Dig Dis Sci DOI 10.1007/s10620-013-3008-z
ORIGINAL ARTICLE
Thiopurine S-methyltransferase (TPMT) Activity Is Better Determined by Biochemical Assay Versus Genotyping in the Jewish Population Yair Kasirer • Rephael Mevorach • Paul Renbaum Nurit Algur • Devora Soiferman • Rachel Beeri • Yelana Rachman • Reeval Segel • Dan Turner
•
Received: 21 October 2013 / Accepted: 17 December 2013 Ó Springer Science+Business Media New York 2014
Abstract Background Thiopurine S-methyltransferase (TPMT) is a key enzyme that deactivates thiopurines, into their inactive metabolite, 6-methylmercaptopurine. Intermediate and low TPMT activity may lead to leukopenia following thiopurine treatment. The aim of this study was to determine TPMT activity and TPMT alleles (genotype–phenotype correlation) in Jews, aiming to develop an evidence-based pharmacogenetic assay for this population. Methods TPMT activity was determined in 228 Jewish volunteers by high performance liquid chromatography. Common allelic variants in the Caucasian population [TPMT*2 (G238C), TPMT *3A (G460A and A719G), TPMT* 3B (G460A) and TPMT*3C (A719G)] were tested.
Yair Kasirer, Rephael Mevorach, Reeval Segel and Dan Turner contributed equally to this work. Y. Kasirer Y. Rachman D. Turner (&) Pediatric Gastroenterology and Nutrition Unit, Shaare Zedek Medical Center, The Hebrew University, P.O. B 3235, 91031 Jerusalem, Israel e-mail: [email protected] R. Mevorach N. Algur Biochemistry Laboratory, Shaare Zedek Medical Center, Jerusalem, Israel P. Renbaum D. Soiferman R. Beeri R. Segel Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel R. Segel D. Turner Shaare Zedek Medical Center, The Hebrew University, Jerusalem, Israel
Phenotype–genotype correlation was examined and discordant cases were fully sequenced to identify novel genetic variants. Results Mean TPMT activity was 15.4 ± 4 U/ml red blood cells (range 1–34). Intermediate activity was found in 33/228 (14 %) subjects and absent activity was found in one sample (0.4 %). Only eight individuals (3.5 % of the entire cohort and 24 % of those with intermediate/low activity) were identified as carriers of a TPMT genetic variant, all of whom had the TPMT*3A allele. Sequencing the entire TPMT coding region and splice junctions in the remainder of the discordant cases did not reveal any novel variants. Conclusion Genotyping TPMT in Jews yields a much lower rate of variants than identified in the general Caucasian population. We conclude that a biochemical assay to determine TPMT enzymatic activity should be performed in Jews before starting thiopurine treatment in order to identify low activity subjects. Keywords TPMT activity Jewish population Phenotype Genotype Thiopurines
Introduction Thiopurines (azathioprine and 6-mercaptopurine) are widely used for treating inflammatory bowel diseases (IBD). After ingestion, azathioprine is rapidly converted to 6-MP, which could be converted into 6-thioguanine (6-TG). The latter is the active metabolite at levels above 230–260 pmol/8 9 108 red blood cells (RBC) [1], but myelotoxicity [2], and possibly also malignancy [3], may be observed at 6-TG levels exceeding 450 pmol/8 9 108 RBC. The enzyme thiopurine S-methyltransferase (TPMT) converts 6-MP to its inactive
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metabolite 6-methylmercaptopurine (6-MMP), which is hepatotoxic at high levels [4]. Approximately 30 % of IBD patients treated with thiopurines will discontinue the drug prematurely due to low effectiveness (*15 %) or toxicity (*15 %) [5, 6]. Certain variants in the TPMT gene can modify its structure leading to decreased activity and excessive drug toxicity by increased 6-TG levels. TPMT half-life may be reduced from 18 h in the wild type to as low as 15 min in the mutated enzyme [7]. In Caucasians, three TPMT variants account for 95 % of all alleles associated with low activity: A719G ? G460A (TPMT*3A), G238C (TPMT*2) and A719G (TPMT*3C) [8]. Approximately 10 % of Caucasians are heterozygous for the mutated allele and roughly 0.3 % are homozygous [9]. On the other extreme, 10–15 % of patients have high TPMT activity [10] associated with high 6-MMP/6-TG ratio [11]. No genetic alteration has been associated with this finding to date. The association between high TPMT activity and 6-MMP/6-TG ratio has been recently challenged by Van Egmond et al. [12] showing that TPMT activity did not explain all preferential 6-MMP producers. These patients might be at an increased risk for hepatic injury and non-response to therapy [10, 13]. Studies concluded that testing TPMT prior to thiopurine treatment and following 6-TG and 6-MMP levels may be cost-effective, preventing life-threatening toxicity and increasing effectiveness [14]. In certain populations there is high concordance between lower activity levels and known TPMT genetic variants [15]. In these groups genotyping can be used as an effective tool for prediction of TPMT activity [16]. While TPMT genotyping prior to thiopurine use is a common practice in the USA, it is not widely employed in other parts of the world, in part due to variations in TPMT alleles between nations and ethnicities [5, 17, 18]. Jews are at increased risk for developing IBD (and thus for using thiopurines) while their distinct inbred genetic background may uniquely affect TPMT pharmacogenetics. However, to date, no phenotype–genotype data regarding TPMT activity are available in Jews. A previous study has shown that 10.4 % of healthy Jews (14/134 volunteers) had intermediate TPMT activity using a cutoff value of B11 U, and 7.5 % when using a cutoff value of B10 U, both previously used to determine lower activity [19]. Two other genetic studies have shown that the three common low-activity alleles were found in only 1.5 and 1.8 % of 531 and 164 healthy Jewish volunteers, respectively [20, 21]. This apparent discrepancy set the stage for this study which aimed to concurrently compare TPMT phenotype and genotype in a large Jewish cohort.
