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Background: 6-Mercaptopurine (6-MP), a widely used anti-metabolite for the maintenance phase of childhood acute lymphoblastic leukemia (ALL), has been observed to cause myelotoxicity due to genetic polymorphism of thiopurine methyl transferase (TPMT), one of the drug-metabolizing enzymes. This study aimed to determine the frequency of thiopurine S-methyl transferase (TPMT) polymorphic variants in a group of Pakistani children with acute lymphoblastic leukemia (ALL) using 6-mercaptopurine (6-MP). Methods: Diagnosed cases (n=100) of ALL, either Pre-B or T Cell, between the ages of 2 to 18 years were randomly selected from OPD of Children Cancer Hospital and National Institute of Child Health (NICH), Karachi. The subjects were under BFM (Berlin Frankfurt Munster) protocol. Expired and relapsed patients (2/100) were excluded. Blood samples were drawn and DNA was extracted from serum to determine the genotype of TPMT (*2, *3a, *3B and *3C) using allele-specific PCR (AS-PCR) and RFLP. PCR assays were done to detect the G238C transversion in TPMT*2 and the G460A and A719G transition in TMPT*3 Alleles. Results: Polymorphism in TMPT was found 100% (98/98) in ALL patients belonging to a heterozygous group. Out of them, 74.5% of ALL patients showed myelotoxicity. Furthermore, *1/*3B was the most prevalent variant TPMT allele observed with the highest frequency reported at 97%. The variant allele *1/*3A was found in only 3 patients, whereas, TMPT*2 and *3C Alleles were not found. Conclusion: The majority of our patients displayed a distinct TPMT genetic polymorphism TPMT*1/*3B which is comparatively rare in other parts of the world. Keywords: 6-Mercaptopurine; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Genetic Polymorphism; Alleles; Pakistan.

Aim:Thiopurine S-methyltransferase (TPMT) is an essential enzyme in the metabolism of thiopurine drugs, and its activity may change due to different polymorphisms in the TPMT gene. The TPMT gene has different genetic polymorphisms in different ethnic groups. This study aimed to determine the frequency of TPMT polymorphisms in children with acute leukemia/non-Hodgkin lymphoma (AL/NHL) and healthy children and to evaluate their association with severe toxicities in the study population.Materials and Methods:Sixty-seven pediatric AL/NHL patients and 84 healthy children were evaluated. Genotyping for the TPMT*2, TPMT*3A, TPMT*3B, TPMT*3C, TPMT*4, TPMT*5, TPMT*6, and TPMT*7 alleles were performed by the real-time polymerase chain reaction technique. The number of grade 3 or higher hematologic and hepatic toxicities were recorded from the patient charts.Results:In the AL/NHL patients, we found that the patients had generally wild-type TPMT*1 allele in 80.6%, whereas TPMT*2 (238G>C) was seen in 1.5%, TPMT*3A (c.460G>A and c.719A>G) in 0%, and TPMT*3B polymorphisms (460G>A) in 17.9%. We found wild-type TPMT*1 allele in 98.8% and TPMT*3B polymorphisms (460G>A) in 1.2% of the healthy volunteers. Grade ≥3 myelosuppression developed in 22/54 patients with the wild type allele, and it developed in 5/12 patients with TPMT*3B allele. Six (8.9%) patients had grade ≥3 aspartate aminotransferase elevations, 17 (25%) patients had grade ≥3 alanine transaminase elevations (1-5 times), and 42 patients had (62.6%) grade ≥3 total bilirubin elevations.Conclusion:TPMT*3B polymorphism was the most common allele detected in our study group. This allele frequency is very high in comparison to other studies from our country and it was over-represented in comparison to the healthy volunteers. We did not find any relationship between severe hematologic/hepatic toxicities and TPMT gene polymorphisms.

