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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 natio...

BACKGROUND Azathioprine (AZA) and its close analog 6-mercaptopurine are thiopurines widely used in the treatment of patients with cancer, organ transplantation, and autoimmune or inflammatory diseases, including systemic lupus erythematosus. Bone marrow inhibition is a common side effect of AZA, and severe bone marrow inhibition is related to decreased thiopurine S-methyltransferase (TPMT) activity. CASE SUMMARY We herein report a patient with proliferative lupus nephritis who was using AZA for maintenance therapy, had no common TPMT pathogenic site mutations, and exhibited severe bone marrow inhibition on the 15th day after oral administration. CONCLUSION This report alerts physicians to the fact that even though the TPMT gene has no common pathogenic site mutation, severe myelosuppression may also occur.

Pediatric Crohn's disease (CD) carries a higher genetic susceptibility and an increased risk of a more aggressive disease course than adult CD. Treatment of CD is based on immunomodulatory drugs, such as thiopurines. The enzyme mainly involved in drug metabolism is thiopurine S-methyltransferase (TPMT). An increased concentration of drug metabolites can cause adverse drug effects, such as myelosuppression and hepatotoxicity; therefore, assessing the activity of TPMT is essential both before and during treatment. TPMT genotyping result is not affected by previous thiopurine dose and currently is the primary component of TPMT activity and disease monitoring. Until now, more than 40 allelic variants of the TPMT gene have been reported, with most of them having an uncertain or no enzyme function. In this article, we report the first case of a novel TPMT allele, TPMT*45, that was identified in a Korean girl with CD whose findings suggested decreased TPMT activity. This newly observed variant is caused by a single nucleotide polymorphism resulting in nonsense mutation (c.676C>T, p.R226*) and the partial loss of amino acids in the TPMT protein. Initially, the patient began azathioprine at a standard dosage (1.5 mg/kg/day), and her laboratory results, including red blood cell (RBC) TPMT activity (6-methylmercaptopurine 2.68 nmol/mL/h and 6-methylmercaptopurine riboside 4.82 nmol/mL/h) along with thiopurine metabolite levels (6-thioguanine nucleotides 479.3 pmol/8×108 RBC), suggested an enzyme deficiency. The thiopurine dose was reduced to half (0.7 mg/kg/day), and the follow-up metabolite results as well as the associated inflammatory markers were continuously within reference ranges. Along with an improvement in the patient's subjective reports and clinical symptoms, the patient demonstrated a good treatment response to the adjusted dose. The results of our report illustrate the importance of TPMT genotyping and pharmacogenetic-based thiopurine dose adjustment. Further research should focus on the functional characterization and impact on this novel allele's treatment effect.

Aim: To assess rare TPMT variants in patients carrying a deficient phenotype not predicted by the four more frequent genotypes (*2, *3A, *3B and *3C). Materials & methods: Next-generation sequencing of TPMT in 39 patients with a discordant genotype. Results: None of the variants identified explained the discordances assuming that they are of uncertain significance according to the Clinical Pharmacogenetics Implementation Consortium classification. Two unknown variants were detected and predicted to result in a splicing defect. We show that TPMT*16 and TMPT*21 are defective alleles, and TPMT*8 and TPMT*24 are associated with a normal activity. Conclusion: Whole-exon sequencing for rare TPMT mutations has a low diagnostic yield. A reassessment of the functional impact of rare variants of uncertain significance is a critical issue.

The common marmoset (Callithrix jacchus) is a New World monkey that is increasingly used in pharmacological and toxicological studies. Thiopurine S-methyltransferase (TPMT) plays roles in the metabolism of widely used anticancer and anti-inflammatory drugs. Here, we report the isolation and molecular characterization of marmoset TPMT cDNA, which was found to contain an open-reading frame of 245 amino acids that was approximately 92% identical to its human ortholog. Marmoset TPMT was phylogenetically closer to other primate orthologs than to its pig, dog, rabbit, or rodent orthologs. Among the five marmoset tissue types analyzed, marmoset TPMT mRNA was most abundant in kidney and liver, just as human TPMT is. These results suggest that marmoset and human TPMT are similar at the molecular level.