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Fig. 1 Distribution of TPMT biochemical activity among 228 volunteers. The eight arrows represent the eight cases with identified mutations (the wide arrow represent homozygosity). No other variants where found in the entire cohort
Methods Design In this prospective study, blood samples were collected from 228 Jewish volunteers ([18 years of age), seen at the emergency department of Shaare Zedek Medical Center for reasons other than IBD. Exclusion criteria were recent blood transfusion and/or treatment with thiopurine, allopurinol or mesalamine compounds during the previous 6 months, which may alter TPMT activity. Basic demographic and phenotypic data were recorded on a standardized case report form. All samples were tested for TPMT activity, and for the three most prevalent alleles in Caucasians (TPMT*2, *3A, *3B and *3C). Since no bimodal distribution was obtained, it was difficult to determine the exact cutoff value that defines low TPMT activity. We thus used the cutoff that included all subjects who were positive for the common genetic mutations associated with low activity, meaning B11 U/ml (nMol/mlPRBC) (Fig. 1). As a sensitivity analysis we also present the intermediate rate utilizing the cutoff of B10 U/ml. The coding sequence and splice junctions of samples with intermediate TPMT activity that were not carriers of the common variants were sequenced in search of novel variants. In addition, all high activity samples were also sequenced to search for specific variants which could explain this finding. There is no definite accepted cutoff for high TPMT activity. While some use a cutoff of [20 U/ml [16], we used the upper 95th percentile of our cohort (i.e. [23 U/ml).
Dig Dis Sci
Biochemical Assay for TPMT Activity TPMT activity was measured by a homemade assay. Whole blood was collected in an EDTA tube, centrifuged at 3,000g for 5 min at 4 °C. After removal of plasma, packed RBC were washed with saline and centrifuged at 10,000g for 10 min. A total of 500 ll of packed RBC were divided equally into two tubes (250 ll each). Packed RBC were lysed with 1.0 ml distilled cold water, centrifuged at 10,000g for 10 min, and 1.0 ml of the supernatant was removed and stored at -80 °C. Enzyme activity was determined by methylation rate of 6-MP to 6-MMP as described elsewhere with some modifications [22]. Briefly, reaction mixture [composed of 100 ll of hemolysate, 20 ll of 1 mM 6-mercaptopurine (6-MP) as a substrate, 20 ll of 3 mM S-adenosyl-L-methionine (SAM, methyl donor) (Sigma, Israel), 315 ll 0.067 M phosphate buffer pH 7.4, and 20 ll of HCl 0.1 M] was incubated at 37 °C for 60 min. For termination of the enzymatic reaction, 25 ll of HClO4 60 % (Sigma, Israel) was added, the tube was centrifuged at 13,000g for 4 min at 4 °C and the supernatant was kept at 4 °C until high performance liquid chromatography (HPLC) analysis. Chromatography was performed at room temperature using RP-HPLC system (Young-lin 9100) with a variable UV–vis detector on a reversed phase column using C-18 125-3 Purospher STAR column (Merck). The column was protected by a guard column containing the same phase. Mobile phase consisted of 0.01 M phosphate buffer adjusted to PH 2.7 with phosphoric acid and acetonitrile (80:20) at a flow rate of 1 ml/min (isocratic mode); 6-MMP was detected at a wavelength of 290 nm. The calibration standards were diluted from homemade stock solution. TPMT activity, expressed in nMol/mlPRBC (herein U/ml), was calculated by Y.L. Clarity software. The intra- and inter-assay coefficient of variance (CV) were 7.41 and 10.6 %, respectively. Genetic Analysis Genotyping DNA was extracted by a high salt extraction technique [23] and analyzed for the common Caucasian variants using allele-specific PCR followed by restriction fragment length polymorphism analysis (PCR–RFLP) [24]. Briefly, PCR was performed using an initial denaturation step at 94 °C for 5 min was followed by 37 cycles consisting of a denaturation step at 94 °C for 45 s, annealing for 45 s at 56 °C for TPMT*2 or 59 °C for TPMT*3A and TPMT*3C, followed by extension at 72 °C for 45 s. The resulting PCR products were digested with restriction enzymes (HpyIII for G238C, MwoI for A460G, and AccI for G719A) and analyzed on a
