Thiopurine S-methyltransferase Phenotype Research Articles

P798 Study of the concordance between the enzymatic activity of Thiopurine S-methyltransferase and polymorphisms in the TPMT gene in patients with Inflammatory Bowel Disease

Abstract Background Thiopurine therapy is associated with an increased risk of serious adverse events in patients with Inflammatory Bowel Disease (IBD). Thiopurine S-methyltransferase (TPMT) is the enzyme responsible for metabolism, and its determination helps identify patients at risk of toxicity. This study aims to explore the level of correspondence between the TPMT genotype and enzymatic activity (phenotype) with the objective of preventing adverse events prior to the commencement of thiopurine therapy. Methods Consecutive patients with IBD, Crohn’s Disease (CD) or Ulcerative Colitis (UC), were included. Genotype was conducted for the most prevalent TPMT variants (TPMT*2, TPMT*3B, TPMT*3C), TPMT enzymatic activity assessment, and quantification of 6-thioguanine nucleotide (6-TGN) levels. Adverse effects were documented, along with the administration of biologics, mesalazine, ACE inhibitors or allopurinol. The primary objective was to evaluate the concordance between TPMT genotype and phenotype. Secondary objectives included determining the prevalence of genotype and phenotype within our patient cohort, 6-TGN serum levels, the prevalence of adverse events and identifying potential pharmacological inducers of the phenotype. Results A total of 218 patients were included (131 with CD and 81 with UC). Thirteen patients did not initiate thiopurines, and 34.63% (71/205) had discontinued treatment. The most observed adverse events were myelotoxicity (15.12%), gastrointestinal toxicity (12.20%), and hepatic toxicity (5.37%). TPMT activity exhibited a trimodal distribution, with 87.61% (191/218) having normal activity, 11.47% having intermediate activity, and 1% having low activity. Genotypically, 90.83% (198/218) had a wild-type genotype, 8.72% (18/218) had a heterozygous genotype, and 0.5% (1/218) had a homozygous genotype. The concordance index between phenotype and genotype was calculated at 0.57. Significantly, myelotoxicity was associated with the presence of genotypic variants (p=0.004). 6-TGN levels did not correlate with TPMT activity and concurrent medications did not affect TPMT activity . Conclusion The proactive assessment of TPMT (phenotype or genotype) before initiating thiopurine therapy effectively mitigates the occurrence of myelotoxicity in IBD patients. Importantly, the level of concordance between TPMT activity and genotype is moderate, with genotyping serving as the more reliable approach for preselecting patients susceptible to myelotoxic reactions.

Determination of Thiopurine S-Methyltransferase (TPMT) Activity in Human Erythrocytes by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the metabolism of thiopurine medications that are used in the treatment of multiple malignant and nonmalignant immunologic conditions. Polymorphisms in the TPMT gene associated with low enzyme activity can produce pronounced pharmacologic effects during therapy. The determination of TPMT erythrocyte activity is a valuable adjunct test to genotyping for the assignment of TPMT phenotype, especially in the presence of indeterminate genotypes. We present a method for the determination TPMT activity in human erythrocytes whereby we utilize LC-MS/MS to measure the amount of 6-methylmercaptopurine formed in red blood cell (RBC) lysates using 6-mercaptopurine as substrate and S-adenosylmethionine (SAM) as methyl donor. This method has been successfully implemented and proven to be reliable for use in human specimens.

Automation of the Whole-Blood Thiopurine S-Methyltransferase (TPMT) Phenotyping Assay Using the Biomek NXP and Biomek i5 Liquid-Handling Workstations.