Mahidol R2R e-Journal, 9, 1, 15-22

Selenium (Se) is an essential trace element in animals; however, the element can become highly toxic in excess amounts beyond the nutritional level. Although Se is mainly excreted into urine as a selenosugar within the nutritional level, excess amounts of Se are transformed as an alternative urinary metabolite, trimethylselenonium ion (TMSe). Se methylation appears to be an important metabolic process for the detoxification of excess Se; however, the biochemical mechanisms underlying the Se methylation have not been elucidated. In this study, we evaluated biochemical characteristics of two human methyltransferases for Se methylation, thiopurine S-methyltransferase (TPMT) and indolethylamine N-methyltransferase (INMT). The first methylation of Se, i.e., a nonmethylated to a monomethylated form, was specifically driven by TPMT, and INMT specifically mediated the third methylation, i.e., dimethylated to trimethylated form. The second methylation, i.e., a monomethylated to dimethylated form, was driven by either TPMT or INMT. Exogenous expression of TPMT, but not INMT, ameliorated the cytotoxicity of inorganic nonmethylated selenium salt, suggesting that only TPMT gave the cellular resistance against selenite exposure. TPMT was ubiquitously expressed in most mouse tissues and preferably expressed in the liver and kidneys, while INMT was specifically expressed in the lung and supplementally expressed in the liver and kidneys. Our results revealed that both TPMT and INMT cooperatively contributed to the TMSe production, enabling urinary excretion of Se and maintenance of homeostasis of this essential yet highly toxic trace element. Thus, TPMT and INMT can be recognized as selenium methyltransferases as a synonym.

Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurines. Mutations in the TPMT gene can affect drug activity, which may have adverse effects in humans. Thus, genotyping can help elucidate genetic determinants of drug response to thiopurines and optimize the selection of drug therapies for individual patients, effectively avoiding palindromia during maintenance treatment caused by insufficient dosing and the serious side effects caused by excessive doses. The current available detection methods used for TPMT*3B and TPMT*3C are complex, costly and time-consuming. Therefore, innovative detection methods for TPMT genotyping are urgently required. The aim of the present study was to establish and optimize a simple, specific and timesaving TPMT genotyping method. Using the principles of Web-based Allele-Specific PCR and competitive real-time fluorescent allele-specific PCR (CRAS-PCR), two pairs of Scorpion primers were designed for the detection of TPMT*3B and *3C, respectively, and a mutation in TPMT*3A was inferred based on data from TPMT*3B and *3C. In total, 226 samples from volunteers living in Chongqing were used for CRAS-PCR to detect TPMT*3 mutations. Results showed that nine (3.98%) were mutant (MT) heterozygotes and none were MT homozygotes for TPMT*3C, and no TPMT*3A and TPMT*3B mutations were found. Three TPMT*3C MT heterozygotes were randomly selected for DNA sequencing, and CRAS-PCR results were consistent with the sequencing results. In conclusion, in order to improve simplicity, specificity and efficiency, the present study established and optimized CRAS-PCR assays for commonly found mutant alleles of TPMT*3A (G460A and A719G), TPMT*3B (G460A), and TPMT*3C (A719G).

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gut. It can be divided into 2 main diseases: Crohn's disease (CD) and ulcerative colitis (UC). In the past 25 years, novel biological therapies have emerged for the treatment of IBD. However, for maintaining remission, conventional thiopurines [azathioprine (AZA) and mercaptopurine (6-MP)] remain important first-line immunosuppressives.1,2 Three to 4 million patients with IBD are known to use thiopurines in daily practice.3 However, approximately 60% of AZA/MP users cease treatment within 5 years mostly because of adverse events or nonresponse.4 Various strategies have been proposed to optimize conventional thiopurine therapies. Common approaches include personalized dosing using the individual geno/phenotype of thiopurine S-methyltransferase (TPMT), reduced dosing with coprescription of allopurinol, and therapeutic drug monitoring (TDM)-directed dosing. The latter approach, that is, TDM, involves measuring 6-thioguanine nucleotides (TGNs), followed by dosing advice.5–7 These approaches are dependent on access to measuring TPMT and TGNs; thus, this could be difficult in countries where such facilities are not readily available. More than 6 decades ago, the Nobel Prize Laureates Elion and Hitchings created thiopurines for the treatment of childhood leukemia,8 heralding the advent of designer drugs. TG was invented before MP and remains part of leukemia treatment regimens.9 The use of TG in IBD started 2 decades ago, and on retrospection, the TG doses, although well tolerated, were markedly high (>120 mg/d) and resulted in high rates of reversible liver injury, limiting its widespread use and potential licensing for IBD.9 With the evolution of analytical chemistry, there is a far better understanding of purine metabolism allowing 6-TGN measurements in erythrocytes, presenting clear benefits such as optimizing TG therapy for leukemia,10 helping determine adherence to thiopurines, and the need for allopurinol co-therapy.11 The enzyme TPMT is responsible for the metabolism of TG and its active TGN metabolites. This results in the formation of inactive methyl-TG and active methyl-TGNs.12 Since its initial use for IBD in 2001, TG has been rediscovered for IBD therapy13 and currently holds a provisional license in the Netherlands.14,15 This has been achieved by administering a lower daily clinical dose of TG in IBD (20 mg/d) than those in initial reports for IBD and leukemia. Similar to other immunosuppressives, adverse events remain an important reason for patients to cease TG treatment in IBD, observed in 11%–20%16,17 of cases; however, these adverse events are reversible with no reported deaths. To further improve safety, TDM and TPMT geno/phenotyping might add value to reduce adverse events if the facility is available at the treatment center. However, to date, no relationship has been identified between 6-TGN levels and abnormal laboratory values.18,19 In this study, we aimed to investigate the role of 6-TGN measurements, TPMT geno/phenotyping, and their mutual relationship with TG therapy in IBD.