Table 1 Basic characteristics Entire cohort (n = 228)
Subjects with low TPMT activitya (n = 34)
Subjects with high TPMT activitya (n = 12)
Males
124 (54 %)
17 (50 %)
6 (50 %)
Age (years)
52 ± 22
39 ± 22
44 ± 19
Origin Ashkenazi
123 (54 %)
17 (50 %)
9 (75 %)
Sephardic
48 (21 %)
11 (31 %)
1 (8 %)
Others
57 (25 %)
6 (19 %)
2 (17 %)
Medians (interquartile range) or mean (±SD) are presented as appropriate for the data distribution a Low activity defined as TPMT B11 U/ml and high as TPMT [23 U/ml
3 % agarose gel by electrophoresis. Direct sequencing of carriers was used to confirm the RFLP results. Sequencing PCR products were cleaned with Exonuclease I and shrimp alkaline phosphatase (EXO-SAP Affymetrix, CA, USA) and 2 ll of the purified amplicon was sequenced using Big Dye Terminator v1.1 (Applied Biosystems, CA, USA). The extension products were purified using hydrated gel filtration matrix columns (Edge Biosystems, Maryland, USA) and separated on an ABI 3130xl DNA Genetic Analyzer (Applied Biosystems, CA, USA). SeqScape v2.5v software was used for analysis and the results were compared to the RefSeq TPMT sequence, NM_000367.2. Statistics Data are presented as means (±standard deviation), or medians (interquartile range) and compared using unpaired Student’s t test or Wilcoxon rank sum test as appropriate for the distribution normality. Categorical variables were compared using v2 or Fisher’s exact, as appropriate. A multivariate linear regression model was constructed to explore the association between gender, age and Jewish ethnicity with TPMT activity. Assumptions of the models were verified using residual plots. Analyses were performed using SPSS V15; P values\0.05 were considered significant. This study was approved by the local and Israeli Ministry of Health Supreme ethical committees. Written informed consent was obtained from all participants.
Results Blood samples were collected from 228 volunteers (Table 1) with the mean TPMT activity of 15.4 ± 4 U/ml
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(range 1–34) (Fig. 1). Thirty-three subjects (14 %) had intermediate TPMT activity, and one subject (0.4 %) showed absent activity (\1 U/ml). When considering a cutoff value of B10 U/ml, 18/228 (8 %) of subjects had low/intermediate activity. Twelve (5 %) subjects demonstrated high TPMT activity ([23 U/ml) and 20 (8.8 %) had [20 U/ml TPMT activity. Univariate (Table 1) and multivariate regression analysis for age, gender and ethnicity revealed no association with TPMT activity (all P [ 0.05; r2 = 0.21). Genotyping of the entire cohort detected only eight carriers of the common Caucasian variants [3.5 % of the entire cohort (8/228) and 24 % of those with low/intermediate activity (8/34)]. When using the lower cutoff value for low/intermediate activity of B10 U/ml, 6/18 (33.3 %) of subjects were variant-carriers. The positive subjects all carried the TPMT*3A allele (i.e. A719G ? G460A); seven were heterozygotes, and the one patient with the absent TPMT activity was homozygote (Fig. 1). Sequencing of the TPMT coding region and splice junctions of the 26 subjects with intermediate activity who did not carry the known variants did not reveal any new mutations. Similarly, sequencing the 12 subjects with high TPMT activity did not reveal any unknown variants.
