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High prevalence of polymorphism and low activity of thiopurine methyltransferase in patients with inflammatory bowel disease
European Journal of Internal Medicine 23 (2012) 273–277
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European Journal of Internal Medicine journal homepage: www.elsevier.com/locate/ejim
Original article
High prevalence of polymorphism and low activity of thiopurine methyltransferase in patients with inflammatory bowel disease Tiziana Larussa a, Evelina Suraci a, Margherita Lentini b, Immacolata Nazionale a, Luigia Gallo b, Ludovico Abenavoli a, Maria Imeneo a, Francesco Saverio Costanzo b, Giovanni Cuda b, Francesco Luzza a,⁎ a b
Department of Health Science, University of Catanzaro "Magna Graecia", Catanzaro, Italy Department of Clinical and Experimental Medicine, and Tommaso Campanella Foundation, University of Catanzaro "Magna Graecia", Catanzaro, Italy
a r t i c l e
i n f o
Article history: Received 1 November 2011 Received in revised form 3 December 2011 Accepted 8 December 2011 Available online 5 January 2012 Keywords: Crohn's disease Inflammatory bowel diseases Thiopurines Ulcerative colitis Azathioprine
a b s t r a c t Background: Gene polymorphism of thiopurine methyltransferase (TPMT) correlates with decreased enzyme activity which determines a significant risk of adverse effect reactions (ADR) in patients treated with thiopurines. The aim of this study was to investigate TPMT genotype and phenotype status in patients with inflammatory bowel diseases (IBD). Methods: Fifty-one consecutive out-patients with IBD were genotyped for the following allelic variants: rs1800462 (referred as TPMT*2 allele), rs1800460 (referred as TPMT *3B allele), and 1142345 (referred as TPMT *3C allele). Red blood cell TPMT activity was measured using a competitive micro-well immunoassay for the semi-quantitative determination of TPMT activity in red blood cells (RBC) by means of a 6-MP substrate. Results: Polymorphism of TPMT was found in 5 out of 51 patients (10%; 95% CI 2%–18%), three heterozygous and two homozygous carriers. Six patients (11.8%; 95% CI 2.4%–19.5%) displayed very low, 12 (23.5%; 95% CI 11.4%– 34.5%) intermediate, and 33 (64.7%; 95% CI 52%–78%) normal/high TPMT activity. There were no differences between TPMT genotype and phenotype groups according to age, type of disease, smoking, and chronic medications. A 71% (95% CI 61%–81%; κ = 0.45) concordance rate was found between genotype and phenotype status. Six out of 27 (22%) current or past users of azathioprine developed ADR, with three (50%) displaying TPMT genotype and/or phenotype alterations. Conclusion: Compared to the general population, IBD patients may have significantly higher prevalence of TPMT polymorphism and, even more, low activity. Phenotypic more than genotypic TPMT analysis could be useful to better manage IBD therapy with thiopurines. © 2011 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
1. Introduction The thiopurine drugs 6-mercaptopurine (6-MP) and its prodrug azathioprine (AZA) are widely used in the treatment of active, steroid-refractory and/or steroid-dependent inflammatory bowel disease (IBD), and have been proven to be effective for inducing and maintaining remission in these patients [1,2]. However, as many as one-third of these patients may develop adverse effect reactions (ADR), the dose of thiopurine often must be reduced or the therapy has to be discontinued in more than 30% of patients [3]. Gastrointestinal symptoms, bone marrow toxicity (BMT), hepatotoxicity and pancreatitis are the most common ADR. Blood tests are performed regularly to monitor blood count and liver function in order to find BMT and hepatotoxicity at an early enough stage and discontinue therapy before the onset of life-threatening toxicity. However, in ⁎ Corresponding author at: Dipartimento di Scienze della Salute, Università di Catanzaro “Magna Graecia”, Campus Universitario di Germaneto Viale Europa 88100 Catanzaro, Italy. Tel.: + 39 0961 3697113; fax: + 39 0961 3697164. E-mail address: [email protected] (F. Luzza).
some cases, ADR might develop unpredictably during the interval between the two tests [4]. Much interest has focused on the metabolism of thiopurines to identify individualized therapeutic pathways, minimizing adverse effects and enhancing clinical response. At date it is assumed that AZA is not enzymatically converted to MP, even if a potential effect of glutathione-S-transferase in the conversion of AZA into MP has been demonstrated [5]. 6MP is then enzymatically converted by competing pathways into the following metabolites: 6-methylmercaptopurine (6-MMP) by the action of thiopurine methyltransferase (TPMT), thought to be inactive or maybe responsible for hepatotoxicity; 6thiouric acid by the action of xanthine oxidase (XO), also an inactive compound; 6-thioguanine nucleotides (6-TGNs), formed by an enzymatic anabolic process dealing with the action of hypoxanthine guanine phosphoribosyl transferase (HGPRT), which produces 6-thioinosine monophosphate (6-TIMP). Then 6MP is transformed by inosine monophosphate dehydrogenase (IMPD) into 6-thioxanthosine monophosphate (6-TXMP), which is metabolized to 6-thioguanine monophosphate (6-TGMP), -diphosphate (6TGDP) and -triphosphate (6 TGTP) [6]. 6-TGNs, comprehensive of the above mentioned products,
0953-6205/$ – see front matter © 2011 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ejim.2011.12.002
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are the active metabolites responsible for pharmacological effect but also associated with dose-related adverse reactions, mainly myelotoxicity [7]. A balance between TPMT, XO, and HGPRT is responsible for final serum concentration of 6-TGNs. Genetic variation of the activity of TPMT and its influence on AZA metabolism is an important pharmacogenetic model. TPMT*1 is the wild-type allele, whereas TPMT*2 and TPMT*3 account for the vast majority (>90%) of mutant alleles among at least 10 which are associated with a decreased TPMT enzymatic activity [8]. Distribution of TPMT mutant alleles differs significantly among ethnic populations. TPMT*3 A, including TPMT*3C (719A > G) and 3*B (460 G > A), is the most frequent occurring mutant allele (3.2–5.7%) in Caucasian populations [9]. There is a trimodal distribution of TPMT activity in Caucasian population: one in 300 subjects (0.3%) has low or undetectable TPMT activity, 8–11% of individuals have intermediate TPMT activity, 89–92% have high or normal enzyme activity [10]. A high degree of correlation between TPMT geno- and phenotype has been ascertained, since data of the trimodal distribution can be considered overlapping prevalence of genetic variants in the general population. Indeed polymorphisms of the gene encoding TPMT enzyme correlate with decreased enzyme activity. In particular, heterozygosity TPMT gene status is associated with intermediate enzyme activity whereas homozygosity status for the mutant alleles determines a significant risk of toxicity in patients receiving standard doses of thiopurines, primarily as a result of unchecked production of TGNs [11]. Recently, screening patients for TPMT polymorphism and/or enzymatic activity before thiopurine treatment in order to predict the risk of toxicity has aroused considerable interest in several clinical centers. The aim of this study was to investigate TPMT genotype and phenotype status in a Southern Italian population of IBD patients. 2. Materials and methods 2.1. Subjects Fifty-one consecutive out-patients followed at our University reference Centre for IBD were enrolled for the study, 19 patients with Crohn's disease (CD) and 32 with ulcerative colitis (UC). Diagnosis of IBD was assessed in accordance with current clinical guidelines and criteria based on endoscopic, radiological and histopathologic examination [12]. Demographic data, clinical history, disease-related features and type of medications were recorded (Table 1). Venous blood sample from each patient was obtained after overnight fasting.