Thiopurines by their glucocorticoid-sparing property help in maintaining remission for patients with inflammatory bowel disease (IBD), when glucocorticoids are reduced and withdrawn. However, due to bone marrow suppression, it cannot be used in various conditions where it is indicated. A 17-year-old patient presented with pancytopenia with neutropenic sepsis and alopecia after 3 weeks of starting azathioprine for her underlying Crohn's disease. Thiopurine S-methyltransferase (TPMT;*2, *3A, *3C) analysis resulted in a wild-type genotype, whereas homozygous Nudix hydrolase 15 (NUDT 15 C415T) variant was positive. Azathioprine was stopped immediately, and she was started on broad-spectrum antibiotics that led to some clinical improvements initially, but later on, the patient developed intestinal obstruction along with postoperative complications leading to death. In this report, we highlight a case of serious hematological toxicity associated with azathioprine use in a patient with Crohn's disease with homozygous NUDT 15 variant, thus favoring the implementation of a pharmacogenomic approach before starting azathioprine, particularly in the Asian population. How to cite this article Debnath P, Nair S, Jain S, Udgirkar S, Contractor Q, Rathi P. Thiopurine-induced Myelosuppression with Severe Sepsis in a Patient with Crohn's Disease: A Case Report. Indian J Crit Care Med 2021;25(2):228-230. Prior presentation of case report at professional meeting The case was presented in abstract form at the American College of Gastroenterology Annual Scientific Meeting, held at San Antonio, TX, USA 2019. Informed consent for publication of case details Obtained from patient's relatives.