Discussion This is the first study in Jews that concurrently measured both biochemical activity and genetic analysis of TPMT, aiming to determine phenotype–genotype correlation. While we found that 8–14 % of our cohort had low-medium TPMT enzymatic activity, only 3.5 % were positive for one of the common Caucasian TPMT variants. This low rate is consistent with the two previous genetic studies among Jews which tested the three common Caucasian variants [8/531 (1.5 %) and 3/164 (1.8 %)] [21, 24]. We hypothesized that the phenotype–genotype discrepancy among Jews could be explained by novel population-specific mutations. However, we failed to identify any additional mutations after sequencing the entire coding sequences and exon flanking regions of the TPMT gene in all discordant genotype–phenotype cases. We speculate that other genetic variants exist in regulatory regions of the TPMT gene, such as in the promoter, alternative splicing sites, non-translated or enhancer regions, or epigenic modifications that influence the enzyme’s activity explaining the discrepancy observed between TPMT genotype and phenotype in the Jewish population. In our cohort, the TPMT*3A variant was the only allele associated with low activity. In the two previous genetic studies in the Jewish population, TPMT*3A has been found in 10 of the 11 positive samples (out of 695 subjects,
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combined) with the only exception being TPMT*3C. The A719G variant is part of both TPMT*3A (G460A and A719G) and TPMT*3C (only A719G). Therefore, we propose that only the A719G variant should be tested among Jews when genotyping is warranted such as during active thiopurine treatment when biochemical assay is technically not feasible. After initial testing of A719G, positive samples could be tested also for G460A, in order to differentiate between TPMT*3A and *3C. Combining the results of this study with the previous two Jewish genetic studies it could be estimated that the likelihood of finding a known variant, other than A719G, is as low as \1:700. One of the advantages of testing TPMT activity over genotyping is the ability to identify those with high enzyme activity, which may be associated with low drug efficacy and higher risk for hepatic toxicity. While we used the upper 5 % of the cohort to define high activity, 8.8 % (20/ 228) subjects showed TPMT activity [20 U/ml. We sequenced coding sequences and exon flanking regions of the TPMT gene in all high-activity subjects; however, no genetic variants were identified to explain this finding. Some authors suggest prescribing higher doses of thiopurines for patients with high activity [25]; however, the association between high activity and efficacy is not established. In practice, close monitoring of liver enzymes is mandatory during treatment, and measuring drug metabolites after commencing thiopurines is advised. The design of this study did not enable us to correlate TPMT activity with clinical outcome, nor was this within the scope of our study. This is the subject of an ongoing continuation study. The fact that all mutations found in the genetic analysis were in subjects with lower TPMT activity provide internal validity of the biochemical and genetic assays. Seven studies considered the economics of TPMT testing prior to thiopurine treatment, and six (except one in ALL) found it cost-effective in IBD [14, 26, 27], rheumatology [28, 29] and ALL [30, 31]. While it may have been preferable to perform both genetic and biochemical assays, this is not a feasible option in most cases. Our results show that molecular analysis is limited in predicting TPMT activity among Jews, and that a biochemical assay should be preferred over the genetic assay. Our conclusion is in accordance with the recent NASPGHAN guidelines, stating that TPMT should be assessed prior to thiopurine treatment and given the comparable costs of the genetic and biochemical assays, the latter should be preferred. In the few patients for whom biochemical assay is not possible (e.g. while already taking thiopurines or taking other medications that affect TPMT activity), screening for only the A719G variant may be sufficient to identify the known variant alleles in Jews. Determining TPMT activity enables introducing the drug in full dose from the outset rather than
Dig Dis Sci
a gradual escalation and modification of the required thiopurine dosage. Thiopurines should not be prescribed in those with very low activity, but they may be cautiously attempted, at half dose, in those patients with intermediately low activity while closely monitoring blood counts and thiopurine levels. Close monitoring for liver enzymes and thiopurine levels is warranted in those with high TPMT activity. The need for close monitoring of blood count, liver enzymes and perhaps also thiopurine metabolites is mandatory also in patients with normal activity, as no pretreatment method is accurate enough to predict all patients at risk. Acknowledgments This study was funded by internal academic grants from Shaare Zedek Medical Center and the Hebrew University of Jerusalem. There was no influence of the sponsors on the study design, implementation or analysis. Conflict of interest
None.