Assessment of thiopurine S-methyltransferase (TPMT) status is required before commencing thiopurine treatment to reduce the potential for adverse drug reactions. Our laboratory has provided a national TPMT phenotyping service since 2003. Our assay uses 6-thioguanine as substrate and detection of 6-methylthioguanine via high-performance liquid chromatography (HPLC) fluorescence. Here, we report the automation of this complex, labor-intensive, manual assay using the Biomek NXP and Biomek i5 liquid-handling workstations. We optimized assay performance and validated for precision, linearity, and lower limit of quantitation. We also compared results from the manual and automated methods. Primary sample mixing and aliquoting were performed on the Biomek NXP. On-board inversions (n = 10) replaced offline mixing. No carryover was observed. Eleven percent of primary sample tubes were incompatible with the Biomek NXP, and these were assayed manually. The Biomek i5 was used to automate the enzyme assay. Optimum vortex mixing was achieved at 2500 rpm for 60 s, and the temperature was set to 37.0 °C for the 60 min enzyme incubation. Intra- and inter-assay precision were excellent, with coefficients of variation (CVs) of ≤2.3% and ≤7.4%, respectively, for patient samples. Linearity was demonstrated up to 199 mIU/L (R2 = 0.992), with a lower limit of quantitation of 3.9 mIU/L. Correlation between the manual and automated methods was good (R2 = 0.979, n = 405), with results being interchangeable. We have successfully developed and validated a novel automated method for the TPMT phenotyping enzyme assay. The two methods are cost-neutral. Automation of other complex enzyme assays may be possible using this approach.

Relationship Between Thiopurine S-Methyltransferase Genotype/Phenotype and 6-Thioguanine Nucleotide Levels in 316 Patients With Inflammatory Bowel Disease on 6-Thioguanine.

Background:In inflammatory bowel disease (IBD), conventional thiopurine users cease treatment in 60% of cases within 5 years, mostly because of adverse events or nonresponse. In this study, the authors aimed to investigate the role of 6-thioguanine nucleotide (TGN) measurements, geno/phenotyping of thiopurine S-methyltransferase (TPMT), and their mutual relationship with TG therapy in IBD.Methods:An international retrospective, multicenter cohort study was performed at 4 centers in the Netherlands (Máxima Medical Centre) and the United Kingdom (Guy's and St. Thomas' Hospital, Queen Elizabeth Hospital, and East Surrey Hospital).Results:Overall, 526 6-TGN measurements were performed in 316 patients with IBD. The median daily dosage of TG was 20 mg/d (range 10–40 mg/d), and the median duration of TG use was 21.1 months (SD, 28.0). In total, 129 patients (40.8%) had a known TPMT status. In the variant-type and wild-type TPMT genotype metabolism groups, median 6-TGN values were 1126 [interquartile range (IQR) 948–1562] and 467.5 pmol/8 × 10E8 red blood cells (RBCs) (IQR 334–593). A significant difference was observed between the 2 groups (P = 0.0001, t test). For TPMT phenotypes, in the slow, fast, and normal metabolism groups, the median 6-TGN values were 772.0 (IQR 459–1724), 296.0 (IQR 200–705), and 774.5 pmol/8 × 10E8 RBCs (IQR 500.5–981.5), with a significant difference observed between groups (P < 0.001, analysis of variance).Conclusions:Our findings indicated that TPMT measurements at TG initiation can be useful but are not necessary for daily practice. TPMT genotypes and phenotypes are both associated with significant differences in 6-TGN levels between metabolic groups. However, the advantage of TG remains that RBC 6-TGN measurements are not crucial to monitor treatments in patients with IBD because these measurements did not correlate with laboratory result abnormalities. This presents as a major advantage in countries where patients cannot access these diagnostic tests.

TPMT Genetic Variability and Its Association with Hematotoxicity in Indonesian Children with Acute Lymphoblastic Leukemia in Maintenance Therapy.

PurposeHematotoxicity monitoring in children with acute lymphoblastic leukemia (ALL) is critical to preventing life-threatening infections and drug discontinuation. The primary drug that causes hematotoxicity in ALL children is 6-mercaptopurine (6-MP). Genetic variability of the drug-metabolizing enzymes of 6-MP, thiopurine S-methyltransferase (TPMT), is one factor that might increase the susceptibility of children to hematotoxicity. The present study aimed to determine the variability in TPMT genotypes and phenotypes and its association with the occurrence of hematotoxicity in ALL children in maintenance therapy.Patients and MethodsA cross-sectional study was conducted at Cipto Mangunkusumo and Dharmais National Cancer Hospital, Jakarta, Indonesia, from June 2017 to October 2018. We included ALL patients, 1–18 years, who were receiving at least one month of 6-MP during maintenance therapy according to the Indonesian protocol for ALL 2013. Direct sequencing was used to determine TPMT*3A, *3B, and *3C genotypes, and LC-MS/MS analysis was performed to measure the plasma concentrations of 6-MP and its metabolites. Association analysis between the TPMT genotype and hematotoxicity was evaluated using the unpaired t-test or Mann–Whitney’s test.ResultsThe prevalence of neutropenia, anemia, and thrombocytopenia in ALL children during maintenance therapy was 51.9%, 44.3%, and 6.6%, respectively. We found a low frequency of TPMT*3C, which is 0.95%. No association was found between hematotoxicity and TPMT genotypes or age, nutritional status, serum albumin levels, risk stratification, the daily dose of 6-MP, and cotrimoxazole co-administration. However, hematotoxicity was associated with 6-methylmercaptopurine (6-MeMP) plasma concentrations and the ratio 6-MeMP/6-thioguanine (6-TGN). We also found no association between TPMT genotypes and TPMT phenotypes.ConclusionThe 6-MeMP/6-TGN ratio is associated with hematotoxicity in ALL children during maintenance therapy but is not strong enough to predict hematotoxicity.

