Search

Purpose: Thiopurine S-methyltransferase (TPMT) is an enzyme that metabolizes purine analogs, agents used in the treatment of acute lymphoblastic leukemia. Improper drug metabolism leads to toxicity in chemotherapy patients and reduces treatment effectiveness. TPMT variants associated with reduced enzymatic activity vary across populations. Therefore, studying these variants in heterogeneous populations, such as Ecuadorians, can help identify molecular causes of deficiency for this enzyme.Methods: We sequenced the entire TPMT coding region in 550 Ecuadorian individuals from Afro-Ecuadorian, Indigenous, Mestizo, and Montubio ethnicities. Moreover, we conducted an ancestry analysis using 46 informative ancestry markers.Results: We identified 8 single nucleotide variants in the coding region of TPMT. The most prevalent alleles were TPMT*3A, TPMT*3B, and TPMT*3C, with frequencies of 0.055, 0.012, and 0.015, respectively. Additionally, we found rare alleles TPMT*4 and TPMT*8 with frequencies of 0.005 and 0.003. Correlating the ancestry proportions with TPMT-deficient genotypes, we observed that the Native American ancestry proportion influenced the distribution of the TPMT*1/TPMT*3A genotype (OR = 5.977, p = 0.002), while the contribution of African ancestral populations was associated with the TPMT*1/TPMT*3C genotype (OR = 9.769, p = 0.003). The rates of TPMT-deficient genotypes observed in Mestizo (f = 0.121) and Indigenous (f = 0.273) groups provide evidence for the influence of Native American ancestry and the prevalence of the TPMT*3A allele. In contrast, although Afro-Ecuadorian groups demonstrate similar deficiency rates (f = 0.160), the genetic factors involved are associated with contributions from African ancestral populations, specifically the prevalent TPMT*3C allele.Conclusion: The distribution of TPMT-deficient variants offers valuable insights into the populations under study, underscoring the necessity for genetic screening strategies to prevent thiopurine toxicity events among Latin American minority groups. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases are chronic relapsing immune-mediated diseases of the intestinal tract with multifaceted manifestations and treatment related morbidity. Faecal and blood tests, radiological, endoscopic and histologic investigations are now widely used for managing both ulcerative colitis and Crohn's disease. Over the years, a number of new investigations have been proposed but not widely adopted yet. Patients with Crohn's disease may have multiple causes of diarrhoea, not always attributable to disease exacerbation, but sometimes linked to bile acid malabsorption; we have a reliable serum test, C4, that allows us to recognize and treat this cause of diarrhoea efficaciously and not empirically, but it is not available or used widely. There is genetic inter-individual variability in drug responses, in terms of both efficacy and toxicity, leading to high rates of therapeutic failure. Patients treated with thiopurine or, more rarely, 5-aminosalicylic acid may suffer from unpredictable and serious adverse events, some of these with pathogenesis related to genetic variants: myelosuppression, acute pancreatitis and nephrotoxicity. The identification of pre-treatment genetic tests can optimize therapeutic choice and avoid adverse events. With regard to biological drugs, patients can experience primary non-response or loss of response due to induction of immune responses to the drugs affecting drug efficacy and determining hypersensitivity reactions. We have specifically reviewed a number of investigations, whose use is currently limited, and highlighted four tests that deserve to be more widely incorporated in clinical practice as these could improve medical decision-making and patient outcomes. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases are chronic relapsing immune-mediated diseases of the intestinal tract with multifaceted manifestations and treatment related morbidity. Faecal and blood tests, radiological, endoscopic and histologic investigations are now widely used for managing both ulcerative colitis and Crohn's disease. Over the years, a number of new investigations have been proposed but not widely adopted yet. Patients with Crohn's disease may have multiple causes of diarrhoea, not always attributable to disease exacerbation, but sometimes linked to bile acid malabsorption; we have a reliable serum test, C4, that allows us to recognize and treat this cause of diarrhoea efficaciously and not empirically, but it is not available or used widely. There is genetic inter-individual variability in drug responses, in terms of both efficacy and toxicity, leading to high rates of therapeutic failure. Patients treated with thiopurine or, more rarely, 5-aminosalicylic acid may suffer from unpredictable and serious adverse events, some of these with pathogenesis related to genetic variants: myelosuppression, acute pancreatitis and nephrotoxicity. The identification of pre-treatment genetic tests can optimize therapeutic choice and avoid adverse events. With regard to biological drugs, patients can experience primary non-response or loss of response due to induction of immune responses to the drugs affecting drug efficacy and determining hypersensitivity reactions. We have specifically reviewed a number of investigations, whose use is currently limited, and highlighted four tests that deserve to be more widely incorporated in clinical practice as these could improve medical decision-making and patient outcomes. [ABSTRACT FROM AUTHOR]