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12. van Egmond R, Chin P, Zhang M, Sies CW, Barclay ML. High TPMT enzyme activity does not explain drug resistance due to preferential 6-methylmercaptopurine production in patients on thiopurine treatment. Aliment Pharmacol Ther. 2012;35:1181–1189. 13. Cuffari C, Dassopoulos T, Turnbough L, Thompson RE, Bayless TM. Thiopurine methyltransferase activity influences clinical response to azathioprine in inflammatory bowel disease. Clin Gastroenterol Hepatol.. 2004;2:410–417. 14. Dubinsky MC, Reyes E, Ofman J, Chiou CF, Wade S, Sandborn WJ. A cost-effectiveness analysis of alternative disease management strategies in patients with Crohn’s disease treated with azathioprine or 6-mercaptopurine. Am J Gastroenterol. 2005;100: 2239–2247. 15. Hindorf U, Lindqvist M, Peterson C, et al. Pharmacogenetics during standardised initiation of thiopurine treatment in inflammatory bowel disease. Gut. 2006;55:1423–1431. 16. Hindorf U, Appell ML. Genotyping should be considered the primary choice for pre-treatment evaluation of thiopurine methyltransferase function. J Crohns Colitis.. 2012;6:655–659. 17. Boson WL, Romano-Silva MA, Correa H, Falcao˜ RP, TeixeiraVidigal PV, De Marco L. Thiopurine methyltransferase polymorphisms in a Brazilian population. Pharmacogenomics J.. 2003;3:178–182. 18. Tai HL, Krynetski EY, Yates CR, 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;58:694–702. 19. Lowenthal A, Meyerstein N, Ben-Zvi Z. Thiopurine methyltransferase activity in the Jewish population of Israel. Eur J Clin Pharmacol. 2001;57:43–46. 20. Fevery J, Henckaerts L, Van Oirbeek R, et al. Malignancies and mortality in 200 patients with primary sclerosering cholangitis: a long-term single-centre study. Liver Int.. 2012;32:214–222. 21. Ronen O, Cohen SB, Rund D. Evaluating frequencies of thiopurine S-methyl transferase (TPMT) variant alleles in Israeli ethnic subpopulations using DNA analysis. Isr Med Assoc J.. 2010;12:721–725. 22. Khalil MN, Erb N, Khalil PN, Escherich G, Janka-Schaub GE. Interference free and simplyfied liquid chromatography-based determination of thiopurine S-methyltransferase activity in erythrocytes. J Chromatogr B Analyt Technol Biomed Life Sci. 2005;821:105–111. 23. Eaton JE, Silveira MG, Pardi DS, et al. High-dose ursodeoxycholic acid is associated with the development of colorectal neoplasia in patients with ulcerative colitis and primary sclerosing cholangitis. Am J Gastroenterol. 2011;106:1638–1645. 24. Efrati E, Adler L, Krivoy N, Sprecher E. Distribution of TPMT risk alleles for thioupurine toxicity in the Israeli population. Eur J Clin Pharmacol. 2009;65:257–262. 25. Dewit O, Starkel P, Roblin X. Thiopurine metabolism monitoring: implications in inflammatory bowel diseases. Eur J Clin Invest. 40:1037–1047. 26. Priest VL, Begg EJ, Gardiner SJ, et al. Pharmacoeconomic analyses of azathioprine, methotrexate and prospective pharmacogenetic testing for the management of inflammatory bowel disease. Pharmacoeconomics.. 2006;24:767–781. 27. Winter J, Walker A, Shapiro D, Gaffney D, Spooner RJ, Mills PR. Cost-effectiveness of thiopurine methyltransferase genotype screening in patients about to commence azathioprine therapy for treatment of inflammatory bowel disease. Aliment Pharmacol Ther. 2004;20:593–599. 28. Marra CA, Esdaile JM, Anis AH. Practical pharmacogenetics: the cost effectiveness of screening for thiopurine s-methyltransferase polymorphisms in patients with rheumatological conditions treated with azathioprine. J Rheumatol. 2002;29:2507–2512.
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ORIGINAL ARTICLE
Thiopurine S-methyltransferase (TPMT) Activity Is Better Determined by Biochemical Assay Versus Genotyping in the Jewish Population Yair Kasirer • Rephael Mevorach • Paul Renbaum Nurit Algur • Devora Soiferman • Rachel Beeri • Yelana Rachman • Reeval Segel • Dan Turner
•
Received: 21 October 2013 / Accepted: 17 December 2013 Ó Springer Science+Business Media New York 2014
Abstract Background Thiopurine S-methyltransferase (TPMT) is a key enzyme that deactivates thiopurines, into their inactive metabolite, 6-methylmercaptopurine. Intermediate and low TPMT activity may lead to leukopenia following thiopurine treatment. The aim of this study was to determine TPMT activity and TPMT alleles (genotype–phenotype correlation) in Jews, aiming to develop an evidence-based pharmacogenetic assay for this population. Methods TPMT activity was determined in 228 Jewish volunteers by high performance liquid chromatography. Common allelic variants in the Caucasian population [TPMT*2 (G238C), TPMT *3A (G460A and A719G), TPMT* 3B (G460A) and TPMT*3C (A719G)] were tested.
Yair Kasirer, Rephael Mevorach, Reeval Segel and Dan Turner contributed equally to this work. Y. Kasirer Y. Rachman D. Turner (&) Pediatric Gastroenterology and Nutrition Unit, Shaare Zedek Medical Center, The Hebrew University, P.O. B 3235, 91031 Jerusalem, Israel e-mail: [email protected] R. Mevorach N. Algur Biochemistry Laboratory, Shaare Zedek Medical Center, Jerusalem, Israel P. Renbaum D. Soiferman R. Beeri R. Segel Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel R. Segel D. Turner Shaare Zedek Medical Center, The Hebrew University, Jerusalem, Israel
Phenotype–genotype correlation was examined and discordant cases were fully sequenced to identify novel genetic variants. Results Mean TPMT activity was 15.4 ± 4 U/ml red blood cells (range 1–34). Intermediate activity was found in 33/228 (14 %) subjects and absent activity was found in one sample (0.4 %). Only eight individuals (3.5 % of the entire cohort and 24 % of those with intermediate/low activity) were identified as carriers of a TPMT genetic variant, all of whom had the TPMT*3A allele. Sequencing the entire TPMT coding region and splice junctions in the remainder of the discordant cases did not reveal any novel variants. Conclusion Genotyping TPMT in Jews yields a much lower rate of variants than identified in the general Caucasian population. We conclude that a biochemical assay to determine TPMT enzymatic activity should be performed in Jews before starting thiopurine treatment in order to identify low activity subjects. Keywords TPMT activity Jewish population Phenotype Genotype Thiopurines