Population-scale predictions of DPD and TPMT phenotypes using a quantitative pharmacogene-specific ensemble classifier

BackgroundInter-individual differences in dihydropyrimidine dehydrogenase (DPYD encoding DPD) and thiopurine S-methyltransferase (TPMT) activity are important predictors for fluoropyrimidine and thiopurine toxicity. While several variants in these genes are known to decrease enzyme activities, many additional genetic variations with unclear functional consequences have been identified, complicating informed clinical decision-making in the respective carriers.MethodsWe used a novel pharmacogenetically trained ensemble classifier to analyse DPYD and TPMT genetic variability based on sequencing data from 138,842 individuals across eight populations.ResultsThe algorithm accurately predicted in vivo consequences of DPYD and TPMT variants (accuracy 91.4% compared to 95.3% in vitro). Further analysis showed high genetic complexity of DPD deficiency, advocating for sequencing-based DPYD profiling, whereas genotyping of four variants in TPMT was sufficient to explain >95% of phenotypic TPMT variability. Lastly, we provided population-scale profiles of ethnogeographic variability in DPD and TPMT phenotypes, and revealed striking interethnic differences in frequency and genetic constitution of DPD and TPMT deficiency.ConclusionThese results provide the most comprehensive data set of DPYD and TPMT variability published to date with important implications for population-adjusted genetic profiling strategies of fluoropyrimidine and thiopurine risk factors and precision public health.

Modeling the Outcome of Systematic TPMT Genotyping or Phenotyping Before Azathioprine Prescription: A Cost-Effectiveness Analysis.

Thiopurine S-methyltransferase (TPMT) testing, either by genotyping or phenotyping, can reduce the incidence of adverse severe myelotoxicity episodes induced by azathioprine. The comparative cost-effectiveness of TPMT genotyping and phenotyping are not known. Our aim was to assess the cost-effectiveness of phenotyping-based dosing of TPMT activity, genotyping-based screening and no screening (reference) for patients treated with azathioprine. A decision tree was built to compare the conventional weight-based dosing strategy with phenotyping and with genotyping using a micro-simulation model of patients with inflammatory bowel disease from the perspective of the French health care system. The time horizon was set up as 1year. Only direct medical costs were used. Data used were obtained from previous reports, except for screening test and admission costs, which were from real cases. The main outcome was the cost-effectiveness ratios, with an effectiveness criterion of one averted severe myelotoxicity episode. The total expected cost of the no screening strategy was €409/patient, the total expected cost of the phenotyping strategy was €427/patient, and the total expected cost of the genotyping strategy was €476/patient. The incremental cost-effectiveness ratio was €2602/severe myelotoxicity averted in using the phenotyping strategy, and €11,244/severe myelotoxicity averted in the genotyping strategy compared to the no screening strategy. At prevalence rates of severe myelotoxicity > 1%, phenotyping dominated genotyping and conventional strategies. The phenotype-based strategy to screen for TPMT deficiency dominates (cheaper and more effective) the genotype-based screening strategy in France. Phenotype-based screening dominates no screening in populations with a prevalence of severe myelosuppression due to azathioprine of > 1%.

Retrospective Data Analysis of the Influence of Age and Sex on TPMT Activity and Its Phenotype-Genotype Correlation.