Table 1 Patient characteristics. Variable
Crohn's disease n = 19
Ulcerative colitis n = 32
Sex (M/F) Agea(years) Disease site – Ileum – Ileocolon – Colon Smoke Medication – Oral mesalamine – Sulphasalazine – Azathioprine (currently users) – Azathioprine (formerly users) – Othersb
9/10 40 (18–65)
19/13 34.5 (18–52)
5 9 5 3
– – 32 0
6 4 6 5 6
24 1 12 4 3
a
Values are shown as median and range. Such as oral steroids, antibiotics, biological therapy, antihypertensive drugs, proton pump inhibitors. b
None of the subjects had undergone blood transfusions during the previous 3 months. All patients were considered Caucasians as assessed by excluding a different origin of their first degree relatives. The study was approved by the local ethics committee and all patients gave written informed consent prior to entering the study. 2.2. TPMT genotyping Total genomic DNA was extracted from peripheral blood lymphocytes using a commercial kit (Wizard Genomic DNA Purification Kit, Promega, Madison, WI, USA) according to the manufacturer's recommendations. Each individual was genotyped at the TPMT*2 (rs1800462), TPMT*3B (rs1800460), and TPMT*3C (rs1142345) alleles by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP)-based assays, using previously described conditions [13–15]. Positive and negative controls were available for each genotype. The PCR products were separated by nondenaturing 8% PAGE. 2.3. TPMT phenotyping Blood samples were collected in EDTA containing tubes and stored at 4 °C. TPMT enzymatic activity was performed within 3 h of time of collection. The TPMT phenotype was assessed by measuring TPMT activity using a competitive micro-well immunoassay for the semi-quantitative determination of TPMT activity in red blood cells (RBC) by means of a 6MP substrate. TPMT catalyzes the S-methylation of 6-mercaptopurine (6-MP) with the presence of S-adenosylmethionine (SAM), the methyl donor, yielding 6-methylmercaptopurine (6-MMP). Then 6-MMP is measured with a micro-well EIA. Biologix TPMT ELISA kit (CE Mark, Biologix Diagnostics, LLC., Lenexa, KS, USA) was used. One unit of enzyme activity is defined as a formation of 1 nmol of 6-MMP per ml of packed RBC per 60 min incubation at 37 °C. Erythrocyte TPMT activity was expressed as units per gram of hemoglobin (U/gHb). The assay interpretation is according to the following criteria: ≤5.5, 5.6–15.5, and ≥15.6 Units as very low, medium, and normal to high TPMT activity level, respectively. Intra and inter-assay coefficients of variation were 8.90% and 9.15%, respectively. 2.4. Statistics Percentages were given along with 95% confidence intervals (CI). Comparison of proportions was performed using chi-square test. Continuous data were compared using the Student t test for independent samples. Multiple testing was assessed by one-way analysis of variance (ANOVA) and, when the F value was significant, by Tukey's multiple comparison test. Difference was considered significant if the p value was b0.05. The relation between TPMT genotype and phenotype was evaluated by means of k statistic. The k statistic, a measure of the agreement between two observers or tests, ranges from − 1 to 1 with 1 indicating perfect agreement, 0 indicating the agreement expected on the basis of chance alone, and values between 0 and 0.4 a poor to fair agreement [16]. The software program Primit (1994, version 3.03) was used to perform the statistical analysis. 3. Results Overall, allelic mutations for TPMT were found in 5 out of the 51 patients (10%; 95% CI 2%–18%). One patient was heterozygous for the TPMT*3B (rs1800460) allele, two (4%) were heterozygous for the TPMT*3C (rs1142345) allele, whereas two more patients (4%) were homozygous for TPMT*3C (rs1142345) and TPMT*2 (rs1800462) allele, respectively. Of them, only one (heterozygous) patient was currently using azathioprine (2 mg/kg/d) but had not experienced any ADR until now. The remaining 46 patients (90%; 95% CI 81%–98%) did not show any of the variant TPMT alleles tested for, and
T. Larussa et al. / European Journal of Internal Medicine 23 (2012) 273–277 Table 2 Patient characteristics according to TPMT polymorphism. Variables
Polymorphism carriers n=5
Wild type patients n = 46
Agea(years) Sex (M/F) Disease Smoke Medications – Oral mesalamine – Sulphasalazine – Azathioprine (past or currently) Othersb
40 (29–64) 3/2 (ratio 1.5:1) 3 (60%) UC; 2 (40%) CD 1
35.5 (18–65) 25/21 (ratio 1.2:1) 29 (63%) UC; 17 (37%) CD 2
2 (40%) 1 (20%) 4 (80%)
28 (60%) 4 (8%) 23 (50%)
1 (20%)
8 (17%)
UC, ulcerative colitis; CD, Crohn's disease. a Values are shown as median and range. b Such as oral steroids, antibiotics, biological therapy, antihypertensive drugs, proton pump inhibitors.