Thiopurine S -methyltransferase (TPMT) is an important enzyme involved in the deactivation of thiopurines and represents a major determinant of thiopurine-related toxicities. Despite its well-known importance in thiopurine metabolism, the understanding of its endogenous role is lacking. In the present study, we aimed to gain insight into the molecular processes involving TPMT by applying a data fusion approach to analyze whole-genome expression data. The RNA profiling was done on whole blood samples from 1017 adult male and female donors to the Estonian biobank using Illumina HTv3 arrays. Our results suggest that TPMT is closely related to genes involved in oxidoreductive processes. The in vitro experiments on different cell models confirmed that TPMT influences redox capacity of the cell by altering S -adenosylmethionine (SAM) consumption and consequently glutathione (GSH) synthesis. Furthermore, by comparing gene networks of subgroups of individuals, we identified genes, which could have a role in regulating TPMT activity. The biological relevance of identified genes and pathways will have to be further evaluated in molecular studies. [Display omitted] [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract Unlabelled Image Highlights • Selenocysteine binds to human recombinant thiopurine S -methyltransferase (TPMT) as determined by STD NMR spetroscopy. • In silico analysis positions Sec into the active site of TPMT. • S -adenosylmethionine is a methyl donor in the enzymatic reaction of selenocysteine methylation by TPMT. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A meta-analysis was conducted to decide whether to reduce an initial 6-mercaptopurine (6-MP) dose in TPMT heterozygote in the case of an initial 6-MP dose of <75 mg/m2/d and to compare the tolerable 6-MP dose among different ethnic groups. The study was undertaken according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The differences in mean values of the tolerable 6-MP dose were calculated by using Comprehensive Meta-Analysis version 3. The results of the meta-analysis indicated that the tolerable 6-MP dose was significantly lower in the TPMT heterozygote group (difference in mean values =11.729, 95% confidence interval =7.617-15.842, P<0.001) even when the initial 6-MP dose was <75 mg/m2/d. The TPMT*3C allele-dominant ethnic group (Asian) needed less reduction in mean 6-MP dose in comparison to the TPMT*3A allele-dominant ethnic group (Caucasian, Mediterranean, South American) (difference in mean values =8.884 vs 15.324). In conclusion, the initial 6-MP dose needs to be reduced in TPMT heterozygote when compared to the wild-type, and ethnic difference might influence the tolerable 6-MP dose in TPMT heterozygotes. [ABSTRACT FROM AUTHOR]

Treatment induced non-traumatic osteonecrosis (ON) has been reported increasingly in children treated for acute lymphoblastic leukemia (ALL). Several risk factors for ON have been identified in childhood cancer patients; however, their diagnostic and prognostic power is limited and the etiology of the disease remains unclear. Therefore, a continuous effort is focused on the identification of additional ON risk factors. We performed a retrospective study of 313 childhood ALL patients to test the association between the ON occurrence in children receiving ALL therapy and common polymorphisms in potential target genes: Thiopurine S-methyltransferase (TPMT; 460G>A, 719A>G), 5,10-methylenetetrahydrofolate reductase (MTHFR; 677C>T, 1298A>C), estrogen receptor alpha 1 (ESR1; XbaI) and collagen type I, α1 (COL1A1; Sp1). In the present cohort, higher age and more recently developed treatment protocols were independent risk factors for ON. In children >14.5 years old, TPMT genotype modulated the risk of ON. Additionally, in children <12.9 years old ESR1 genotypes were also implicated in the pathogenesis of ON. Besides greater age and more recent treatment protocols, genetic factors (polymorphisms in ESR1 and TPMT genes) were suggested to be implicated in the pathogenesis of ON and could be potentially used as genetic prognostic markers for ON. [ABSTRACT FROM AUTHOR]