Introduction Thiopurines (azathioprine and 6-mercaptopurine) are widely used for treating inflammatory bowel diseases (IBD). After ingestion, azathioprine is rapidly converted to 6-MP, which could be converted into 6-thioguanine (6-TG). The latter is the active metabolite at levels above 230–260 pmol/8 9 108 red blood cells (RBC) [1], but myelotoxicity [2], and possibly also malignancy [3], may be observed at 6-TG levels exceeding 450 pmol/8 9 108 RBC. The enzyme thiopurine S-methyltransferase (TPMT) converts 6-MP to its inactive
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metabolite 6-methylmercaptopurine (6-MMP), which is hepatotoxic at high levels [4]. Approximately 30 % of IBD patients treated with thiopurines will discontinue the drug prematurely due to low effectiveness (*15 %) or toxicity (*15 %) [5, 6]. Certain variants in the TPMT gene can modify its structure leading to decreased activity and excessive drug toxicity by increased 6-TG levels. TPMT half-life may be reduced from 18 h in the wild type to as low as 15 min in the mutated enzyme [7]. In Caucasians, three TPMT variants account for 95 % of all alleles associated with low activity: A719G ? G460A (TPMT*3A), G238C (TPMT*2) and A719G (TPMT*3C) [8]. Approximately 10 % of Caucasians are heterozygous for the mutated allele and roughly 0.3 % are homozygous [9]. On the other extreme, 10–15 % of patients have high TPMT activity [10] associated with high 6-MMP/6-TG ratio [11]. No genetic alteration has been associated with this finding to date. The association between high TPMT activity and 6-MMP/6-TG ratio has been recently challenged by Van Egmond et al. [12] showing that TPMT activity did not explain all preferential 6-MMP producers. These patients might be at an increased risk for hepatic injury and non-response to therapy [10, 13]. Studies concluded that testing TPMT prior to thiopurine treatment and following 6-TG and 6-MMP levels may be cost-effective, preventing life-threatening toxicity and increasing effectiveness [14]. In certain populations there is high concordance between lower activity levels and known TPMT genetic variants [15]. In these groups genotyping can be used as an effective tool for prediction of TPMT activity [16]. While TPMT genotyping prior to thiopurine use is a common practice in the USA, it is not widely employed in other parts of the world, in part due to variations in TPMT alleles between nations and ethnicities [5, 17, 18]. Jews are at increased risk for developing IBD (and thus for using thiopurines) while their distinct inbred genetic background may uniquely affect TPMT pharmacogenetics. However, to date, no phenotype–genotype data regarding TPMT activity are available in Jews. A previous study has shown that 10.4 % of healthy Jews (14/134 volunteers) had intermediate TPMT activity using a cutoff value of B11 U, and 7.5 % when using a cutoff value of B10 U, both previously used to determine lower activity [19]. Two other genetic studies have shown that the three common low-activity alleles were found in only 1.5 and 1.8 % of 531 and 164 healthy Jewish volunteers, respectively [20, 21]. This apparent discrepancy set the stage for this study which aimed to concurrently compare TPMT phenotype and genotype in a large Jewish cohort.
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Fig. 1 Distribution of TPMT biochemical activity among 228 volunteers. The eight arrows represent the eight cases with identified mutations (the wide arrow represent homozygosity). No other variants where found in the entire cohort
Methods Design In this prospective study, blood samples were collected from 228 Jewish volunteers ([18 years of age), seen at the emergency department of Shaare Zedek Medical Center for reasons other than IBD. Exclusion criteria were recent blood transfusion and/or treatment with thiopurine, allopurinol or mesalamine compounds during the previous 6 months, which may alter TPMT activity. Basic demographic and phenotypic data were recorded on a standardized case report form. All samples were tested for TPMT activity, and for the three most prevalent alleles in Caucasians (TPMT*2, *3A, *3B and *3C). Since no bimodal distribution was obtained, it was difficult to determine the exact cutoff value that defines low TPMT activity. We thus used the cutoff that included all subjects who were positive for the common genetic mutations associated with low activity, meaning B11 U/ml (nMol/mlPRBC) (Fig. 1). As a sensitivity analysis we also present the intermediate rate utilizing the cutoff of B10 U/ml. The coding sequence and splice junctions of samples with intermediate TPMT activity that were not carriers of the common variants were sequenced in search of novel variants. In addition, all high activity samples were also sequenced to search for specific variants which could explain this finding. There is no definite accepted cutoff for high TPMT activity. While some use a cutoff of [20 U/ml [16], we used the upper 95th percentile of our cohort (i.e. [23 U/ml).