Crohnova bolezen je ena od najpogostejših oblik avtoimunske kronične vnetne črevesne bolezni. Pomemben terapevtski pristop za vzdrževanje bolezni v fazi remisije je zdravljenje z azatioprinom. Zaradi ozkega terapevtskega okna te imunosupresivne zdravilne učinkovine se lahko med terapijo razvijejo hujši neželeni učinki, kot sta mielosupresija in hepatotoksičnost. V izogib temu je pomembno načrtovanje učinkovite in varne terapije z določitvijo ustreznega individualnega odmerka azatioprina za bolnika na osnovi sledečih diagnostičnih pristopov: genotipizacija ter merjenje aktivnosti encima tiopurin S-metiltransferaze (TPMT) pred zdravljenjem ter določitev koncentracije glavnih presnovkov azatioprina – 6-tiogvanozinskih nukleotidov (6-TG) in 6-metilmerkaptopuina (6-MMP) – 4–6 tednov po začetku terapije. Magistrsko delo je zajemalo analize na vzorcih krvi bolnikov s Crohnovo boleznijo, ki so bili napoteni iz UKC Ljubljana, oddelka za gastroenterologijo, v Laboratorij za molekularno diagnostiko na Fakulteti za farmacijo z namenom določitve genotipa in aktivnosti TPMT ter analize glavnih presnovkov zdravila. Po izolaciji DNA iz krvi bolnikov smo izvedli genotipizacijo najpogostejših alelov TPMT (TPMT *2, *3A, *3C), ki je temeljila na reakciji PCR z uporabo hidrolizirajočih sond. Na delu vzorcev smo v lizatih rdečih krvničk na osnovi pretvorbe 6-merkaptopurina v 6-MMP s pomočjo postavljene metode HPLC nadalje določili aktivnost encima TPMT. Identificirali smo prisotnost najpogostejših alelnih variant TPMT*3A in TPMT*3C, in sicer pri 6,25 % oz. 1,56 % preiskovancev. Na predhodno določenih presnovkih azatioprina (6-TG in 6-MMP) smo preučili tudi vpliv genotipa TPMT na glavna presnovka ter na njuno razmerje. Prisotnost variantnega alela TPMT je bila značilno povezana z višjimi koncentracijami 6-TG ter z nizkim razmerjem 6-MMP : 6-TG. Nadalje smo preverili napovedno vrednost za pojav neželenih učinkov, ki je temeljila na koncentracijah 6-TG, 6-MMP ter njunega razmerja. Po literaturnih podatkih koncentracija 6-TG > 450 pmol x (8 x 10^8 eritrocitov)^-1 korelira s povečanim tveganjem za mielosupresijo, koncentracija 6-MMP > 5700 pmol x (8 x 10^8 eritrocitov)^-1 ter razmerje 6-MMP : 6-TG > 11 pa s hepatotoksičnostjo. Na naši populaciji je imel vsak tretji preiskovanec prisoten vsaj en dejavnik tveganja za pojav neželenih učinkov azatioprina pri prvem odvzemu vzorca. Mielosupresija bi se lahko izrazila pri 20,31 % preiskovancev, hepatotoksičnosti pa bi bilo podvrženih 23,44 % preiskovancev. Z merjenjem metabolitov tekom terapije je te vrednosti pri večini bolnikov ob uvedbi ustreznih ukrepov v terapijo zdravnikom uspelo znižati. Pomen pravega odmerjanja in spremembe zdravljenja na osnovi genotipizacije TPMT oz. merjenja metabolitov smo ponazorili na dveh kliničnih primerih. V primeru mielotoksičnosti se je odmerjanje azatioprina zamenjalo z biološkim zdravilom, pri hepatotoksičnosti pa se je po smernicah ob uvedbi alopurinola znižal odmerek azatioprina. Rezultati tega magistrskega dela poudarjajo pomen določanja genotipa in aktivnosti TPMT in podrobno spremljanje metabolitov tiopurinov tekom zdravljenja. S temi diagnostičnimi pristopi lahko preprečimo resne neželene učinke ali sprejmemo ustrezne ukrepe, ki vodijo v remisijo bolezni brez resnejših zapletov. Crohn's disease is one of the most common forms of autoimmune chronic inflammatory bowel diseases. An important therapeutic approach to maintain the disease in remission is azathioprine therapy. Due to the narrow therapeutic window of this immunosuppressive drug, severe side effects, such as myelosuppression and hepatotoxicity, may develop during therapy. To avoid this, it is important to plan an effective and safe therapy by determining the appropriate individual dose of azathioprine for the patient based on the following diagnostic approaches: genotyping and measurement of the activity of the enzyme thiopurine S-methyltransferase (TPMT) prior to treatment, and determination of the levels of the main metabolites of azathioprine - 6-thioguanosine nucleotides (6-TG) and 6-methylmercaptopurine (6-MMP) – 4–6 weeks after the initiation of therapy. The master's thesis included analyses on blood samples from Crohn's disease patients referred from the UKC Ljubljana, Department of Gastroenterology to the Laboratory of Molecular Diagnostics at the Faculty of Pharmacy in order to determine the genotype and activity of TPMT and to analyse the major metabolites of the drug. After DNA isolation from the patients' blood, genotyping of the most frequent TPMT alleles (TPMT *2, *3A, *3C) was performed based on PCR using hydrolysable probes. In a subset of samples, TPMT enzyme activity was further determined in red blood cell lysates based on the conversion of 6-mercaptopurine to 6-MMP using an established HPLC method. The presence of the most common allelic variants TPMT*3A and TPMT*3C was identified in 6.25% and 1.56% of the subjects, respectively the most common allelic variants TPMT*3A and TPMT*3C were identified in 6.25 % and 1.56 % of the subjects. We also investigated the influence of the TPMT genotype on the major metabolites and their ratio on previously determined azathioprine metabolites (6-TG and 6-MMP). The presence of a variant TPMT allele was typically associated with higher 6-TG concentrations and a low 6-MMP : 6-TG ratio. We further tested the predictive value for the occurrence of adverse effects based on 6-TG, 6-MMP concentrations and their ratio. According to the literature, a 6-TG concentration > 450 pmol x (8 x 10^8 erythrocytes)^-1 correlates with an increased risk of myelosuppression, and a 6-MMP concentration > 5700 pmol x (8 x 10^8 erythrocytes)^-1 and a 6-MMP : 6-TG ratio > 11 with hepatotoxicity. In our population, one in three subjects had at least one risk factor for azathioprine adverse effects present at the first sample collection. Myelosuppression could be manifested in 20.31 % of subjects and hepatotoxicity in 23.44 % of subjects. By measuring metabolites during the course of therapy, physicians were able to lower these values in most patients when appropriate measures were introduced into the therapy. The importance of correct dosing and treatment modification based on TPMT genotyping or metabolite measurement was illustrated in two clinical cases. In the case of myelotoxicity, the dosage of azathioprine was switched to a biologic, and in the case of hepatotoxicity, the dose of azathioprine was reduced according to guidelines when allopurinol was introduced. The results of this Master thesis underline the importance of genotyping and activity determination of TPMT and close monitoring of thiopurine metabolites during treatment. These diagnostic approaches can prevent serious side effects or take appropriate measures leading to disease remission without serious complications.