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., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Thiopurines are essential medications in Acute Lymphoblastic Leukemia (ALL) treatment protocols as anti-cancer agents since long time; however, their use might result in unexpected toxicities in ALL children due to the low thiopurine S-methyltransferase (TPMT) activity, a major enzyme involved in 6- mercaptopurine metabolism, which strongly correlates to the genetic polymorphism of the TPMT gene in those patients. Objective: To identify the most common TPMT polymorphisms in children with ALL and its frequencies. Methods: A cross sectional study enrolling eighty-one ALL children receiving mercaptopurine drug during their maintenance course of treatment according to UKALL – 2011 protocol, were enrolled in this study. After DNA extraction from whole blood TPMT genetic polymorphisms were detected by allele-specific multiplex-PCR analysis. Results: A total of 51 children with allele frequencies of (62.96%) were homozygous for the wild-type allele TPMT*1, 30 children with allelic frequency of (37.03%) were heterozygous for one of the two mutant alleles (TPMT*3A or TPMT*3C) with allele frequencies of 29.62% and 7.4% respectively, while no result was found homozygous for two mutant alleles or TPMT*3B allele. Conclusions: This is the first study in Iraq to identify the genetic polymorphism of TPMT in a group of ALL children being treated for ALL. The study revealed the presence of TPMT*3A and TPMT*3C genetic polymorphisms among the study sample, no TPMT*3B was identified in the study sample.

Changhee Ha,1 Eun Sil Kim,2 Yiyoung Kwon,2 Yon Ho Choe,2 Mi Jin Kim,2 Soo-Youn Lee1,3 1Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; 2Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; 3Department of Clinical Pharmacology and Therapeutics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, KoreaCorrespondence: Mi Jin KimDepartment of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 06351, KoreaTel +82 2 3410 0951Fax +82 2 3410 0043Email mijin1217.kim@samsung.comSoo-Youn LeeDepartment of Laboratory Medicine and Genetics, Department of Clinical Pharmacology and Therapeutics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 06351, KoreaTel +82 2 3410 1834Fax +82 2 3410 2719Email suddenbz@skku.eduAbstract: 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.Keywords: TPMT*45, Crohn’s disease, thioupurine, single nucleotide polymorphism, TPMT activity, drug metabolism

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: 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.

Abstract Objectives This study assessed the activity of thiopurine S-methyltransferase (TPMT) in Nigerians with a view to providing data on susceptibility to thiopurine toxicity, and as well generate reference activity values for clinical use. Results TPMT activity, expressed as the amount of 6MMP in ng/mL after 1 h incubation at 37 °C per haemoglobin (U/g Hb), varied between 2.34 and 63.50 U/g Hb in the study population. Poor metabolic phenotypes, characterised by an activity values below 8.41 U/g Hb, were observed in 20% of the study subjects. Intermediate metabolizers had activity values between 8.41 and 16.13 U/g Hb. Fast and very fast metabolizers were characterised by activity values of 16.20–56.22 and > 56.22 U/g Hb, respectively. These findings suggest that a potentially huge discordance between TPMT phenotype and genotype exist in Nigerians, and emphasizes the superiority of a prior determination of TPMT metabolic phenotype in ensuring the safety of thiopurine drug administration in the population.

Objective: Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurinedrugs. Low activity phenotypes are correlated with several mutations in the TPMTgene and adverse drug reactions. The molecular basis for dissimilar enzymatic activityof TPMT has been established in Caucasians, African-Americans and Southwest Asians,but it remains to be elucidated in Iranian population. Until present, no study on Iranianpopulation has been performed on the known alleles of TPMT. The aim of this study wasto investigate the frequencies of four of the most common variants of this gene.Materials and Methods: This study was conducted during 2007 at the Department of Hematology,Tarbiat Modares University, Tehran, Iran. Using PCR-RFLP and allele specificPCR techniques, allelic variants of the TPMT gene TPMT*2(G238C), TPMT*3B (G460A),TPMT*3C (A719G) and TPMT*3A (G460A and A719G) were genotyped in a normal populationof 127 Iranians.Results: In this study TPMT*2 showed a prevalence of 7.08%. TPMT*3C and *3A werefound in 2.47% and 2.18% of the samples, respectively. TPMT*3B variant was not detectedin Iranian subjects. 112 out of 127 participants showed homozygote wild type allele.Conclusion: This study is the first to analyze TPMT allele frequencies in a sample ofIranian population and indicates that TPMT*2 is the most common allele (7.08%) in thispopulation. These results can help to organize national pretreatment strategies in patientswith acute lympho blastic leukemia (ALL) or other diseases requiring thiopurine medicationin their standard therapy.