so were considered to be wild type (TPMT*1/*1). There were no significant differences (p > 0.05) between carriers of mutant alleles and wild type patients according to age, sex, type of disease, smoking, and chronic medications (Table 2). Six patients (11.8%; 95% CI 2.4%–19.5%) displayed very low TPMT enzyme activity levels. However, only one of them carried an allelic mutation, therefore showing association between genotype and phenotype status. Twelve patients (23.5%; 95% CI 11.4%–34.5%) had an intermediate enzyme activity, even if just 3 showed allelic variants of TPMT (two heterozygous and one homozygous for TPMT*3C and TPMT*2 respectively) while 9 of them were genotyped as TPMT wild type. Finally, the remaining 33 patients (64.7%; 95% CI 52%– 78%) displayed normal or high enzyme activity and this agreed with their detected wild type status, except for one patient who was heterozygous for the allelic variant TPMT*3B. According to TPMT enzymatic activity, no differences were found (p > 0.05) among the three groups of patients as regards age, type of disease, smoking, and chronic medications while a significantly higher male/female ratio has been found in the intermediate TPMT activity group (p = 0.008; Table 3). As regards smoking, three patients were smokers and one of them had an intermediate TPMT activity and was a carrier of heterozygous allelic variant, while the other two were characterized as wild-type and showed normal/high TPMT activity. As an agreement between TPMT allelic mutation and enzymatic activity was found in 4 (TPMT allelic mutation and low/intermediate activity) and 32 (TPMT wild type and normal/higher activity) patients, a 71% (95% CI 61%–81%) concordance rate between genotype and phenotype status was calculated, leading to k statistic of 0.45.
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Table 4 Adverse effect reactions (ADR) in 6 out of 27 (22%) patients with inflammatory bowel disease who underwent therapy with azathioprine, according to TPMT genotype and phenotype. ADR
Patient characteristics
TPMT genotype
TPMT phenotype
Pancreatitis n=1 Hypertransaminasemia n=2 Bone marrow toxicity n=3
CD, 24 yrs, male
WT
Very low
UC, UC, UC, CD, CD,
TPMT*2 homozygous WT WT TPMT*3A Homozygous WT
Intermediate Normal Normal Very low Normal
64 yrs, male 52 yrs, female 49 yrs, female 29 yrs, female 40 yrs, male
UC, ulcerative colitis; CD, Crohn's disease; WT, wild type.
In the previous clinical history, among the 6 patients with very low TPMT activity levels, only two were administered AZA in order to maintain remission at the dosage of 1.5 mg/kg/d. Both cases displayed serious ADR, and it was necessary to discontinue the drug immediately. One patient, homozygous for TPMT*3C (A719G) mutant allele, developed myelotoxicity abruptly, despite a 4 year period of drug tolerance without any adverse event. AZA was stopped and the bone marrow function slowly ameliorated, also supported by the parenteral use of granulocyte–macrophage colony-stimulating factor. The second, TPMT wild type according to genotype evaluation, showed significant raising levels of serum pancreatic enzymes after 4 weeks since AZA was introduced, without any abdominal painful symptomatology but slight increase in diameter of the Wirsung's pancreatic duct at ultrasound evaluation. Hospitalization and the withdrawal of the drug were necessary but monitoring clinical and biochemical parameters showed normalization within the following two weeks. Among the 12 subjects considered intermediate AZA metabolizers, two resulted heterozygous respectively for TPMT*3B and *3C and one homozygous for TPMT*2. Formerly 3 of these intermediate metabolizers used AZA (1.5–2 mg/kg/d) but 2 subjects had to withdraw the drug for lack of efficacy (one was wild-type and one heterozygous for TPMT*3C) and one for sudden onset of hypertransaminasemia. It occurred a few days since AZA introduction and values returned to normal within a few weeks after drug withdrawal. This subject resulted homozygous for TPMT*2 variant allele according to genotype evaluation. None of the current 5 AZA users (1.5–2 mg/kg/d) of this group had never developed ADR due to AZA, although one of them was just an heterozygous allelic variant carrier for TPMT*3B. The remaining 4 patients identified as intermediate metabolizers were all wild-type and had never used AZA. In the 33 patients with normal/ higher TPMT activity, one showed an allelic variant for TPMT*3C in heterozygosis and was AZA-naive. All the others were identified as
Table 3 Patient characteristics according to TPMT activity. Variables
Very low TPMT activity n=6
Intermediate TPMT activity n = 12
Normal/high TPMT activity n = 33
Agea (years) Sex⁎ (M/F)
28 (24–41) 3/3 (ratio 1:1) 3 (50%) UC 3 (50%) CD 0
41 (18–64) 8/4 (ratio 2:1) 6 (50%) UC 6 (50%) CD 1
37 (18–65) 17/16 (ratio 1:1) 23 (70%) UC 10 (30%) CD 2
4 1 0 2 1
10 1 5 3 3
21 4 13 4 3
Disease Smoke Medications – Oral mesalamine – Sulphasalazine – Azathioprine currently – Azathioprine formerly – Othersb
UC, ulcerative colitis; CD, Crohn's disease. a Values are shown as median and range. b Such as oral steroids, antibiotics, biological therapy, antihypertensive drugs, proton pump inhibitors. ⁎ p = 0.008.