Therapeutic efficacy and toxicity of thiopurine drugs (used as anticancer and immunosuppressant agents) are affected by thiopurine S-methyltransferase (TPMT) enzyme activity. TPMT genotype and/or phenotype is used to predict the risk for adverse effects before drug administration. Inosine triphosphate pyrophosphatase (ITPA) is another enzyme involved in thiopurine metabolism. In this study, we aimed to evaluate (a) frequency of various TPMT phenotypes and genotypes, (b) correlations between them, (c) influence of age and sex on TPMT activity, and (d) distribution of ITPA variants among various TPMT subgroups. TPMT enzyme activity was determined by LC-MS/MS. TPMT (*2,*3A-C) and ITPA (rs1127354, rs7270101) genotypes were determined using a customized TaqMan® OpenArray®. TPMT enzyme activity varied largely (6.3-90 U/mL). The frequency of low, intermediate, normal, and high activity was 0.5% (n = 230), 13.1% (n = 5998), 86.1% (n = 39448), and 0.28% (n = 126), respectively. No significant difference in TPMT activity in relation to age and sex was found. Genotype analysis revealed the frequency of variant TPMT alleles was 6.73% (*3A, n = 344), 0.05% (*3B, n = 2), 2.22% (*3C, n = 95), and 0.42% (*2, n = 19). Analysis of paired phenotype and genotype showed that TPMT activity in samples with variant allele(s) was significantly lower than those without variant alleles. Lastly, an equal distribution of ITPA variants was found among normal and abnormal TPMT activity. This retrospective data analysis demonstrated a clustering of variant TPMT genotypes with phenotypes, no significant influence of age and sex on TPMT activity, and an equal distribution of ITPA variants among various TPMT subgroups.

Validation of a high-performance liquid chromatography method for thiopurine S-methyltransferase activity in whole blood using 6-mercaptopurine as substrate.

Variation in metabolism, toxicity and therapeutic efficacy of thiopurine drugs is largely influenced by genetic polymorphisms in the thiopurine S-methyltransferase (TPMT) gene. Determination of TPMT activity is routinely performed in patients to adjust drug therapy. We further optimized a previously established high-performance liquid chromatography (HPLC) method by measuring TPMT activity in whole blood instead of isolated erythrocytes, which is based on conversion of 6-mercaptopurine to 6-methylmercaptopurine using S-adenosyl-methionine as methyl donor. The simplified TPMT whole-blood method showed similar or better analytical and diagnostic performance compared with the former erythrocyte assay. The whole-blood method was linear for TPMT activities between 0 and 40 nmol/(mL·h) with a quantification limit of 0.1 nmol/(mL·h). Within-day imprecision and between-day imprecision were ≤5.1% and ≤8.5%, respectively. The optimized method determining TPMT activity in whole blood (y) showed agreement with the former method determining TPMT activity in erythrocytes (x) (n=45, y=1.218+0.882x; p>0.05). Phenotype-genotype concordance (n=300) of the whole-blood method was better when TPMT activity was expressed per volume of whole blood (specificity 92.2%), whereas correction for hematocrit resulted in lower genotype concordance (specificity 86.9%). A new cutoff for the whole-blood method to distinguish normal from reduced TPMT activity was determined at ≤6.7 nmol/(mL·h). This optimized TPMT phenotyping assay from whole blood using 6-MP as substrate is suitable for research and routine clinical analysis.

Genomewide Approach Validates Thiopurine Methyltransferase Activity Is a Monogenic Pharmacogenomic Trait

We performed a genomewide association study (GWAS) of primary erythrocyte thiopurine S-methyltransferase (TPMT) activity in children with leukemia (n = 1,026). Adjusting for age and ancestry, TPMT was the only gene that reached genomewide significance (top hit rs1142345 or 719A>G; P = 8.6 × 10-61 ). Additional genetic variants (in addition to the three single-nucleotide polymorphisms [SNPs], rs1800462, rs1800460, and rs1142345, defining TPMT clinical genotype) did not significantly improve classification accuracy for TPMT phenotype. Clinical mercaptopurine tolerability in 839 patients was related to TPMT clinical genotype (P = 2.4 × 10-11 ). Using 177 lymphoblastoid cell lines (LCLs), there were 251 SNPs ranked higher than the top TPMT SNP (rs1142345; P = 6.8 × 10-5 ), revealing a limitation of LCLs for pharmacogenomic discovery. In a GWAS, TPMT activity in patients behaves as a monogenic trait, further bolstering the utility of TPMT genetic testing in the clinic.

Thiopurine S-methyltransferase testing for averting drug toxicity: a meta-analysis of diagnostic test accuracy.