Objective: To determine thiopurine S-methyltransferase (TPMT) activity and the range of TPMT activity values for the Indian population. Methods: An isocratic reversed-phase high-performance liquid chromatography method with ultraviolet detection for the determination of TPMT activity in red blood cells was developed and validated. TPMT activity (nmol of 6-methylmercaptopurine [6-MMP]/h/ml erythrocytes) was measured based on the conversion of 6-mercaptopurine to 6-MMP with S-adenosyl-l-methionine. The stability of erythrocyte lysate at −20°C and the whole-blood specimens for TPMT activity were determined at 4°C. TPMT enzyme activity was measured in 150 patients who were not on azathioprine (AZA), and population difference in TPMT enzyme activity was evaluated. Results: The assay was linear from 1.0 to 60.14 nmol 6-MMP concentrations. The chromatogram peaks obtained were baseline separated for 6-MMP and the internal standard. The bias and precision of low-, medium-, and high-quality controls (QC) were within acceptable limits. Stock and lysate specimens were found to be stable in −20°C. The Indian populations have a considerable number of patients with low- and intermediate-TPMT activity. The wide range of TPMT activity in the study population (3.85–35.68 nmol/h/ml erythrocytes) is suggestive of high inter-individual variability in the formation of 6-thioguanine nucleotide formation, and therefore clinical efficacy and safety of AZA. Conclusion: A simple isocratic method was developed and validated for measuring TPMT activity in erythrocytes. Measuring TPMT activity before initiating AZA may help to decide the initial dose of AZA in patient management.

Purpose of review The diagnosis of drug-induced interstitial lung disease (DI-ILD) is challenging and mainly made by exclusion of other possible causes. Toxicity can occur as a cause of drug(s) or drug-drug interactions. In this review, we summarize the possible role of pharmacogenetics of metabolizing enzymes in DI-ILD. Recent findings Knowledge of the genetic predispositions of enzymes involved in drug metabolization and their relation with proposed cytotoxic mechanisms of DI-ILD, in particular direct cell toxicity and free oxygen radical production is increasing. The cytochrome P450 enzyme family and other enzymes play an important role in the metabolism of all sorts of ingested, injected, or inhaled xenobiotic substances. The liver is the major site for metabolism. Metabolic cytotoxic mechanisms have however also been detected in lung tissue. Polymorphisms in genes coding for enzymes that influence metabolic activity may lead to localized (toxic) reactions and tissue damage. This knowledge may be helpful in preventing the risk of DI-ILD. Drug toxicity can be the consequence of absence or very poor enzyme activity, especially if no other metabolic route is available. In the case of reduced enzyme activity, it is recommended to reduce the dose or to prescribe an alternative drug, which is metabolized by a different, unaffected enzyme system to prevent toxic side effects. However, enhanced enzyme activity may lead to excessive formation of toxic and sometimes reactive metabolites. Therefore, knowing a patient's drug-metabolizing profile before drug prescription is a promising way to prevent or explain DI-ILD.