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wild-type. Five of them had taken AZA in the past at a dose of 1.5– 2 mg/kg/d. It has been suspended for lack of efficacy in 3 patients, for the occurrence of hypertransaminasemia in one patient and for bone marrow toxicity in the last patient. Thirteen individuals among normal/higher azathioprine metabolizers currently used AZA at a dose of 1.5–2 mg/kg/d and at present nobody had showed AZArelated side effects with the exception of one patient who experienced slight leucopoenia in a previous approach with the drug but this did not reoccurred when we reintroduced the drug at lower dose. This subject had a wild type TPMT status and normal enzyme activity, even if with a trend toward the intermediate activity (16.7 units). All the ADR are summarized in Table 4. 4. Discussion Even though since 1980s several studies have shown that low TPMT activity is associated with ADR, the efficacy of a screening strategy for TPMT gene mutation and/or enzymatic activity in patients undergoing treatment with thiopurine drugs has not yet been firmly established [17]. In this study both genotype and phenotype TPMT status has been investigated and a high percentage of genotypic TPMT changes and phenotypic even more have been found in a series of IBD patients from Southern Italy. Genotypic TPMT findings are consistent with the few currently available data suggesting a significantly higher prevalence of TPMT polymorphism in Caucasian IBD population setting compared to non-Caucasian [18,19]. Nevertheless, data significantly differ from the results of a recent large healthy Italian population study [20]. However, the peculiar homogeneity of the genotypic background of the study population together with the disease status may account for the apparent inconsistency with a larger nation-wide series. TPMT phenotype findings of this study significantly differ from the 0.3%, 8–11%, and 89–92% trimodal distribution which has been recognized in healthy Caucasian populations as well as from data of a large sample of Spanish IBD patients [21,22]. Considering that the coadministration of other drugs is a potential source of interference on TPMT activity, several studies have focused on the role of aminosalicylates, conventional drugs for IBD patients, for inducing ADR by reducing TPMT activity and increasing 6-TGNs serum levels [23,24]. In this study, 16 (88%) patients among those displaying a very low/intermediate TPMT activity currently used salicylic acid preparations. Nevertheless, 25 (78%) patients displaying normal/high TPMT activity were salicylate-drug users too. Thus, it seems unlikely that standard dose of aminosalicylates significantly decreased TPMT activity and this is consistent with more recent findings [25,26]. The median age of patients with intermediate and normal/high TPMT activity has been found to be higher than in patients with very low TPMT activity. Likewise, males did not show higher TPMT activity than females and a 2:1 male/female ratio has been found in the intermediate TPMT activity group. Both findings contrast with other studies showing increased TPMT activity in children compared to adults as well as in males compared to females [27,28]. Overall, 27 (52%) patients had taken AZA, including current and past users. Among them, 6 (22%) patients developed ADR but 3 (50%) of them (one displaying hypertransaminasemia and two myelotoxicity) did not show nor decreased TPMT activity neither polymorphism. In the remaining 3 patients, one who developed pancreatitis was TPMT wild-type but had low TPMT activity. The other two, who experienced hypertransaminasemia and myelotoxicity respectively, were both homozygous for TPMT allelic mutation, the former showing the TMPT*2 polymorphism and an intermediate TPMT activity, the latter being a TPMT*3C polymorphism carrier who had very low TPMT activity. Therefore, in this series, 50% of patients who developed AZA-related ADR had TPMT normal activity
and wild type status. Findings of this study are consistent with a recent meta-analysis which suggests that the TPMT polymorphisms are associated with thiopurine induced overall ADRs and BMT, but not with hepatotoxicity and pancreatitis [29]. It has been demonstrated that TPMT activity may significantly increase during thiopurine treatment as a result of enzyme induction [30]. According to this finding, it has been here found that 13 (39%) patients displaying normal/high and 5 (41%) displaying intermediate TPMT activity currently used AZA, while patients displaying very low TPMT activity were not current but past users of thiopurines. Therefore, that TPMT activity has been measured during AZA treatment in patients who currently tolerate the drug while after AZA discontinuation in patients showing ADR could lead to an overestimation or underestimation, respectively, of TPMT activity. Nevertheless, the lack of dose standardization in this study could affect findings of association between TPMT alterations and AZA toxicity. In conclusion, TPMT genetic polymorphism and even more TPMT enzymatic activity should be considered relevant hot topics in IBD therapeutic management, particularly taking into account the observed variability of clinical response to thiopurines [31]. Learning points • A higher prevalence and lower activity of TPMT could be found in IBD patients compared to the general population. • In IBD patients, a 50% association of AZA-related ADR with TPMT alterations could be expected. Conflict of interest statement The authors have nothing to declare, and there are no financial obligations to any of the author. Acknowledgments The authors would like to thank Dr. Heather Mandy Bond for revision of English language. References [1] Prefontaine E, Sutherland LR, Macdonald JK, Cepoiu M. Azathioprine or 6mercaptopurine for maintenance of remission in Crohn's disease. Cochrane Database Syst Rev 2009;21:CD000067. [2] Prefontaine E, Macdonald JK, Sutherland LR. Azathioprine or 6-mercaptopurine for induction of remission in Crohn's disease. Cochrane Database Syst Rev 2010;16:CD000545. [3] Jharap B, Seinen ML, de Boer NK, van Ginkel JR, Linskens RK, Kneppelhout JC, et al. Thiopurine therapy in inflammatory bowel disease patients: analyses of two 8-year intercept cohorts. Inflamm Bowel Dis 2010;16:1541–9. [4] Warman JI, Korelitz BI, Fleisher MR, Janardhanam R. Cumulative experience with short- and long-term toxicity to 6-mercaptopurine in the treatment of Crohn's disease and ulcerative colitis. J Clin Gastroenterol 2003;37:220–5. [5] Eklund BI, Moberg M, Bergquist J, Mannervik B. Divergent activities of human glutathione transferases in the bioactivation of azathioprine. Mol Pharmacol 2006;70:747–54. [6] van Asseldonk DP, Sanderson J, de Boer NK, Sparrow MP, Lémann M, Ansari A, et al. Thiopurine Task Force Interest Group. Difficulties and possibilities with thiopurine therapy in inflammatory bowel disease—proceedings of the first Thiopurine Task Force meeting. Dig Liver Dis 2011;43:270–6. [7] Ding L, Zhang FB, Liu H, Gao X, Bi HC, Wang XD, et al. Hypoxanthine guanine phosphoribosyltransferase activity is related to 6-thioguanine nucleotide concentrations and thiopurine-induced leukopenia in the treatment of inflammatory bowel disease. Inflamm Bowel Dis 2011, doi:10.1002/ibd.21676 [Epub ahead of print]. [8] Relling MV, Gardner EE, Sandborn WJ, Schmiegelow K, Pui CH, Yee SW, et al. Clinical pharmacogenetics implementation consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing. Clin Pharmacol Ther 2011;89:387–91. [9] Roberts-Thomson IC, Butler WJ. Azathioprine, 6-mercaptopurine and thiopurine S-methyltransferase. J Gastroenterol Hepatol 2005;20:955. [10] Weinshilboum RM, Sladek SL. Mercaptopurine pharmacogenetics: monogenic inheritance of erythrocyte thiopurine methyltransferase activity. Am J Hum Genet 1980;32:651–62.