Thiopurine S-methyltransferase (TPMT) deficiency increases the risk of serious adverse events in persons receiving thiopurines. The objective was to synthesize reported sensitivity and specificity of TPMT phenotyping and genotyping using a latent class hierarchical summary receiver operating characteristic meta-analysis. In 27 studies, pooled sensitivity and specificity of phenotyping for deficient individuals was 75.9% (95% credible interval (CrI), 58.3-87.0%) and 98.9% (96.3-100%), respectively. For genotype tests evaluating TPMT*2 and TPMT*3, sensitivity and specificity was 90.4% (79.1-99.4%) and 100.0% (99.9-100%), respectively. For individuals with deficient or intermediate activity, phenotype sensitivity and specificity was 91.3% (86.4-95.5%) and 92.6% (86.5-96.6%), respectively. For genotype tests evaluating TPMT*2 and TPMT*3, sensitivity and specificity was 88.9% (81.6-97.5%) and 99.2% (98.4-99.9%), respectively. Genotyping has higher sensitivity as long as TPMT*2 and TPMT*3 are tested. Both approaches display high specificity. Latent class meta-analysis is a useful method for synthesizing diagnostic test performance data for clinical practice guidelines.The Pharmacogenomics Journal advance online publication, 24 May 2016; doi:10.1038/tpj.2016.37.

Association between Thiopurine S-Methyltransferase Polymorphisms and Azathioprine-Induced Adverse Drug Reactions in Patients with Autoimmune Diseases: A Meta-Analysis.

PurposeAzathioprine (AZA) is widely used as an immunosuppressive drug in autoimmune diseases, but its use is limited by significant adverse drug reactions (ADRs). Thiopurine S-methyltransferase (TPMT) is an important enzyme involved in AZA metabolism. Several clinical guidelines recommend determining TPMT genotype or phenotype before initiating AZA therapy. Although several studies have investigated the association between TPMT polymorphisms and AZA-induced ADRs, the results are inconsistent. The purpose of this study is to evaluate whether there is an association between TPMT polymorphisms and AZA-induced ADRs using meta-analysis.MethodsWe explored PubMed, Web of Science and Embase for articles on TPMT polymorphisms and AZA-induced ADRs. Studies that compared TPMT polymorphisms with-ADRs and without-ADRs in patients with autoimmune diseases were included. Relevant outcome data from all the included articles were extracted and the pooled odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated using Revman 5.3 software.ResultsEleven published studies, with a total of 651 patients with autoimmune diseases, investigated associations between TPMT polymorphisms and AZA-induced ADRs, were included in this meta-analysis. Our meta-analysis demonstrated that TPMT polymorphisms were significantly associated with AZA-induced overall ADRs, bone marrow toxicity and gastric intolerance; pooled ORs were 3.12 (1.48–6.56), 3.76 (1.97–7.17) and 6.43 (2.04–20.25), respectively. TPMT polymorphisms were not associated with the development of hepatotoxicity; the corresponding pooled OR was 2.86 (95%CI: 0.32–25.86). However, the association in GI subset could be driven by one single study. After this study was excluded, the OR was 2.11 (95%CI: 0.36–12.42); namely, the association became negative.ConclusionsOur meta-analysis demonstrated an association of TPMT polymorphisms with overall AZA-induced ADRs, bone marrow toxicity and gastric intolerance, but not with hepatotoxicity. The presence of the normal TPMT genotypes cannot preclude the development of ADRs during AZA treatment, TPMT genotyping prior to commencing AZA therapy cannot replace, may augment, the current practice of regular monitoring of the white blood cell. Because of small sample sizes, large and extensive exploration was required to validate our findings.

Association between thiopurine S-methyltransferase polymorphisms and thiopurine-induced adverse drug reactions in patients with inflammatory bowel disease: a meta-analysis.