Abstract: Introduction: Genetic polymorphism of thiopurine S-methyltransferase, the key enzyme in metabolism of thiopurines (azathioprine and 6-mercaptopurine) used in the treatment of inflammatory bowel disease, results in different enzyme activities. Decreased enzyme activity causes myelosuppression whereas abnormally high activity results in hepatotoxicity at standard thiopurine doses. Four allele variants (TMPT*2, TMPT*3A, TPMT*3B and TPMT*3C) account for decreased activity in more than 95% of cases. Aim: To evaluate the frequency of severe side effects, such as myelosuppression and hepatotoxicity, at standard or decreased azathioprine doses in children with inflammatory bowel disease who do not exhibit any of the four most common variant alleles. Method: Retrospective analysis of children with inflammatory bowel disease treated with azathioprine at a single tertiary referral center. Results: 51 patients were identified (44: Crohn’s disease, 7: ulcerative colitis; male ratio: 28/51; mean age at diagnosis: 12.4 years). Two patients discontinued azathioprine arbitrarily whereas in one patient it was stopped due to serious pancreatitis and in another one because of severe flu-like symptoms. None of the remaining 47 patients exhibited hepatotoxicity suggesting abnormally high thiopurine S-methyltransferase activity. Four patients (8.5%) had profound myelosuppression on less than 1 mg/kg/day azathioprine requiring discontinuation of the drug, and all of them showed complete bone marrow recovery subsequently. No myelosuppression occurred in the remaining 43 patients on 2.17 ± 0.31 mg/kg/day (mean ± SD) azathioprine treatment. Conclusions: Regular blood tests are necessary on thiopurine therapy despite normal thiopurine S-methyltransferase genotype because of the risk of myelosuppression. The four most common variant alleles were identified in routine genotyping only, therefore most likely rare variant allele(s) or polymorphism of other enzymes involved in thiopurine metabolism account for the aforementioned four cases with profound bone marrow suppression. Thiopurine metabolite monitoring is the key for individualized treatment when optimal dosing can be achieved with the least side effects. Orv Hetil. 2019; 160(5): 179–185.

المقدمة : ان سرطان الدم اللمفاوي الحاد هو اكثر الامراض السرطانيه حدوثا في الاطفال، الذي يعالج باستخدام العلاج الكيمياوي فقط. الثايوبيورين هو دواء اساسي في كل الطرق الحديثة المتفق عليها لمعالجة سرطان الدم اللمفاوي، حيث يعمل كعامل مضاد للسرطان ولفتره طويله من العلاج، وقد يؤدي هذا الى التسمم الدوائي عند هؤلاء الاطفال ويعود السبب الى قلة فعالية انزيم الثايوبيورين ميثايل ترانسفيريز والذي هو احد الانزيمات المسؤوله عن العملية الايضيه لدواء الميركابتوبيورين المستخدم في العلاج، ان هذا النقص في الفعاليه الانزيميه يعود بصوره كبيره الى الطفرات الجينيه الحاصله في جين هذا الانزيم. الهدف: الكشف عن الطفرات الاكثر تكرارا لانزيم ثايوبيورين ميثايل ترانسفيريز في الاطفال العراقيين المصابين بسرطان الدم اللمفاوي الحاد وتحديد النسب المئويه لتكراراتها. طرق العمل: شملت هذه الدراسة 81 طفل عراقي مصاب بسرطان الدم اللمفاوي الحاد في مرحلة الـ (maintenance) من العلاج الكيمياوي من ضمن البرنامج العلاجي البريطاني (UKALL) لسنة 2011،وبعد اجراء عملية الاستخلاص للحمض النووي من دم هؤلاء الاطفال اجري الكشف عن وجود الطفرات في هذا الانزيم بواسطة تفاعل التضاعف المتسلسل (PCR). النتائج : اظهرت النتائج ان الانزيم لـ 51 طفل كان طبيعي لايحوي اي طفره جينيه وبتردد تكراري (62.96%) و 30 طفل كان لديهم طفره جينيه في الانزيم وبتردد تكراري (37.03%) اما للاليل (TPMT*3A) بتردد تكراري (29.62%) او للاليل (TPMT*3C) وبتردد تكراري (7.4%) . واما الطفره الجينيه بالاليل (TPMT*3B) فلم تظهر في هذه المجموعه من المرضى. لقد وجدنا في هذه الدراسة الطفرات الجينيه (TPMT*3A) و (TPMT*3C) في جين الانزيم الناقل لمجموعة المثيل (TPMT) ولم نجد الطفره الجينية (TPMT*3B). هذه الدراسة هي الاولى في تحليل الطفرات الجينيه للانزيم ثايوبيورين مثايل ترانسفيريز في عينه من اطفال المجتمع العراقي المصابين بسرطان الدم اللمفاوي اثناء مرحلة العلاج الكيمياوي.