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European Journal of Internal Medicine journal homepage: www.elsevier.com/locate/ejim
Original article
High prevalence of polymorphism and low activity of thiopurine methyltransferase in patients with inflammatory bowel disease Tiziana Larussa a, Evelina Suraci a, Margherita Lentini b, Immacolata Nazionale a, Luigia Gallo b, Ludovico Abenavoli a, Maria Imeneo a, Francesco Saverio Costanzo b, Giovanni Cuda b, Francesco Luzza a,⁎ a b
Department of Health Science, University of Catanzaro "Magna Graecia", Catanzaro, Italy Department of Clinical and Experimental Medicine, and Tommaso Campanella Foundation, University of Catanzaro "Magna Graecia", Catanzaro, Italy
a r t i c l e
i n f o
Article history: Received 1 November 2011 Received in revised form 3 December 2011 Accepted 8 December 2011 Available online 5 January 2012 Keywords: Crohn's disease Inflammatory bowel diseases Thiopurines Ulcerative colitis Azathioprine
a b s t r a c t Background: Gene polymorphism of thiopurine methyltransferase (TPMT) correlates with decreased enzyme activity which determines a significant risk of adverse effect reactions (ADR) in patients treated with thiopurines. The aim of this study was to investigate TPMT genotype and phenotype status in patients with inflammatory bowel diseases (IBD). Methods: Fifty-one consecutive out-patients with IBD were genotyped for the following allelic variants: rs1800462 (referred as TPMT*2 allele), rs1800460 (referred as TPMT *3B allele), and 1142345 (referred as TPMT *3C allele). Red blood cell TPMT activity was measured using a competitive micro-well immunoassay for the semi-quantitative determination of TPMT activity in red blood cells (RBC) by means of a 6-MP substrate. Results: Polymorphism of TPMT was found in 5 out of 51 patients (10%; 95% CI 2%–18%), three heterozygous and two homozygous carriers. Six patients (11.8%; 95% CI 2.4%–19.5%) displayed very low, 12 (23.5%; 95% CI 11.4%– 34.5%) intermediate, and 33 (64.7%; 95% CI 52%–78%) normal/high TPMT activity. There were no differences between TPMT genotype and phenotype groups according to age, type of disease, smoking, and chronic medications. A 71% (95% CI 61%–81%; κ = 0.45) concordance rate was found between genotype and phenotype status. Six out of 27 (22%) current or past users of azathioprine developed ADR, with three (50%) displaying TPMT genotype and/or phenotype alterations. Conclusion: Compared to the general population, IBD patients may have significantly higher prevalence of TPMT polymorphism and, even more, low activity. Phenotypic more than genotypic TPMT analysis could be useful to better manage IBD therapy with thiopurines. © 2011 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
1. Introduction The thiopurine drugs 6-mercaptopurine (6-MP) and its prodrug azathioprine (AZA) are widely used in the treatment of active, steroid-refractory and/or steroid-dependent inflammatory bowel disease (IBD), and have been proven to be effective for inducing and maintaining remission in these patients [1,2]. However, as many as one-third of these patients may develop adverse effect reactions (ADR), the dose of thiopurine often must be reduced or the therapy has to be discontinued in more than 30% of patients [3]. Gastrointestinal symptoms, bone marrow toxicity (BMT), hepatotoxicity and pancreatitis are the most common ADR. Blood tests are performed regularly to monitor blood count and liver function in order to find BMT and hepatotoxicity at an early enough stage and discontinue therapy before the onset of life-threatening toxicity. However, in ⁎ Corresponding author at: Dipartimento di Scienze della Salute, Università di Catanzaro “Magna Graecia”, Campus Universitario di Germaneto Viale Europa 88100 Catanzaro, Italy. Tel.: + 39 0961 3697113; fax: + 39 0961 3697164. E-mail address: [email protected] (F. Luzza).
some cases, ADR might develop unpredictably during the interval between the two tests [4]. Much interest has focused on the metabolism of thiopurines to identify individualized therapeutic pathways, minimizing adverse effects and enhancing clinical response. At date it is assumed that AZA is not enzymatically converted to MP, even if a potential effect of glutathione-S-transferase in the conversion of AZA into MP has been demonstrated [5]. 6MP is then enzymatically converted by competing pathways into the following metabolites: 6-methylmercaptopurine (6-MMP) by the action of thiopurine methyltransferase (TPMT), thought to be inactive or maybe responsible for hepatotoxicity; 6thiouric acid by the action of xanthine oxidase (XO), also an inactive compound; 6-thioguanine nucleotides (6-TGNs), formed by an enzymatic anabolic process dealing with the action of hypoxanthine guanine phosphoribosyl transferase (HGPRT), which produces 6-thioinosine monophosphate (6-TIMP). Then 6MP is transformed by inosine monophosphate dehydrogenase (IMPD) into 6-thioxanthosine monophosphate (6-TXMP), which is metabolized to 6-thioguanine monophosphate (6-TGMP), -diphosphate (6TGDP) and -triphosphate (6 TGTP) [6]. 6-TGNs, comprehensive of the above mentioned products,
0953-6205/$ – see front matter © 2011 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ejim.2011.12.002
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are the active metabolites responsible for pharmacological effect but also associated with dose-related adverse reactions, mainly myelotoxicity [7]. A balance between TPMT, XO, and HGPRT is responsible for final serum concentration of 6-TGNs. Genetic variation of the activity of TPMT and its influence on AZA metabolism is an important pharmacogenetic model. TPMT*1 is the wild-type allele, whereas TPMT*2 and TPMT*3 account for the vast majority (>90%) of mutant alleles among at least 10 which are associated with a decreased TPMT enzymatic activity [8]. Distribution of TPMT mutant alleles differs significantly among ethnic populations. TPMT*3 A, including TPMT*3C (719A > G) and 3*B (460 G > A), is the most frequent occurring mutant allele (3.2–5.7%) in Caucasian populations [9]. There is a trimodal distribution of TPMT activity in Caucasian population: one in 300 subjects (0.3%) has low or undetectable TPMT activity, 8–11% of individuals have intermediate TPMT activity, 89–92% have high or normal enzyme activity [10]. A high degree of correlation between TPMT geno- and phenotype has been ascertained, since data of the trimodal distribution can be considered overlapping prevalence of genetic variants in the general population. Indeed polymorphisms of the gene encoding TPMT enzyme correlate with decreased enzyme activity. In particular, heterozygosity TPMT gene status is associated with intermediate enzyme activity whereas homozygosity status for the mutant alleles determines a significant risk of toxicity in patients receiving standard doses of thiopurines, primarily as a result of unchecked production of TGNs [11]. Recently, screening patients for TPMT polymorphism and/or enzymatic activity before thiopurine treatment in order to predict the risk of toxicity has aroused considerable interest in several clinical centers. The aim of this study was to investigate TPMT genotype and phenotype status in a Southern Italian population of IBD patients. 2. Materials and methods 2.1. Subjects Fifty-one consecutive out-patients followed at our University reference Centre for IBD were enrolled for the study, 19 patients with Crohn's disease (CD) and 32 with ulcerative colitis (UC). Diagnosis of IBD was assessed in accordance with current clinical guidelines and criteria based on endoscopic, radiological and histopathologic examination [12]. Demographic data, clinical history, disease-related features and type of medications were recorded (Table 1). Venous blood sample from each patient was obtained after overnight fasting.