PurposeThiopurine drugs are well established treatments in the management of inflammatory bowel disease (IBD), but their use is limited by significant adverse drug reactions (ADRs). Thiopurine S-methyltransferase (TPMT) is an important enzyme involved in thiopurine metabolism. Several clinical guidelines recommend determining TPMT genotype or phenotype before initiating thiopurine therapy. Although several studies have investigated the association between TPMT polymorphisms and thiopurine-induced ADRs, the results are inconsistent. The purpose of this study is to evaluate whether there is an association between TPMT polymorphisms and thiopurine-induced ADRs using meta-analysis.MethodsWe explored PubMed, Web of Science and Embase for articles on TPMT polymorphisms and thiopurine-induced ADRs. Studies that compared TPMT polymorphisms with-ADRs and without-ADRs in IBD patients were included. Relevant outcome data from all the included articles were extracted and the pooled odds ratio (OR) with corresponding 95% confidence intervals were calculated using Revman 5.3 software.ResultsFourteen published studies, with a total of 2,206 IBD patients, which investigated associations between TPMT polymorphisms and thiopurine-induced ADRs were included this meta-analysis. Our meta-analysis demonstrated that TPMT polymorphisms were significantly associated with thiopurine-induced overall ADRs and bone marrow toxicity; pooled ORs were 3.36 (95%CI: 1.82–6.19) and 6.67 (95%CI: 3.88–11.47), respectively. TPMT polymorphisms were not associated with the development of other ADRs including hepatotoxicity, pancreatitis, gastric intolerance, flu-like symptoms and skin reactions; the corresponding pooled ORs were 1.27 (95%CI: 0.60–2.71), 0.97 (95%CI: 0.38–2.48), 1.82 (95%CI: 0.93–3.53), 1.28 (95%CI: 0.47–3.46) and 2.32 (95%CI: 0.86–6.25), respectively.ConclusionsOur meta-analysis demonstrated an association of TPMT polymorphisms with overall thiopurine-induced ADRs and bone marrow toxicity, but not with hepatotoxicity, pancreatitis, flu-like symptoms, gastric intolerance and skin reactions. These findings suggest that pretesting the TPMT genotype could be helpful in clinical practice before initiating thiopurine therapy. However, white blood cell count analysis should be the mainstay for follow-up.

P-176 Thiopurine S-methyltransferase Analysis Does Not Correctly Predict the Risk of Toxicity in Hispanic Children with Inflammatory Bowel Disease

Thiopurine S-methyltransferase (TPMT) catalyzes the methlyation of thiopurine compounds, and is measured prior to using medications such as 6-mercaptopurine or Azathioprine. TPMT can be measured as blood enzyme levels or allelic variations (phenotype or genotype assay), both of which are important in distinguishing the possibility of desired clinical effects versus toxicity. The aim of our study was to evaluate the prevalence of variations in TPMT activity in pediatric Hispanic Inflammatory Bowel Disease (IBD) patients in our South Florida based Pediatric Gastroenterology practice, and to evaluate the reliability of TPMT levels in predicting toxicity risk in our studied population. Sixty-four pediatric Hispanic patients treated with either 6-mercaptopurine or Azathioprine for IBD were retrospectively studied. Either a radiochemical method to measure TPMT phenotype or TPMT genotype was analyzed to detect allelic variations and predisposition to myelotoxicity, hepatotoxicity, and pancreatotoxicity. Of the 64 Hispanic patients included (mean age: 16 years): 54.6% with Crohn's disease, 37.5% with Ulcerative Colitis, 4.6% with nonspecific colitis, and 3.1% with other unspecified abdominal complaints. TPMT phenotypes were documented in 50 patients and Genotype analysis was documented in 14 patients. TPMT phenotype was distributed as: Normal level 80% (40/50), Intermediate 18% (9/50), Low 2% (1/50). TPMT genotypic variations were distributed as: Normal 92.8% (13/14), Intermediate 7.1% (1/14), Low 0% (0/14). Toxicity was characterized by leukopenia with or without neutropenia, anemia, pancytopenia, elevated transaminases, or pancreatitis. The percentage of patients with toxicity regardless of normal or abnormal TPMT levels or alleles were 37.5%. This was further evaluated to find that 12.5% had liver toxicity, 15% had bone marrow toxicity, and 10% had both. The percentage of patients with toxicity despite normal TPMT phenotype was 22.5%. The percentage of patients with toxicity despite normal genotype was 23.07%. Our study demonstrates that 17% (11/64) of pediatric Hispanic patients in our practice have either an abnormal phenotype or genotype TPMT enzyme analysis, which is greater than previously documented in other ethnic groups. Additionally, the risk of toxicity despite normal TPMT enzyme levels and alleles was higher in our studied group compared to other previously studied populations, indicating that pediatric Hispanic patients must be monitored carefully at regular intervals to monitor for adverse reactions to thiopurine based medications.

