5′-GCCTTACACCCAGGTCTCTG-3′ 
10P719F5′-AAGTGTTGGGATTACAGGTG-3′273
P719R5′-TCCTCAAAAACATGTCAGTGTG-3′ 
image

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

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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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

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. 13.54 ± 2.89 U ml−1 RBC, P = 0.280) [Table 2]. The total mean TPMT activity of both racial groups was 13.32 ± 3.49 U ml−1 RBC. There was a 5.3-fold interindividual variation in the TPMT activity, ranging from 4.54 to 24.03 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 = 0.1581, 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. 13.15 ± 3.43 U ml−1 RBC, Table 2), but the difference was not statistically significant (P = 0.197).

Table 2.  TPMT activity (U/ml RBC) in Chinese children according to racial and gender groups
GenderHan ChineseYao ChineseTotal
nMean ± s.d.nMean ± s.d.nMean ± s.d.
Male2612.79 ± 2.77 6613.30 ± 3.66 9213.15 ± 3.43
Female6113.10 ± 2.83 6013.80 ± 4.1412113.45 ± 3.54
Total8713.01 ± 2.8012613.54 ± 3.8921313.32 ± 3.49
image

Figure 2. Frequency distribution histogram of erythrocyte TPMT activity in 213 Chinese children. The heavy line represents the model predicted activity distribution. RBC, erythrocyte

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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 Chinese 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 12.36 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

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 10–43 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 [2.3–3.0%] 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−1RBC.

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 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 12.36 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.