INTRODUCTION Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyses the inactivation of mercaptopurine (MP) which is widely used in the treatment of acute lymphoblastic leukaemia (ALL) of childhood. Potentially fatal myelotoxicity may develop after standard doses of MP in TPMT deficient patients. OBJECTIVES To establish if individually tailored doses of MP can reduce myelotoxicity in ALL patients carrying mutations in the TPMT gene. To establish if variable number of tandem repeats (VNTR) genotype influences the treatment effects of MP. METHOD Fifty randomly selected patients treated according to ALL IC-BFM 2002 protocol were tested for most frequent TPMT gene mutations using PCR based methods. VNTR genotype was determined in 20 children by PCR methods. During the maintenance phase, we recorded the number of weeks when therapy was applied in either full doses, reduced doses or when patients were without any therapy. RESULTS Fifty children were examined, 29 boys (58%) and 21 girls (42%); age ranged from 1.8-17.3 years (median 6.2 years). Four patients (8%) were heterozygous for TPMT mutations, all of them carrying the TPMT*3A variant. After 12, 14, 16 and 19 weeks of therapy with reduced doses of MP, the patients switched to full doses due to good tolerance. There was no therapy omission. Cumulative dose of MP was reduced for 7.8%, 7.4%, 11.2% and 16.6%, respectively, in patients with TPMT mutations. No significant difference was found between children with no mutations and TPMT heterozygotes regarding full dose therapy (53.6 vs. 55.7 weeks, respectively) and reduced dose therapy (19.9 vs. 15.2 weeks respectively). The number of detected VNTRs ranged from four to seven. The majority of patients had different number of VNTRs on homologous chromosomes. Most frequently detected polymorphism was VNTR*5. No correlation was found between TPMT and VNTR genotype inheritance. CONCLUSION Obeying pharmacogenetic principles in the treatment of childhood ALL may improve the tolerance of therapy with MP.