Pharmacogenetics is a study of how genetic variation of an individual affects the drug response. We report a case of recurrent pancytopenia resulting from maintenance chemotherapy in a patient with acute promyelocytic leukemia and two pharmacogenetic mutations, namely, methylene tetrahydrofolate reductase C677T homozygous mutation and thiopurine methyltransferase mutation. [ABSTRACT FROM AUTHOR]

Objectives Thiopurine compounds are commonly used in the treatment of childhood acute lymphoblastic leukemia, and as immunosuppressants following organ transplantation or for treatment of various autoimmune disorders. Thiopurine S-methyltransferase (TPMT) is required for detoxification, through S-methylation, of 6-thioguanine nucleotides (TGNs), a byproduct of thiopurine metabolism. Single nucleotide polymorphisms (SNPs) in the TPMT gene have been shown to affect its function, with some variants associated with serious clinical manifestations including severe to fatal myelosuppression and organ transplant rejection following treatment with standard thiopurine doses. In this study, we describe a TaqMan real time PCR allelic discrimination assay requiring minimal DNA input for TPMT genotyping. Design and methods We designed controls for the homozygous wild type and allelic variants of TPMT*2, *3B, and *3C. Genomic DNA was extracted from an additional 412 human blood samples. The samples were tested for the TPMT *2, *3B, *3C, and *3A polymorphisms by TaqMan genotyping assays using the AB 7500 FAST Real-Time PCR instrument. Allelic discrimination plots were used to identify each mutation. Results The TaqMan assay correctly genotyped all custom control DNA samples. Of the 412 tested samples, our assay identified 375 samples as wild-type *1/*1 (91.02%), 3 as *1/*2 (0.73%), 1 as *1/*3B (0.24%), 3 as *1/*3C (0.73%), 27 presumed to be *1/*3A (6.55%), and 3 as *3B/*3A (0.73%). Conclusions The clinical implications of TPMT genotyping, along with the simplicity and specificity of the TaqMan genotyping assays make this test highly suitable for use in a clinical laboratory. [ABSTRACT FROM AUTHOR]

Pediatric acute lymphoblastic leukemia (ALL) is treated with combination chemotherapy including mercaptopurine (6MP) as an important component. Upon its uptake, 6MP undergoes a complex metabolism involving many enzymes and active products. The prognostic value of all the factors engaged in this pathway still remains unclear. This study attempted to determine which components of 6MP metabolism in leukemic blasts and red blood cells are important for 6MP's sensitivity and toxicity. In addition, changes in the enzymatic activities and metabolite levels during the treatment were analyzed. In a cohort (N = 236) of pediatric ALL patients enrolled in the Dutch ALL-9 protocol, we studied the enzymes inosine-5′-monophosphate dehydrogenase (IMPDH), thiopurine S-methyltransferase (TPMT), hypoxanthine guanine phosphoribosyl transferase (HGPRT), and purine nucleoside phosphorylase (PNP) as well as thioguanine nucleotides (TGN) and methylthioinosine nucleotides (meTINs). Activities of selected enzymes and levels of 6MP derivatives were measured at various time points during the course of therapy. The data obtained and the toxicity related parameters available for these patients were correlated with each other. We found several interesting relations, including high concentrations of two active forms of 6MP—TGN and meTIN—showing a trend toward association with better in vitro antileukemic effect of 6MP. High concentrations of TGN and elevated activity of HGPRT were found to be significantly associated with grade III/IV leucopenia. However, a lot of data of enzymatic activities and metabolite concentrations as well as clinical toxicity were missing, thereby limiting the number of assessed relations. Therefore, although a complex study of 6MP metabolism in ALL patients is feasible, it warrants more robust and strict data collection in order to be able to draw more reliable conclusions. [ABSTRACT FROM AUTHOR]