DNA controls/DNA reference samples were kindly provided by Ms. Szumlanski, Ms. Prondzinski and Dr Weinshilboum, Mayo Clinic (Rochester) and their assistance is gratefully acknowledged.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
  • 1
    Krynetski EY, Tai HL, Yates CR, Fessing MY, Loennechen T, Schuetz JD, et al. Genetic polymorphism of thiopurine S-methyltransferase: clinical importance and molecular mechanisms. Pharmacogenetics 1996; 6: 27990.
  • 2
    McLeod HL, Krynetski EY, Relling MV, Evans WE. Genetic polymorphism of thiopurine methyltransferase and its clinical relevance for childhood acute lymphoblastic leukemia. Leukemia 2000; 14: 56772.
  • 3
    Weinshilboum RM, Sladek SL. Mercaptopurine pharmacogenetics: monogenic inheritance of erythrocyte thiopurine methyltransferase activity. Am J Human Genet 1980; 32: 6512.
  • 4
    McLeod HL, Lin JS, Scott EP, Pui CH, Evans WE. Thiopurine methyltransferase activity in American white subjects and black subjects. Clin Pharmacol Ther 1994; 55: 1520.
  • 5
    Krynetski EY, Evans WE. Genetic polymorphism of thiopurine S-methyltransferase: molecular mechanisms and clinical importance. Pharmacology 2000; 61: 13646.
  • 6
    Dubinsky MC, Yang H, Hassard PV, Seidman EG, Kam LY, Abreu MT, et al. 6-MP metabolite profiles provide a biochemical explanation for 6-MP resistance in patients with inflammatory bowel disease. Gastroenterology 2002; 122: 90415.
  • 7
    Klemetsdal B, Tollefsen E, Loennechen T, Johnsen K, Utsi E, Gisholt K, et al. Interethnic difference in thiopurine methyltransferase activity. Clin Pharmacol Ther 1992; 51: 2431.
  • 8
    Krynetski EY, Schuetz JD, Galpin AJ, Pui CH, Relling MV, Evans WE. A single point mutation leading to loss of catalytic activity in human thiopurine S-methyltransferase. Proc Natl Acad Sci USA 1995; 92: 94953.
  • 9
    Tai HL, Krynetski EY, Yates CR, Loennechen T, Fessing MY, Krynetskaia NF, et al. Thiopurine S-methyltransferase deficiency: two nucleotide transitions define the most prevalent mutant allele associated with loss of catalytic activity in Caucasians. Am J Hum Genet 1996 April; 58 (694–702): 1996.
  • 10
    Loennechen T, Yates CR, Fessing MY, Relling MV, Krynetski EY, Evans WE. Isolation of a human thiopurine S-methyltransferase (TPMT) complementary DNA with a single nucleotide transition A719G (TPMT*3C) and its association with loss of TPMT protein and catalytic activity in humans. Clin Pharmacol Ther 1998; 64: 4651.
  • 11
    Yates CR, Krynetski EY, Loennechen T, Fessing MY, Tai HL, Pui CH, et al. Molecular diagnosis of thiopurine S-methyltransferase deficiency: genetic basis for azathioprine and mercaptopurine intolerance. Ann Intern Med 1997; 126: 60814.
  • 12
    Wood AJJ. Racial differences in the response to drugs – Pointers to genetic differences. N Engl J Med 2001; 344 (18): 13936.
  • 13
    McLeod HL, Pritchard SC. Githang’a J, Indalo A, Ameyaw MM, Powrie RH, et al. Ethnic differences in thiopurine methyltransferase pharmacogenetics: evidence for allele specificity in Caucasian and Kenyan individuals. Pharmacogenetics 1999; 9: 7736.
  • 14
    Ameyaw MM, Collie-Duguid ES, Powrie RH, Ofori-Adjei D, McLeod HL. Thiopurine methyltransferase alleles in British and Ghanaian populations. Hum Mol Genet 1999; 8: 36770.
  • 15
    Hon YY, Fessing MY, Pui CH, Relling MV, Krynetski EY, Evans WE. Polymorphism of the thiopurine S-methyltransferase gene in African-Americans. Hum Mol Genet 1999; 8: 3716.
  • 16
    Collie-Duguid ES, Pritchard SC, Powrie RH, Sludden J. Collier DA, Li T, et al. The frequency and distribution of thiopurine methyltransferase alleles in Caucasian and Asian populations. Pharmacogenetics 1999; 9: 3742.
  • 17
    Huang M, Jiang WQ, Lou YL, Cheng MX. Comparison of thiopurine methyltransferase activity between Chinese and Caucasian Populations. Chin J Cancer 2000; 19: 85861.
  • 18
    , YeQ, Gu L, Zhao J, Liang A, , YeY. [The study on hereditary polymorphism of thiopurine S-methyltransferasein Chinese Han population of Shanghai area]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2000; 17: 4213.
  • 19
    Lennard L, Singleton HJ. High-performance liquid chromatographic assay of human red blood cell Thiopurine methyltransferase activity. J Chromatogr B Biomed Appl 1994; 661 (1): 2533.
  • 20
    Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 1569.
  • 21
    Lee EJ, Kalow W. Thiopurine S-methyltransferase activity in a Chinese population. Clin Pharmacol Ther 1993; 54: 2833.
  • 22
    Menor C, Fueyo JA, Escribano O, Cara C, Fernandez-Moreno MD, Roman ID, et al. Determination of thiopurine methyltransferase activity in human erythrocytes by high-performance liquid chromatography: comparison with the radiochemical method. Ther Drug Monit 2001; 23: 53641.
  • 23
    Park-Hah JO, Klemetsdal B, Lysaa R, Choi KH, Aarbakke J. Thiopurine methyltransferase activity in a Korean population sample of children. Clin Pharmacol Ther 1996; 60: 6874.
  • 24
    Klemetsdal B, Wist E, Aarbakke J. Gender difference in red blood cell thiopurine methyltransferase activity. Scand J Clin Laboratory Invest 1993; 53: 7479.
  • 25
    Szumlanski CL, Honchel R, Scott MC, Weinshilboum RM. Human liver thiopurine methyltransferase pharmacogenetics. biochemical properties, liver-erythrocyte correlation and presence of isozymes. Pharmacogenetics 1992; 2: 14859.
  • 26
    Kham SK, Tan PL, Tay AH, Heng CK, Yeoh AE, Quah TC. Thiopurine methyltransferase polymorphisms in a multiracial asian population and children with acute lymphoblastic leukemia. J Pediatr Hematol Oncol 2002; 24: 3539.
  • 27
    Otterness D, Szumlanski C, Lennard L, Klemetsdal B, Aarbakke J, Park-Hah JO, et al. Human thiopurine methyltransferase pharmacogenetics: gene sequence polymorphisms. Clin Pharmacol Ther 1997; 62: 6073.
  • 28
    Hiratsuka M, Inoue T, Omori F, Agatsuma Y, Mizugaki M. Genetic analysis of thiopurine methyltransferase polymorphism in a Japanese population. Mutat Res 2000; 448: 915.
  • 29
    Spire-Vayron de la Moureyre C, Debuysere H, Sabbagh N, Marez D, Vinner E, Chevalier ED, et al. Detection of known and new mutations in the thiopurine S-methyltransferase gene by single-strand conformation polymorphism analysis. Hum Mutat 1998; 12: 17785.
  • 30
    Otterness DM, Szumlanski CL, Wood TC, Weinshilboum RM. Human thiopurine methyltransferase pharmacogenetics. Kindred with a terminal exon splice junction mutation that results in loss of activity. J Clin Invest 1998; 101 103644.
  • 31
    Colombel JF, Ferrari N, Debuysere H, Marteau P, Gendre JP, Bonaz B, et al. Genotypic analysis of thiopurine S-methyltransferase in patients with Crohn's disease and severe myelosuppression during azathioprine therapy. Gastroenterology 2000; 118: 102530.
  • 32
    Yan L, Zhang S, Eiff B, Szumlanski CL, Powers M, O'Brien JF, et al. Thiopurine methyltransferase polymorphic tandem repeat: genotype-phenotype correlation analysis. Clin Pharmacol Ther 2000; 68: 2109.
  • 33
    Weinshilboum R. Thiopurine pharmacogenetics: clinical and molecular studies of thiopurine methyltransferase. Drug Metab Dispos 2001; 29 (4 Part 2): 6015.
  • 34
    Marinaki AM, Arenas M, Khan ZH, Lewis CM, Shobowale-Bakre el M, Escuredo E, et al. Genetic determinants of the thiopurine methyltransferase intermediate activity phenotype in British Asians and Caucasians. Pharmacogenetics 2003; 13: 97105.
  • 35
    Coulthard SA, Rabello C, Robson J, Howell C, Minto L, Middleton PG, et al. A comparison of molecular and enzyme-based assays for the detection of thiopurine methyltransferase mutations. Br J Haematol 2000; 110: 599604.
  • 36
    Dubinsky MC, Hassard PV, Seidman EG, Kam LY, Abreu MT, Targan SR, et al. An open-label pilot study using thioguanine as a therapeutic alternative in Crohn's disease patients resistant to 6-mercaptopurine therapy. Inflamm Bowel Dis 2001; 7: 1819.
  • 37
    Krynetskaia NF, Cai X, Nitiss JL, Krynetski EY, Relling MV. Thioguanine substitution alters DNA cleavage mediated by topoisomerase II. FASEB J 2000; 14: 233944.