Background Methylation driven by thiopurine S-methylatransferase (TPMT) is crucial for deactivation of cytostatic and immunosuppressant thiopurines. Despite its remarkable integration into clinical practice, the endogenous function of TPMT is unknown. Methods To address the role of TPMT in methylation of selenium compounds, we established the research on saturation transfer difference (STD) and 77Se NMR spectroscopy, fluorescence measurements, as well as computational molecular docking simulations. Results Using STD NMR spectroscopy and fluorescence measurements of tryptophan residues in TPMT, we determined the binding of selenocysteine (Sec) to human recombinant TPMT. By comparing binding characteristics of Sec in the absence and in the presence of methyl donor, we confirmed S-adenosylmethionine (SAM)-induced conformational changes in TPMT. Molecular docking analysis positioned Sec into the active site of TPMT with orientation relevant for methylation reaction. Se-methylselenocysteine (MeSec), produced in the enzymatic reaction, was detected by 77Se NMR spectroscopy. A direct interaction between Sec and SAM in the active site of rTPMT and the formation of both products, MeSec and S-adenosylhomocysteine, was demonstrated using NMR spectroscopy. Conclusions The present study provides evidence on in vitro methylation of Sec by rTPMT in a SAM-dependant manner. General Significance. Our results suggest novel role of TPMT and demonstrate new insights into enzymatic modifications of the 21st amino acid. Bibliografija: str. 188-190. Abstract. ARRS ARRS ARRS

Background and aim Thiopurines are often used in combination with mesalazine for the treatment of ulcerative colitis (UC). Mesalazine formulations are delivered to the digestive tract by various delivery systems and absorbed as 5-aminosalicylic acid (5-ASA). 5-ASA is known to inhibit thiopurine S-methyltransferase (TPMT) activity and to affect thiopurine metabolism. There have been no studies comparing TPMT inhibition by multimatrix mesalazine (MMX) with other formulations. We investigated the difference in TPMT inhibition by different mesalazine formulations and prospectively confirmed the clinical relevance. Methods Plasma concentrations of 5-ASA, N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA), and TPMT activities were measured in UC patients receiving various mesalazine formulations (time-dependent or pH-dependent mesalazine or MMX) as monotherapy. Patients already on both time-dependent or pH-dependent mesalazine and thiopurines switched their mesalazine to MMX, examining 6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine (6-MMP) 0 and 8 weeks after switching. Clinical relapse after switching was also monitored for 24 weeks. Results Plasma 5-ASA and N-Ac-5-ASA levels were significantly higher in patients receiving time-dependent mesalazine (n = 12) compared with pH-dependent mesalazine (n = 12) and MMX (n = 15), accompanied by greater TPMT inhibition. Prospective switching from time-dependent mesalazine to MMX decreased 6-TGN levels, increased those of 6-MMP, and increased 6-MMP/6-TGN ratios. Furthermore, this resulted in significantly more relapses than switching from pH-dependent mesalazine to MMX. Conclusions Time-dependent mesalazine has higher plasma 5-ASA and N-Ac-5-ASA levels and greater TPMT inhibition than MMX. Therefore, switching from time-dependent mesalazine to MMX may lead to an increase of 6-MMP/6-TGN, which may reduce the clinical effectiveness of thiopurines, warranting close monitoring after switch.

Background & objective Thiopurine drugs are considered as a treatment modality in various autoimmune disorders including pemphigus vulgaris (PV). These drugs are metabolized by an enzyme "Thiopurine S-methyl transferase" (TPMT). Various variants of this enzyme may have decreased activity leading to serious drug side effects. To investigate the phenotype and genotype of TPMT in PV patients receiving thiopurine drugs. Methods A total of 50 patients (29 women and 21 men) with pemphigus vulgaris treating with standard dose of Thiopurine drugs were selected. Sex, age, result of liver function test and complete blood count were recorded. Genotyping of two common non-functional allele (TPMT*2 and TPMT*3C) by Allele-specific and RFLP-PCR was performed. TPMT enzymatic level was determined by an ELISA based method. Results Of patients, 36 (72%) were found to have normal TPMT level; and 12, (24%) had higher level of enzyme and 2, 4% had low TPMT enzyme, but none of the patients showed mutant TPMT*2 and TPMT*3C alleles. None of the patients showed hepatotoxicity and bone marrow suppression. Conclusion The phenotypic assay based on ELISA method may have false positive and misleading results but genotyping using PCR-RFLP and allele specific PCR is accurate, simple and cost-effective and can be used in patients decided to undergo thiopurine treatment.