Azathioprine (AZA) is an important immunosuppressant drug used in heart transplantation (HTX). Consensus guidelines recommend that patients with thiopurine S-methyltransferase (TPMT) genetic variants be started on lower AZA dose because of higher active metabolite levels and risk of adverse events. However, in-vitro lymphocyte proliferation assays performed in participants with inactive TPMT alleles have suggested that AZA use may result in decreased immunosuppressant efficacy as compared with wild-type (WT) individuals. The objective of this study was therefore to determine the effect of TPMT genetic variation on AZA efficacy or prevention of rejection in HTX recipients treated with AZA.We genotyped 93 HTX recipients treated with AZA and measured erythrocyte TPMT enzyme activity. Acute rejection was monitored by routine endomyocardial biopsies.There were 83 WT and 10 heterozygote (HZ) HTX recipients. TPMT activity level was lower in HZ compared with WT (13.1±2.8 vs. 21±4.5 U/ml red blood cell, P<0.001). Despite similar AZA dose, HZ developed severe rejection earlier (P<0.001), and the total rejection score was higher (P=0.02) than WT. AZA was discontinued more frequently in HZ (P=0.01) because of rejection. The incidence of leukopenia was similar between the groups (40 vs. 43%, P=1.0).HTX recipients with TPMT genetic variant alleles who are treated with AZA develop acute rejection earlier, more frequently, and of greater severity. These patients, despite having lower TPMT enzymatic activity, should be monitored carefully for possible increased risk of acute rejection. [ABSTRACT FROM AUTHOR]

Purpose: Important drugs in the treatment of childhood acute lymphoblastic leukaemia (ALL) are 6-mercaptopurine (6-MP) and methotrexate (MTX). Thiopurine methyltransferase (TPMT) is a polymorphic enzyme causing variability in 6-MP response and toxicity. The aim of this study was to investigate the fluctuation in TPMT enzyme activity over time and the effect of high-dose MTX infusions on TPMT enzyme activity and 6-MP metabolites in paediatric ALL patients. Methods: Fifty-three children with ALL treated according to the NOPHO-ALL 2000 protocol were included in the study. TPMT enzyme activity was measured at six different times starting from diagnosis until after the end of maintenance treatment. TPMT and 6-MP metabolites were measured before the initiation of high-dose MTX (HD-MTX) infusions and at 66 h post-infusion. The interaction between MTX and TPMT was investigated in vitro using recombinant TPMT protein and a leukaemic cell line. Results: Forty percent of TPMT wild-type individuals had deceptively low TPMT enzyme activity according to genotype at the time of diagnosis. TPMT activity had decreased significantly 66 h after the start of HD-MTX infusions (−9.2 %; p = 0.013). MTX bound to recombinant TPMT protein severely inhibiting TPMT enzyme activity (remaining activity 16 %). Conclusions: Our results show that TPMT genotyping should be performed in children with ALL, since 40 % of the children in our study who carried the wild-type TPMT gene were at risk of initial underdosing of 6-MP in cases where only TPMT enzyme activity was determined. MTX inhibits the TPMT enzyme activity after HD-MTX infusions due to protein binding. [ABSTRACT FROM AUTHOR]

Thiopurine methyltransferase (TPMT) is a key component in thiopurine metabolism. There is an insufficient evidence about the distribution of the genotype frequencies of TPMT variants and frequencies of TPMT alleles associated with intermediate and deficient activity in a healthy Slovak population and pediatric patients with inflammatory bowel disease (IBD). TPMT variant alleles (*1,*2, *3A, *3B, and *3C) were determined in 114 children treated for IBD and in 281 healthy volunteers. Mutant alleles were present in 9/114 (7.89%) in the IBD patients and in 23/281 (8.19%) of probands. The distribution of the most frequent variants of TPMT gene was similar in a healthy population and patients with IBD. [ABSTRACT FROM PUBLISHER]

Background and Aims: Thiopurine S-methyltransferase (TPMT) is responsible for inactivation of thiopurine drugs which are commonly used in leukemia, organ transplantation and autoimmune diseases. The gene encoding TPMT is polymorphic, and both phenotyping and genotyping studies have shown ethnic variations in gene sequence and enzyme activity worldwide. The aim of this study is to identify the most common genetic polymorphisms of TPMT in healthy Jordanian volunteers and patients with rheumatoid arthritis (RA). Methods: A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to identify the frequency of TPMT (*2, *3A, *3B, and *3C) polymorphisms in 250 healthy Jordanian volunteers and 110 RA patients. Results: Only four healthy subjects (1.6%) and one RA patient (0.9%) with variant alleles were identified in this study. Two healthy subjects had the TPMT*3A allele and the other two had the TPMT*3B allele, whereas the one RA patient had the TPMT*3A allele. No homozygous polymorphisms were detected and all genotypes detected were heterozygous (*1/*3A) (*1/*3B). None of the subjects had TPMT*2 or TPMT*3C variant alleles. Conclusions: Mutant alleles identified in this study have a low frequency. TPMT (*3A and *3B) were the only detected heterozygous alleles. No homozygous variant allele was detected. Further studies are necessary to identify other variant alleles that might uniquely occur in Jordanians. [Copyright &y& Elsevier]