Table 1 Patient characteristics. Variable
Crohn's disease n = 19
Ulcerative colitis n = 32
Sex (M/F) Agea(years) Disease site – Ileum – Ileocolon – Colon Smoke Medication – Oral mesalamine – Sulphasalazine – Azathioprine (currently users) – Azathioprine (formerly users) – Othersb
9/10 40 (18–65)
19/13 34.5 (18–52)
5 9 5 3
– – 32 0
6 4 6 5 6
24 1 12 4 3
a
Values are shown as median and range. Such as oral steroids, antibiotics, biological therapy, antihypertensive drugs, proton pump inhibitors. b
None of the subjects had undergone blood transfusions during the previous 3 months. All patients were considered Caucasians as assessed by excluding a different origin of their first degree relatives. The study was approved by the local ethics committee and all patients gave written informed consent prior to entering the study. 2.2. TPMT genotyping Total genomic DNA was extracted from peripheral blood lymphocytes using a commercial kit (Wizard Genomic DNA Purification Kit, Promega, Madison, WI, USA) according to the manufacturer's recommendations. Each individual was genotyped at the TPMT*2 (rs1800462), TPMT*3B (rs1800460), and TPMT*3C (rs1142345) alleles by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP)-based assays, using previously described conditions [13–15]. Positive and negative controls were available for each genotype. The PCR products were separated by nondenaturing 8% PAGE. 2.3. TPMT phenotyping Blood samples were collected in EDTA containing tubes and stored at 4 °C. TPMT enzymatic activity was performed within 3 h of time of collection. The TPMT phenotype was assessed by measuring TPMT activity using a competitive micro-well immunoassay for the semi-quantitative determination of TPMT activity in red blood cells (RBC) by means of a 6MP substrate. TPMT catalyzes the S-methylation of 6-mercaptopurine (6-MP) with the presence of S-adenosylmethionine (SAM), the methyl donor, yielding 6-methylmercaptopurine (6-MMP). Then 6-MMP is measured with a micro-well EIA. Biologix TPMT ELISA kit (CE Mark, Biologix Diagnostics, LLC., Lenexa, KS, USA) was used. One unit of enzyme activity is defined as a formation of 1 nmol of 6-MMP per ml of packed RBC per 60 min incubation at 37 °C. Erythrocyte TPMT activity was expressed as units per gram of hemoglobin (U/gHb). The assay interpretation is according to the following criteria: ≤5.5, 5.6–15.5, and ≥15.6 Units as very low, medium, and normal to high TPMT activity level, respectively. Intra and inter-assay coefficients of variation were 8.90% and 9.15%, respectively. 2.4. Statistics Percentages were given along with 95% confidence intervals (CI). Comparison of proportions was performed using chi-square test. Continuous data were compared using the Student t test for independent samples. Multiple testing was assessed by one-way analysis of variance (ANOVA) and, when the F value was significant, by Tukey's multiple comparison test. Difference was considered significant if the p value was b0.05. The relation between TPMT genotype and phenotype was evaluated by means of k statistic. The k statistic, a measure of the agreement between two observers or tests, ranges from − 1 to 1 with 1 indicating perfect agreement, 0 indicating the agreement expected on the basis of chance alone, and values between 0 and 0.4 a poor to fair agreement [16]. The software program Primit (1994, version 3.03) was used to perform the statistical analysis. 3. Results Overall, allelic mutations for TPMT were found in 5 out of the 51 patients (10%; 95% CI 2%–18%). One patient was heterozygous for the TPMT*3B (rs1800460) allele, two (4%) were heterozygous for the TPMT*3C (rs1142345) allele, whereas two more patients (4%) were homozygous for TPMT*3C (rs1142345) and TPMT*2 (rs1800462) allele, respectively. Of them, only one (heterozygous) patient was currently using azathioprine (2 mg/kg/d) but had not experienced any ADR until now. The remaining 46 patients (90%; 95% CI 81%–98%) did not show any of the variant TPMT alleles tested for, and
T. Larussa et al. / European Journal of Internal Medicine 23 (2012) 273–277 Table 2 Patient characteristics according to TPMT polymorphism. Variables
Polymorphism carriers n=5
Wild type patients n = 46
Agea(years) Sex (M/F) Disease Smoke Medications – Oral mesalamine – Sulphasalazine – Azathioprine (past or currently) Othersb
40 (29–64) 3/2 (ratio 1.5:1) 3 (60%) UC; 2 (40%) CD 1
35.5 (18–65) 25/21 (ratio 1.2:1) 29 (63%) UC; 17 (37%) CD 2
2 (40%) 1 (20%) 4 (80%)
28 (60%) 4 (8%) 23 (50%)
1 (20%)
8 (17%)
UC, ulcerative colitis; CD, Crohn's disease. a Values are shown as median and range. b Such as oral steroids, antibiotics, biological therapy, antihypertensive drugs, proton pump inhibitors.