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Biochemical Assay for TPMT Activity TPMT activity was measured by a homemade assay. Whole blood was collected in an EDTA tube, centrifuged at 3,000g for 5 min at 4 °C. After removal of plasma, packed RBC were washed with saline and centrifuged at 10,000g for 10 min. A total of 500 ll of packed RBC were divided equally into two tubes (250 ll each). Packed RBC were lysed with 1.0 ml distilled cold water, centrifuged at 10,000g for 10 min, and 1.0 ml of the supernatant was removed and stored at -80 °C. Enzyme activity was determined by methylation rate of 6-MP to 6-MMP as described elsewhere with some modifications [22]. Briefly, reaction mixture [composed of 100 ll of hemolysate, 20 ll of 1 mM 6-mercaptopurine (6-MP) as a substrate, 20 ll of 3 mM S-adenosyl-L-methionine (SAM, methyl donor) (Sigma, Israel), 315 ll 0.067 M phosphate buffer pH 7.4, and 20 ll of HCl 0.1 M] was incubated at 37 °C for 60 min. For termination of the enzymatic reaction, 25 ll of HClO4 60 % (Sigma, Israel) was added, the tube was centrifuged at 13,000g for 4 min at 4 °C and the supernatant was kept at 4 °C until high performance liquid chromatography (HPLC) analysis. Chromatography was performed at room temperature using RP-HPLC system (Young-lin 9100) with a variable UV–vis detector on a reversed phase column using C-18 125-3 Purospher STAR column (Merck). The column was protected by a guard column containing the same phase. Mobile phase consisted of 0.01 M phosphate buffer adjusted to PH 2.7 with phosphoric acid and acetonitrile (80:20) at a flow rate of 1 ml/min (isocratic mode); 6-MMP was detected at a wavelength of 290 nm. The calibration standards were diluted from homemade stock solution. TPMT activity, expressed in nMol/mlPRBC (herein U/ml), was calculated by Y.L. Clarity software. The intra- and inter-assay coefficient of variance (CV) were 7.41 and 10.6 %, respectively. Genetic Analysis Genotyping DNA was extracted by a high salt extraction technique [23] and analyzed for the common Caucasian variants using allele-specific PCR followed by restriction fragment length polymorphism analysis (PCR–RFLP) [24]. Briefly, PCR was performed using an initial denaturation step at 94 °C for 5 min was followed by 37 cycles consisting of a denaturation step at 94 °C for 45 s, annealing for 45 s at 56 °C for TPMT*2 or 59 °C for TPMT*3A and TPMT*3C, followed by extension at 72 °C for 45 s. The resulting PCR products were digested with restriction enzymes (HpyIII for G238C, MwoI for A460G, and AccI for G719A) and analyzed on a
Table 1 Basic characteristics Entire cohort (n = 228)
Subjects with low TPMT activitya (n = 34)
Subjects with high TPMT activitya (n = 12)
Males
124 (54 %)
17 (50 %)
6 (50 %)
Age (years)
52 ± 22
39 ± 22
44 ± 19
Origin Ashkenazi
123 (54 %)
17 (50 %)
9 (75 %)
Sephardic
48 (21 %)
11 (31 %)
1 (8 %)
Others
57 (25 %)
6 (19 %)
2 (17 %)
Medians (interquartile range) or mean (±SD) are presented as appropriate for the data distribution a Low activity defined as TPMT B11 U/ml and high as TPMT [23 U/ml
3 % agarose gel by electrophoresis. Direct sequencing of carriers was used to confirm the RFLP results. Sequencing PCR products were cleaned with Exonuclease I and shrimp alkaline phosphatase (EXO-SAP Affymetrix, CA, USA) and 2 ll of the purified amplicon was sequenced using Big Dye Terminator v1.1 (Applied Biosystems, CA, USA). The extension products were purified using hydrated gel filtration matrix columns (Edge Biosystems, Maryland, USA) and separated on an ABI 3130xl DNA Genetic Analyzer (Applied Biosystems, CA, USA). SeqScape v2.5v software was used for analysis and the results were compared to the RefSeq TPMT sequence, NM_000367.2. Statistics Data are presented as means (±standard deviation), or medians (interquartile range) and compared using unpaired Student’s t test or Wilcoxon rank sum test as appropriate for the distribution normality. Categorical variables were compared using v2 or Fisher’s exact, as appropriate. A multivariate linear regression model was constructed to explore the association between gender, age and Jewish ethnicity with TPMT activity. Assumptions of the models were verified using residual plots. Analyses were performed using SPSS V15; P values\0.05 were considered significant. This study was approved by the local and Israeli Ministry of Health Supreme ethical committees. Written informed consent was obtained from all participants.
Results Blood samples were collected from 228 volunteers (Table 1) with the mean TPMT activity of 15.4 ± 4 U/ml
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(range 1–34) (Fig. 1). Thirty-three subjects (14 %) had intermediate TPMT activity, and one subject (0.4 %) showed absent activity (\1 U/ml). When considering a cutoff value of B10 U/ml, 18/228 (8 %) of subjects had low/intermediate activity. Twelve (5 %) subjects demonstrated high TPMT activity ([23 U/ml) and 20 (8.8 %) had [20 U/ml TPMT activity. Univariate (Table 1) and multivariate regression analysis for age, gender and ethnicity revealed no association with TPMT activity (all P [ 0.05; r2 = 0.21). Genotyping of the entire cohort detected only eight carriers of the common Caucasian variants [3.5 % of the entire cohort (8/228) and 24 % of those with low/intermediate activity (8/34)]. When using the lower cutoff value for low/intermediate activity of B10 U/ml, 6/18 (33.3 %) of subjects were variant-carriers. The positive subjects all carried the TPMT*3A allele (i.e. A719G ? G460A); seven were heterozygotes, and the one patient with the absent TPMT activity was homozygote (Fig. 1). Sequencing of the TPMT coding region and splice junctions of the 26 subjects with intermediate activity who did not carry the known variants did not reveal any new mutations. Similarly, sequencing the 12 subjects with high TPMT activity did not reveal any unknown variants.