Background: Approximately 10-28 % of patients experience adverse drug reactions related to treatment with thiopurines. The most serious reaction is myelosuppression, typically manifested as leucopenia, which occurs in approximately 2-5 % of patients. Other adverse drug reactions that often accompany thiopurine therapy are pancreatitis, hepatotoxicity, allergic reactions, digestive intolerance, arthralgia, febrile conditions, and rash. Objective: The objective of this study was to assess the relationship between variant alleles of thiopurine S-methyltransferase (SNPs 238G > C, 460G > A and 719A > G), inosine triphosphate diphosphatase (SNPs 94C > A and IVS2 + 21A > C), and xanthine dehydrogenase (837C > T) and the occurrence of adverse drug reactions to azathioprine therapy. Methods: Genotype was determined for 188 Caucasians diagnosed with inflammatory bowel disease treated with a standard dose of azathioprine (1.4-2.0 mg/kg/day). Allelic variants were determined by PCR-REA and real-time PCR methods. Results were statistically evaluated by use of Fisher's test and by odds ratio calculation. Results: Variant genotype thiopurine S-methyltransferase predisposes to development of leucopenia ( P = 0.003, OR = 5, CI 95 %, 1.8058-13.8444). Although not statistically significant, we observed a trend that suggested correlation between the occurrence of digestive intolerance and the variant genotype inosine triphosphate diphosphatase ( P = 0.1102; OR 15.63, CI 95 %, 1.162-210.1094), and between the occurrence of pancreatitis and the variant allele xanthine dehydrogenase 837T ( P = 0.1124; OR 12,1, CI 95 %, 1.15-126.37). Conclusion: The variant genotype thiopurine S-methyltransferase has been associated with the occurrence of leucopenia. The involvement of polymorphisms in inosine triphosphate diphosphatase and xanthine dehydrogenase genes in the development of digestive intolerance and pancreatitis will require further verification. [ABSTRACT FROM AUTHOR]

Acute leukaemia (AL) has been observed in association with Crohn's disease (CD) notably in patients treated with azathioprine (AZA), which is an immunosuppressant known for its carcinogenicity and in particular known to induce therapy-related acute myeloid leukaemia according to the 2008 WHO classification. Whereas the link between inflammatory bowel disease and AL has been well established, the exact role of AZA remains controversial. In this paper, we report the case of a 71-year-old white Caucasian male with CD treated for 7 years with AZA who developed an acute leukaemia. Chemotherapy was administered unsuccessfully and the patient died from this haematological disorder 9 months after diagnosis. We reviewed the current evidence on the interactions between CD, AL and AZA as well as the potential underlying mechanisms of leukaemia in AZA-treated patients. From this review, we concluded that AL should be questioned when facing cytopenia in a patient with CD. The nature of the association between AZA and AL in CD patients warrants further investigation. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) plays a pivotal role in thiopurine treatment outcomes. However, little has been known about its intracellular regulation. Here, we describe the effect of fluctuations in physiological levels of S-adenosyl-l-methionine (SAM) and related metabolites on TPMT activity levels in cell lines and erythrocytes from healthy donors. We determined higher TPMT activity in wild-type TPMT*1/*1 individuals with high SAM concentrations (n =96) compared to the low SAM level group (n =19; P <0.001). These findings confirm the results of our in vitro studies, which demonstrated that the restriction of l-methionine (Met) in cell growth media reversibly decreased TPMT activity and protein levels. Selective inhibition of distinct components of Met metabolism was used to demonstrate that SAM is implicitly responsible for direct post-translational TPMT stabilization. The greatest effect of SAM-mediated TPMT stabilization was observed in the case of wild-type TPMT*1 and variant *3C allozymes. In addition to TPMT genotyping, SAM may serve as an important biochemical marker in individualization of thiopurine therapy. [Copyright &y& Elsevier]