so were considered to be wild type (TPMT*1/*1). There were no significant differences (p > 0.05) between carriers of mutant alleles and wild type patients according to age, sex, type of disease, smoking, and chronic medications (Table 2). Six patients (11.8%; 95% CI 2.4%–19.5%) displayed very low TPMT enzyme activity levels. However, only one of them carried an allelic mutation, therefore showing association between genotype and phenotype status. Twelve patients (23.5%; 95% CI 11.4%–34.5%) had an intermediate enzyme activity, even if just 3 showed allelic variants of TPMT (two heterozygous and one homozygous for TPMT*3C and TPMT*2 respectively) while 9 of them were genotyped as TPMT wild type. Finally, the remaining 33 patients (64.7%; 95% CI 52%– 78%) displayed normal or high enzyme activity and this agreed with their detected wild type status, except for one patient who was heterozygous for the allelic variant TPMT*3B. According to TPMT enzymatic activity, no differences were found (p > 0.05) among the three groups of patients as regards age, type of disease, smoking, and chronic medications while a significantly higher male/female ratio has been found in the intermediate TPMT activity group (p = 0.008; Table 3). As regards smoking, three patients were smokers and one of them had an intermediate TPMT activity and was a carrier of heterozygous allelic variant, while the other two were characterized as wild-type and showed normal/high TPMT activity. As an agreement between TPMT allelic mutation and enzymatic activity was found in 4 (TPMT allelic mutation and low/intermediate activity) and 32 (TPMT wild type and normal/higher activity) patients, a 71% (95% CI 61%–81%) concordance rate between genotype and phenotype status was calculated, leading to k statistic of 0.45.
275
Table 4 Adverse effect reactions (ADR) in 6 out of 27 (22%) patients with inflammatory bowel disease who underwent therapy with azathioprine, according to TPMT genotype and phenotype. ADR
Patient characteristics
TPMT genotype
TPMT phenotype
Pancreatitis n=1 Hypertransaminasemia n=2 Bone marrow toxicity n=3
CD, 24 yrs, male
WT
Very low
UC, UC, UC, CD, CD,
TPMT*2 homozygous WT WT TPMT*3A Homozygous WT
Intermediate Normal Normal Very low Normal
64 yrs, male 52 yrs, female 49 yrs, female 29 yrs, female 40 yrs, male
UC, ulcerative colitis; CD, Crohn's disease; WT, wild type.
In the previous clinical history, among the 6 patients with very low TPMT activity levels, only two were administered AZA in order to maintain remission at the dosage of 1.5 mg/kg/d. Both cases displayed serious ADR, and it was necessary to discontinue the drug immediately. One patient, homozygous for TPMT*3C (A719G) mutant allele, developed myelotoxicity abruptly, despite a 4 year period of drug tolerance without any adverse event. AZA was stopped and the bone marrow function slowly ameliorated, also supported by the parenteral use of granulocyte–macrophage colony-stimulating factor. The second, TPMT wild type according to genotype evaluation, showed significant raising levels of serum pancreatic enzymes after 4 weeks since AZA was introduced, without any abdominal painful symptomatology but slight increase in diameter of the Wirsung's pancreatic duct at ultrasound evaluation. Hospitalization and the withdrawal of the drug were necessary but monitoring clinical and biochemical parameters showed normalization within the following two weeks. Among the 12 subjects considered intermediate AZA metabolizers, two resulted heterozygous respectively for TPMT*3B and *3C and one homozygous for TPMT*2. Formerly 3 of these intermediate metabolizers used AZA (1.5–2 mg/kg/d) but 2 subjects had to withdraw the drug for lack of efficacy (one was wild-type and one heterozygous for TPMT*3C) and one for sudden onset of hypertransaminasemia. It occurred a few days since AZA introduction and values returned to normal within a few weeks after drug withdrawal. This subject resulted homozygous for TPMT*2 variant allele according to genotype evaluation. None of the current 5 AZA users (1.5–2 mg/kg/d) of this group had never developed ADR due to AZA, although one of them was just an heterozygous allelic variant carrier for TPMT*3B. The remaining 4 patients identified as intermediate metabolizers were all wild-type and had never used AZA. In the 33 patients with normal/ higher TPMT activity, one showed an allelic variant for TPMT*3C in heterozygosis and was AZA-naive. All the others were identified as
Table 3 Patient characteristics according to TPMT activity. Variables
Very low TPMT activity n=6
Intermediate TPMT activity n = 12
Normal/high TPMT activity n = 33
Agea (years) Sex⁎ (M/F)
28 (24–41) 3/3 (ratio 1:1) 3 (50%) UC 3 (50%) CD 0
41 (18–64) 8/4 (ratio 2:1) 6 (50%) UC 6 (50%) CD 1
37 (18–65) 17/16 (ratio 1:1) 23 (70%) UC 10 (30%) CD 2
4 1 0 2 1
10 1 5 3 3
21 4 13 4 3
Disease Smoke Medications – Oral mesalamine – Sulphasalazine – Azathioprine currently – Azathioprine formerly – Othersb
UC, ulcerative colitis; CD, Crohn's disease. a Values are shown as median and range. b Such as oral steroids, antibiotics, biological therapy, antihypertensive drugs, proton pump inhibitors. ⁎ p = 0.008.