Discussion This is the first study in Jews that concurrently measured both biochemical activity and genetic analysis of TPMT, aiming to determine phenotype–genotype correlation. While we found that 8–14 % of our cohort had low-medium TPMT enzymatic activity, only 3.5 % were positive for one of the common Caucasian TPMT variants. This low rate is consistent with the two previous genetic studies among Jews which tested the three common Caucasian variants [8/531 (1.5 %) and 3/164 (1.8 %)] [21, 24]. We hypothesized that the phenotype–genotype discrepancy among Jews could be explained by novel population-specific mutations. However, we failed to identify any additional mutations after sequencing the entire coding sequences and exon flanking regions of the TPMT gene in all discordant genotype–phenotype cases. We speculate that other genetic variants exist in regulatory regions of the TPMT gene, such as in the promoter, alternative splicing sites, non-translated or enhancer regions, or epigenic modifications that influence the enzyme’s activity explaining the discrepancy observed between TPMT genotype and phenotype in the Jewish population. In our cohort, the TPMT*3A variant was the only allele associated with low activity. In the two previous genetic studies in the Jewish population, TPMT*3A has been found in 10 of the 11 positive samples (out of 695 subjects,
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combined) with the only exception being TPMT*3C. The A719G variant is part of both TPMT*3A (G460A and A719G) and TPMT*3C (only A719G). Therefore, we propose that only the A719G variant should be tested among Jews when genotyping is warranted such as during active thiopurine treatment when biochemical assay is technically not feasible. After initial testing of A719G, positive samples could be tested also for G460A, in order to differentiate between TPMT*3A and *3C. Combining the results of this study with the previous two Jewish genetic studies it could be estimated that the likelihood of finding a known variant, other than A719G, is as low as \1:700. One of the advantages of testing TPMT activity over genotyping is the ability to identify those with high enzyme activity, which may be associated with low drug efficacy and higher risk for hepatic toxicity. While we used the upper 5 % of the cohort to define high activity, 8.8 % (20/ 228) subjects showed TPMT activity [20 U/ml. We sequenced coding sequences and exon flanking regions of the TPMT gene in all high-activity subjects; however, no genetic variants were identified to explain this finding. Some authors suggest prescribing higher doses of thiopurines for patients with high activity [25]; however, the association between high activity and efficacy is not established. In practice, close monitoring of liver enzymes is mandatory during treatment, and measuring drug metabolites after commencing thiopurines is advised. The design of this study did not enable us to correlate TPMT activity with clinical outcome, nor was this within the scope of our study. This is the subject of an ongoing continuation study. The fact that all mutations found in the genetic analysis were in subjects with lower TPMT activity provide internal validity of the biochemical and genetic assays. Seven studies considered the economics of TPMT testing prior to thiopurine treatment, and six (except one in ALL) found it cost-effective in IBD [14, 26, 27], rheumatology [28, 29] and ALL [30, 31]. While it may have been preferable to perform both genetic and biochemical assays, this is not a feasible option in most cases. Our results show that molecular analysis is limited in predicting TPMT activity among Jews, and that a biochemical assay should be preferred over the genetic assay. Our conclusion is in accordance with the recent NASPGHAN guidelines, stating that TPMT should be assessed prior to thiopurine treatment and given the comparable costs of the genetic and biochemical assays, the latter should be preferred. In the few patients for whom biochemical assay is not possible (e.g. while already taking thiopurines or taking other medications that affect TPMT activity), screening for only the A719G variant may be sufficient to identify the known variant alleles in Jews. Determining TPMT activity enables introducing the drug in full dose from the outset rather than
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a gradual escalation and modification of the required thiopurine dosage. Thiopurines should not be prescribed in those with very low activity, but they may be cautiously attempted, at half dose, in those patients with intermediately low activity while closely monitoring blood counts and thiopurine levels. Close monitoring for liver enzymes and thiopurine levels is warranted in those with high TPMT activity. The need for close monitoring of blood count, liver enzymes and perhaps also thiopurine metabolites is mandatory also in patients with normal activity, as no pretreatment method is accurate enough to predict all patients at risk. Acknowledgments This study was funded by internal academic grants from Shaare Zedek Medical Center and the Hebrew University of Jerusalem. There was no influence of the sponsors on the study design, implementation or analysis. Conflict of interest
None.
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