OC-015 The Influence of Gender and Haemoglobin on TPMT Activity

IntroductionPre-treatment measurement of red blood cell (RBC) thiopurine-S-methyltransferase (TPMT) activity is recommended to guide initial dosing of azathioprine (AZA) and mercaptopurine (MP). TPMT exhibits a trimodal distribution, with low and...

Analysis of thiopurine S-methyltransferase phenotype–genotype in a Tunisian population with Crohn’s disease

This study was conducted to investigate the thiopurine S-methyltransferase TPMT activity distribution and gene mutations in Tunisian population with positive diagnostic for Crohn's disease. TPMT activity was measured in Tunisian population (n = 88) by a high performance liquid chromatography assay. Polymerase chain reaction-based methods were used to determine the frequency of TPMT mutant alleles TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C. TPMT activity was normally distributed, ranging from 4.58 to 35.27 nmol/(h ml) RBC with a mean of 18.67 ± 7.10 nmol/(h ml) RBC. Seven TPMT*3A heterozygotes and one TPMT*3C homozygote were found in 88 patients, with allele frequencies of 0.039 and 1.13, respectively. TPMT*3A and the TPMT*3C, which cause the largest decrease in enzyme activity, were both variant alleles detected in the Tunisian population.

Gene Expression and Thiopurine Metabolite Profiling in Inflammatory Bowel Disease – Novel Clues to Drug Targets and Disease Mechanisms?

Background and AimsThiopurines are effective to induce and maintain remission in inflammatory bowel disease (IBD). The methyl thioinosine monophosphate (meTIMP)/6-thioguanine nucleotide (6-TGN) concentration ratio has been associated with drug efficacy. Here we explored the molecular basis of differences in metabolite profiles and in relation to disease activity.MethodsTranscriptional profiles in blood samples from an exploratory IBD-patient cohort (n = 21) with a normal thiopurine S-methyltransferase phenotype and meTIMP/6-TGN ratios >20, 10.0–14.0 and ≤4, respectively, were assessed by hybridization to microarrays. Results were further evaluated with RT qPCR in an expanded patient cohort (n = 54). Additionally, 30 purine/thiopurine related genes were analysed separately.ResultsAmong 17 genes identified by microarray-screening, there were none with a known relationship to pathways of purines/thiopurines. For nine of them a correlation between expression level and the concentration of meTIMP, 6-TGN and/or the meTIMP/6-TGN ratio was confirmed in the expanded cohort. Nine of the purine/thiopurine related genes were identified in the expanded cohort to correlate with meTIMP, 6-TGN and/or the meTIMP/6-TGN ratio. However, only small differences in gene expression levels were noticed over the three different metabolite profiles. The expression levels of four genes identified by microarray screening (PLCB2, HVCN1, CTSS, and DEF8) and one purine/thiopurine related gene (NME6) correlated significantly with the clinical activity of Crohn’s disease. Additionally, 16 of the genes from the expanded patient cohort interacted in networks with candidate IBD susceptibility genes.ConclusionsSeventeen of the 18 genes which correlated with thiopurine metabolite levels also correlated with disease activity or participated in networks with candidate IBD susceptibility genes involved in processes such as purine metabolism, cytokine signaling, and functioning of invariant natural killer T cells, T cells and B cells. Therefore, we conclude that the identified genes to a large extent are related to drug targets and disease mechanisms of IBD.

Thiopurine S-Methyltransferase Phenotype and Genotype in Pediatric Patients with Inflammatory Bowel Disease; Implication for Azathioprine Treatment