Therapy of molybdenum cofactor (Moco) deficiency has received US Food and Drug Administration (FDA) approval in 2021. Whereas urothione, the urinary excreted catabolite of Moco, is used as diagnostic biomarker for Moco-deficiency, its catabolic pathway remains unknown. Here, we identified the urothione-synthesizing methyltransferase using mouse liver tissue by anion exchange/size exclusion chromatography and peptide mass fingerprinting. We show that the catabolic Moco S-methylating enzyme corresponds to thiopurine S-methyltransferase (TPMT), a highly polymorphic drug-metabolizing enzyme associated with drug-related hematotoxicity but unknown physiological role. Urothione synthesis was investigated in vitro using recombinantly expressed human TPMT protein, liver lysates from Tpmt wild-type and knock-out (Tpmt -/- ) mice as well as human liver cytosol. Urothione levels were quantified by liquid-chromatography tandem mass spectrometry in the kidneys and urine of mice. TPMT-genotype/phenotype and excretion levels of urothione were investigated in human samples and validated in an independent population-based study. As Moco provides a physiological substrate (thiopterin) of TPMT, thiopterin-methylating activity was associated with TPMT activity determined with its drug substrate (6-thioguanin) in mice and humans. Urothione concentration was extremely low in the kidneys and urine of Tpmt -/- mice. Urinary urothione concentration in TPMT-deficient patients depends on common TPMT polymorphisms, with extremely low levels in homozygous variant carriers (TPMT*3A/*3A) but normal levels in compound heterozygous carriers (TPMT*3A/*3C) as validated in the population-based study. Our work newly identified an endogenous substrate for TPMT and shows an unprecedented link between Moco catabolism and drug metabolism. Moreover, the TPMT example indicates that phenotypic consequences of genetic polymorphisms may differ between drug- and endogenous substrates., (© 2022 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Rachid Abaji,1 Maja Krajinovic2 1Department of Pharmacology, 2Departments of Pediatrics and Pharmacology, CHU Sainte-Justine Research Center, University of Montreal, Montreal, QC, Canada Abstract: The thiopurine S-methyltransferase (TPMT) gene encodes for the TPMT enzyme that plays a crucial role in the metabolism of thiopurine drugs. Genetic polymorphisms in this gene can affect the activity of the TPMT enzyme and have been correlated with variability in response to treatment with thiopurines. Advances in the pharmacogenetics of TPMT allowed the development of dosing recommendations and treatment strategies to optimize and individualize prescribing thiopurine in an attempt to enhance treatment efficacy while minimizing toxicity. The influence of genetic polymorphisms in the TPMT gene on clinical outcome has been well-documented and replicated in many studies. In this review, we provide an overview of the evolution, results, conclusions and recommendations of selected studies that investigated the influence of TPMT pharmacogenetics on thiopurine treatment in acute lymphoblastic leukemia, inflammatory bowel disease and autoimmune disorders. We focus mainly on prospective studies that explored the impact of individualized TPMT-based dosing of thiopurines on clinical response. Together, these studies demonstrate the importance of preemptive TPMT genetic screening and subsequent dose adjustment in mitigating the toxicity associated with thiopurine treatment while maintaining treatment efficacy and favorable long-term outcomes. In addition, we briefly address the cost-effectiveness of this pharmacogenetics approach and its impact on clinical practice as well as the importance of recent breakthrough advances in sequencing and genotyping techniques in refining the TPMT genetic screening process. Keywords: TPMT, pharmacogenetics, thiopurine, 6-mercaptopurine, azathioprine, ADRs

Introduction: Acute Lymphoblastic Leukaemia (ALL) treatment protocols widely used thiopurine drugs as an anti-cancer agent, which over the course of time results in drug toxicity. Thiopurine S-Methyltransferase Enzyme (TPMT) is responsible for the activation of 6-Mercaptopurine (6-MP) to Thioguanine Nucleotides (TGNs) that are incorporated into DNA and trigger cell death. Low TPMT activity is strongly correlated to TPMT genetic polymorphism. Aim: To identify the level of TPMT activity and the most common TPMT polymorphism (TPMT*3A, TPMT*3B and TPMT*3C) and its frequencies in a sample of Iraqi ALL paediatric patients. Materials and Methods: Eighty-one Iraqi ALL paediatric patients receiving 6-MP in the maintenance phase of treatment were enrolled in the study. TPMT activity in the serum was measured by using Enzyme-Linked Immunosorbent Assay Technique (ELISA) in the serum and TPMT genetic polymorphism (TPMT*3A, TPMT*3B and TPMT*3C) was detected by allele-specific multiplex-PCR analysis. Statistical analysis was performed by using a two-sample t-test to evaluate the difference in allele frequencies proportion in TPMT polymorphism. Pearson’s correlation analysis was done to determine the correlation between TPMT enzyme genotype and phenotype. Results: There were 51 paediatric ALL patients carrying the wildtype allele with allele frequencies of (62.96%), 30 paediatric ALL patients carrying the mutant alleles either TPMT*3A or TPMT*3C with allele frequencies of 29.62% and 7.4% respectively. The mutant allele TPMT*3B was not detected in the patients under study. The difference in mean of the TPMT enzyme activity between the ALL patients carrying the wild-type allele and the mutant allele was highly significant with p-value ≤0.001. A highly significant positive co-relation (r=0.939) was found between TPMT low activity and presence of genetic mutation across the TPMT gene (p-value≤0.001). Conclusion: TMPT genotyping and phenotyping is an essential tool to reduce the cytotoxic effects of the anti-cancer drug 6-MP in Iraqi paediatric patients with ALL for a successful recovery.