Thiopurine drugs play an important role in the treatment of acute lymphoblastic leukemia by inhibiting the proliferation of cell. It is done by blocking various pathways of nucleic acid synthesis. Because of this typical property of these drugs, they are used in cancer treatment. Mercaptopurine (6-MP) and thioguanine (6-TG) act synergistically in inhibiting the synthesis of purine, ultimately inhibiting DNA synthesis. Incorporation of 6-TGN (thioguanine nucleotides) into DNA is responsible for anticancer activity of thiopurine drugs. Two-thirds of children suffering from acute lymphoblastic leukemia (ALL) can be cured by using 6-MP. Thiopurine drugs concentration in the body is regulated by an enzyme called thiopurine S-methyltransferase (TPMT). Studies on red blood cells suggest that TPMT activity is trimodal in its distribution. Ninety percent of individuals possess normal TPMT activity, but in the remaining 10% of individuals, low TPMT activity is observed. This is because of presence of genetic polymorphism in TPMT gene. Metabolism of thiopurine drugs in our body depends upon the genetic variant of TPMT gene we possess. Before initiating thiopurine drug therapy, it is necessary to determine TPMT status of the patient. Usually TPMT genotyping is used for this purpose. Standard dose of thiopurine drugs is used for patients with high TPMT enzyme activity. In patients, exhibiting low TPMT activity dosage of thiopurine drugs is reduced, or an alternate therapy is considered to avoid adverse drug reactions like myelosuppression, hematologic toxicity, and hypoplasia of the bone marrow.

All live vaccines are contraindicated in children who are immunosuppressed secondary to disease or immunosuppressive therapy Calcineurin inhibitors are commonly used immunosuppressive agents and act via cyclophilin and inhibit IL-2 production The thiopurine S-methyltransferase pharmacogenetic testing is recommended before using thiopurine drugs for immunosuppression Tacrolimus therapy is associated with subsequent cancer risk Febrile neutropenia in low risk patients is treated with empirical antibiotics Colony-stimulating factors are used to treat prolonged neutropenia secondary to immunosuppressive therapy

Purpose Results of a study of prescribers’ responses to a pharmacogenomics-based clinical decision support (CDS) alert designed to prompt thiopurine S -methyltransferase (TPMT) status testing are reported. Methods A single-center, retrospective, chart review–based study was conducted to evaluate prescriber compliance with a pretest CDS alert that warned of potential thiopurine drug toxicity resulting from deficient TPMT activity due to TPMT gene polymorphism. The CDS alert was triggered when prescribers ordered thiopurine drugs for patients whose records did not indicate TPMT status or when historical thiopurine use was documented in the electronic health record. The alert pop-up also provided a link to online educational resources to guide thiopurine dosing calculations. Results During the 9-month study period, 500 CDS alerts were generated: in 101 cases (20%), TPMT phenotyping or TPMT genotyping was ordered; in 399 cases (80%), testing was not ordered. Multivariable regression analysis indicated that documentation of historical thiopurine use was the only independent predictor of test ordering. Among the 99 patients tested subsequent to CDS alerts, 70 (71%) had normal TPMT activity, 29 (29%) had intermediate activity, and none had deficient activity. The online resources provided thiopurine dosing recommendations applicable to 24 patients, but only 3 were prescribed guideline-supported doses after CDS alerts. Conclusion The pretest CDS rule resulted in a large proportion of neglected alerts due to poor alerting accuracy and consequent alert fatigue. Prescriber usage of online thiopurine dosing resources was low.

Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are treated with immunosuppressive purine analogs, 6-mercaptopurine/6-thioguanine/azathiopurine, which are inactivated by thiopurine S-methyltransferase (TPMT). Non-synonymous polymorphisms in TPMT are associated with increased risk of adverse effects in patients treated with thiopurines. This study aimed to determine the frequency of the most common mutant TPMT alleles in Mexican patients with SLE (a prototype autoimmune disease) and RA (one of the most common autoimmune diseases in Mexico). Five hundred fifty-three consecutive patients from Central Mexico with SLE (178) and RA (375) were included. Subjects were genotyped to identify TPMT*2 (rs1800462), TPMT*3A (rs1800460 and rs1142345), TPMT*3B (rs1800460), and TPMT*3C (rs1142345) mutant alleles. DNA samples were assayed with the 5' exonuclease technique and TaqMan probes. Mutant alleles were detected in 6.2 and 5.2% of SLE and RA cases, respectively. Of note, 12.4% of SLE cases and 10.1% of RA cases carried mutant genotypes. Among those, the null genotype (TPMT*2/*3A, 0.3%) and the TPMT*3B (0.5%) and TPMT*3C (1.0%) alleles were found in RA, but not SLE cases. Mexican SLE cases displayed the highest frequency of mutant TPMT genotypes worldwide. TPMT genotyping should be performed for Mexican patients with SLE and RA before prescribing purine analogs.