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wild-type. Five of them had taken AZA in the past at a dose of 1.5– 2 mg/kg/d. It has been suspended for lack of efficacy in 3 patients, for the occurrence of hypertransaminasemia in one patient and for bone marrow toxicity in the last patient. Thirteen individuals among normal/higher azathioprine metabolizers currently used AZA at a dose of 1.5–2 mg/kg/d and at present nobody had showed AZArelated side effects with the exception of one patient who experienced slight leucopoenia in a previous approach with the drug but this did not reoccurred when we reintroduced the drug at lower dose. This subject had a wild type TPMT status and normal enzyme activity, even if with a trend toward the intermediate activity (16.7 units). All the ADR are summarized in Table 4. 4. Discussion Even though since 1980s several studies have shown that low TPMT activity is associated with ADR, the efficacy of a screening strategy for TPMT gene mutation and/or enzymatic activity in patients undergoing treatment with thiopurine drugs has not yet been firmly established [17]. In this study both genotype and phenotype TPMT status has been investigated and a high percentage of genotypic TPMT changes and phenotypic even more have been found in a series of IBD patients from Southern Italy. Genotypic TPMT findings are consistent with the few currently available data suggesting a significantly higher prevalence of TPMT polymorphism in Caucasian IBD population setting compared to non-Caucasian [18,19]. Nevertheless, data significantly differ from the results of a recent large healthy Italian population study [20]. However, the peculiar homogeneity of the genotypic background of the study population together with the disease status may account for the apparent inconsistency with a larger nation-wide series. TPMT phenotype findings of this study significantly differ from the 0.3%, 8–11%, and 89–92% trimodal distribution which has been recognized in healthy Caucasian populations as well as from data of a large sample of Spanish IBD patients [21,22]. Considering that the coadministration of other drugs is a potential source of interference on TPMT activity, several studies have focused on the role of aminosalicylates, conventional drugs for IBD patients, for inducing ADR by reducing TPMT activity and increasing 6-TGNs serum levels [23,24]. In this study, 16 (88%) patients among those displaying a very low/intermediate TPMT activity currently used salicylic acid preparations. Nevertheless, 25 (78%) patients displaying normal/high TPMT activity were salicylate-drug users too. Thus, it seems unlikely that standard dose of aminosalicylates significantly decreased TPMT activity and this is consistent with more recent findings [25,26]. The median age of patients with intermediate and normal/high TPMT activity has been found to be higher than in patients with very low TPMT activity. Likewise, males did not show higher TPMT activity than females and a 2:1 male/female ratio has been found in the intermediate TPMT activity group. Both findings contrast with other studies showing increased TPMT activity in children compared to adults as well as in males compared to females [27,28]. Overall, 27 (52%) patients had taken AZA, including current and past users. Among them, 6 (22%) patients developed ADR but 3 (50%) of them (one displaying hypertransaminasemia and two myelotoxicity) did not show nor decreased TPMT activity neither polymorphism. In the remaining 3 patients, one who developed pancreatitis was TPMT wild-type but had low TPMT activity. The other two, who experienced hypertransaminasemia and myelotoxicity respectively, were both homozygous for TPMT allelic mutation, the former showing the TMPT*2 polymorphism and an intermediate TPMT activity, the latter being a TPMT*3C polymorphism carrier who had very low TPMT activity. Therefore, in this series, 50% of patients who developed AZA-related ADR had TPMT normal activity
and wild type status. Findings of this study are consistent with a recent meta-analysis which suggests that the TPMT polymorphisms are associated with thiopurine induced overall ADRs and BMT, but not with hepatotoxicity and pancreatitis [29]. It has been demonstrated that TPMT activity may significantly increase during thiopurine treatment as a result of enzyme induction [30]. According to this finding, it has been here found that 13 (39%) patients displaying normal/high and 5 (41%) displaying intermediate TPMT activity currently used AZA, while patients displaying very low TPMT activity were not current but past users of thiopurines. Therefore, that TPMT activity has been measured during AZA treatment in patients who currently tolerate the drug while after AZA discontinuation in patients showing ADR could lead to an overestimation or underestimation, respectively, of TPMT activity. Nevertheless, the lack of dose standardization in this study could affect findings of association between TPMT alterations and AZA toxicity. In conclusion, TPMT genetic polymorphism and even more TPMT enzymatic activity should be considered relevant hot topics in IBD therapeutic management, particularly taking into account the observed variability of clinical response to thiopurines [31]. Learning points • A higher prevalence and lower activity of TPMT could be found in IBD patients compared to the general population. • In IBD patients, a 50% association of AZA-related ADR with TPMT alterations could be expected. Conflict of interest statement The authors have nothing to declare, and there are no financial obligations to any of the author. Acknowledgments The authors would like to thank Dr. Heather Mandy Bond for revision of English language. References [1] Prefontaine E, Sutherland LR, Macdonald JK, Cepoiu M. Azathioprine or 6mercaptopurine for maintenance of remission in Crohn's disease. Cochrane Database Syst Rev 2009;21:CD000067. [2] Prefontaine E, Macdonald JK, Sutherland LR. Azathioprine or 6-mercaptopurine for induction of remission in Crohn's disease. Cochrane Database Syst Rev 2010;16:CD000545. [3] Jharap B, Seinen ML, de Boer NK, van Ginkel JR, Linskens RK, Kneppelhout JC, et al. Thiopurine therapy in inflammatory bowel disease patients: analyses of two 8-year intercept cohorts. Inflamm Bowel Dis 2010;16:1541–9. [4] Warman JI, Korelitz BI, Fleisher MR, Janardhanam R. Cumulative experience with short- and long-term toxicity to 6-mercaptopurine in the treatment of Crohn's disease and ulcerative colitis. J Clin Gastroenterol 2003;37:220–5. [5] Eklund BI, Moberg M, Bergquist J, Mannervik B. Divergent activities of human glutathione transferases in the bioactivation of azathioprine. Mol Pharmacol 2006;70:747–54. [6] van Asseldonk DP, Sanderson J, de Boer NK, Sparrow MP, Lémann M, Ansari A, et al. Thiopurine Task Force Interest Group. Difficulties and possibilities with thiopurine therapy in inflammatory bowel disease—proceedings of the first Thiopurine Task Force meeting. Dig Liver Dis 2011;43:270–6. [7] Ding L, Zhang FB, Liu H, Gao X, Bi HC, Wang XD, et al. Hypoxanthine guanine phosphoribosyltransferase activity is related to 6-thioguanine nucleotide concentrations and thiopurine-induced leukopenia in the treatment of inflammatory bowel disease. Inflamm Bowel Dis 2011, doi:10.1002/ibd.21676 [Epub ahead of print]. [8] Relling MV, Gardner EE, Sandborn WJ, Schmiegelow K, Pui CH, Yee SW, et al. Clinical pharmacogenetics implementation consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing. Clin Pharmacol Ther 2011;89:387–91. [9] Roberts-Thomson IC, Butler WJ. Azathioprine, 6-mercaptopurine and thiopurine S-methyltransferase. J Gastroenterol Hepatol 2005;20:955. [10] Weinshilboum RM, Sladek SL. Mercaptopurine pharmacogenetics: monogenic inheritance of erythrocyte thiopurine methyltransferase activity. Am J Hum Genet 1980;32:651–62.
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