Inflammatory Bowel Disease (IBD) is more prevalent in children than adults, and the incidence is increasing. IBD is treated with thiopurines, metabolized by thiopurine S-methyltransferase (TPMT) and inter-individual variability in activity of TPMT affecting therapy efficiency and drug toxicity arises from genetic polymorphisms, mainly TPMT*2, *3A, and *3C. The aim was to investigate the frequency distribution of TPMT activity, determine the penetration rate of TPMT*2, *3A, and *3C alleles in children, and compare TPMT activity in children and adults with IBD. The study included 85 children, 45% with Crohn’s disease (CD) and 55% with ulcerative colitis (UC), and 31 adults with IBD. TPMT activity was measured with radiochemistry. TPMT*2, *3A, and *3C alleles were investigated with PCR and restriction fragment length polymorphism analyses. Children showed median TPMT activities of 13.12 and 13.19 U/ml RBC in CD and UC, respectively, with 4.8-fold variability (range, 4.74 - 22.56 U/ml RBC). TPMT activity was similar in children and adults; ranges: 5.56-21.34 vs. 9.61-17.84 U/ml RBC, respectively, in CD; and 4.74-22.56 vs. 5.19-21.98 U/ml RBC, respectively, in UC. Patients with CD and UC treated with azathioprine displayed similar TPMT activities, similar adverse event frequencies, and similar numbers of non-responders. One out of 85 patients (1.18%) was heterozygous with TPMT*1/TPMT*2 (TPMT activity: 5.19 ± 0.05 U/ml RBC). Individuals with low-intermediate TPMT activity (<8 U/ ml RBC) did not carry mutant alleles *3A or *3C. TPMT phenotypes were similar in children and adults with inflammatory bowel disease.

Genotyping should be considered the primary choice for pre-treatment evaluation of thiopurine methyltransferase function

Background and aimsA pre-treatment determination of the thiopurine S-methyltransferase (TPMT) genotype or phenotype can identify patients at risk of developing severe adverse reactions from thiopurine treatment. The risk of misclassifying a patient might be dependent on the method used. The aim of this study was to investigate the concordance between TPMT genotyping and phenotyping. MethodsThe data consist of 7195 unselected and consecutive TPMT genotype and phenotype determinations sent to the division of Clinical Pharmacology, Linköping, Sweden. TPMT activity was measured in red blood cells (RBC) and the genotype determined by pyrosequencing for the three most common TPMT variants (TPMT *2, *3A, *3C). ResultsTPMT genotyping identified 89% as TPMT wild type (*1/*1), 10% as TPMT heterozygous and 0.5% as TMPT defective. The overall concordance between genotyping and phenotyping was 95%, while it was 96% among IBD patients (n=4024). Genotyping would have misclassified 8% of the TPMT defectives as heterozygous as compared to 11% if only TPMT activity had been measured. 11% of the heterozygous patients had a normal TPMT activity (>8.9U/ml RBC) and 3% of the TPMT wild-type patients had an intermediate TPMT activity (2.5–8.9U/ml RBC). ConclusionsThere is a risk for TPMT misclassification when only genotyping or phenotyping is used, but it is not reasonable to check both in all patients. Since TPMT genotyping is the more reliable test, especially in TPMT heterozygotes, we suggest that genotyping should be considered the primary choice for the pre-treatment evaluation of TPMT function before initiation of thiopurine therapy.

Use of thiopurine testing in the management of inflammatory bowel diseases in clinical practice: A worldwide survey of experts

We performed a worldwide survey to evaluate the extent to which gastroenterologists who are experts in the field of inflammatory bowel diseases (IBDs) are utilizing thiopurine metabolism in practice. This was a Web-based cross-sectional survey consisting of 12 multiple-choice and open-ended questions. Between December 2009 and April 2010, 175 questionnaires were received. The proportion of practitioners with access and reimbursement for thiopurine S-methyltransferase (TPMT) genotype, TPMT phenotype, 6-thioguanine nucleotides (6-TGN) levels, and 6-methylmercaptopurine ribonucleotides (6-MMP) levels was 48%, 54%, 44%, and 35%, respectively. Before azathioprine initiation, TPMT genotype and phenotype were performed by only 30% and 43% of responders, respectively. In patients on thiopurine therapy, 6-TGN and 6-MMP levels were determined by 54% and 44% of responders, respectively. Only 27% of physicians always wait for TMPT activity/genotype results before initiating azathioprine and 81% do not routinely recheck metabolite levels after dose escalation or reduction. In cases of very high or low TPMT activity, 75% and 74% of practitioners take into account TMPT activity result, respectively. If access to all azathioprine metabolite measurements was available and if all these tests were reimbursed by public health insurance, 47% of responders would use these tests more often in their practice. The availability and reimbursement of TPMT status and azathioprine metabolites strongly influenced experts' attitudes. Thiopurine testing is relatively underutilized by IBD gastroenterologists. The availability and reimbursement of TPMT status and azathioprine metabolites strongly influence the management of IBD patients treated with thiopurines.