Abstract: Objectives: Low thiopurine S-methyltransferase (TPMT) enzyme activity is associated with increased thiopurine drug toxicity, particularly myelotoxicity. Pre-analytic and analytic variables for TPMT genotype and phenotype (enzyme activity) testing were reviewed. Design and methods: A systematic literature review was performed, and diagnostic laboratories were surveyed. Results: Thirty-five studies reported relevant data for pre-analytic variables (patient age, gender, race, hematocrit, co-morbidity, co-administered drugs and specimen stability) and thirty-three for analytic variables (accuracy, reproducibility). TPMT is stable in blood when stored for up to 7days at room temperature, and 3months at −30°C. Pre-analytic patient variables do not affect TPMT activity. Fifteen drugs studied to date exerted no clinically significant effects in vivo. Enzymatic assay is the preferred technique. Radiochemical and HPLC techniques had intra- and inter-assay coefficients of variation (CVs) below 10%. Conclusion: TPMT is a stable enzyme, and its assay is not affected by age, gender, race or co-morbidity. [Copyright &y& Elsevier]

Abstract: The prevalence of thiopurine S-methyltransferase (TPMT) polymorphism and its association with clinical and hematological toxicities was retrospectively analyzed in 71 Indian children with acute lymphoblastic leukemia (ALL). Only heterozygous TPMT alleles were observed (10%, 7/71) with relative frequencies being *1/*3C (4.2%), *1/*2 (4.2%) and *1/*3A (1.4%). The median 6-mercaptopurine dose administered during the maintenance therapy was 31% lower among patients with heterozygous TPMT alleles versus the rest (32.1mg/m2/day and 46.2mg/m2/day, p =0.005), though the myelosuppression and toxicities were similar in both the groups. Identification of TPMT genotype appears to be important in making the ALL treatment more effective and less toxic. [Copyright &y& Elsevier]

Abstract: A novel human thiopurine S-methyltransferase (TPMT) variant allele, (319 T>G, 107Tyr>Asp, *27), was identified in a Thai renal transplantation recipient with reduced erythrocyte TPMT activity. The TPMT*27 variant allozyme showed a striking decrease in both immunoreactive protein level and enzyme activity after transient expression in a mammalian cell line. We set out to explore the mechanism(s) responsible for decreased expression of this novel variant of an important drug-metabolizing enzyme. We observed accelerated degradation of TPMT*27 protein in a rabbit reticulocyte lysate. TPMT*27 degradation was slowed by proteasome inhibition and involved chaperone proteins—similar to observations with regard to the degradation of the common TPMT*3A variant allozyme. TPMT*27 aggresome formation was also observed in transfected mammalian cells after proteasome inhibition. Inhibition of autophagy also decreased TPMT*27 degradation. Finally, structural analysis and molecular dynamics simulation indicated that TPMT*27 was less stable than was the wild type TPMT allozyme. In summary, TPMT*27 serves to illustrate the potential importance of protein degradation – both proteasome and autophagy-mediated degradation – for the pharmacogenetic effects of nonsynonymous SNPs. [Copyright &y& Elsevier]

Thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) plays a key role in the metabolism of thioprine drugs. Subjects with intermediate or no TPMT activity are at risk of azathioprines toxicity treated with conventional dosages of thiopurine drugs. While TPMT polymorphisms have been extensively studied in many countries, there is insufficient data in Iranian populations. In the present study, we aimed to identify the common functional TPMT alleles in southeast Iranian population. The TPMT allele frequencies were determined by multiplexed allele-specific polymerase chain reaction. Among 832 samples of Iranian population, the frequency for the TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C, were 2.16%, 1.68%, 1.62%, and 0.54%, respectively. The distribution of the TPMT genotypes were 87.98% for TPMT*1/*1, 4.33% for TPMT*1/*2, 3.36% for TPMT*1/*3A, 3.24% for TPMT*1/*3B, and 1.08% for TPMT*1/*3C. This functional analysis of common TPMT alleles in an Iranian population could provide useful information for thioprine drugs therapy. [ABSTRACT FROM AUTHOR]

Thiopurine S-methyltransferase (TPMT) is the rate-limiting step in the conversion of thiopurine drugs including azathioprine (AZA) to inactive metabolites. Heritable deficiency of TPMT activity increases risk for adverse events, most notably, myelosuppression leading to leukopenia and neutropenic sepsis. The reported European Commission study was undertaken to identify current evidence for the clinical utility of testing for TPMT status and extent of uptake, by either genotyping or phenotyping, in the clinical setting. Data presented here for the UK and Spain indicate that there has been a considerable increase in the uptake of TPMT testing in recent years. There are some data that support routine TPMT testing before AZA prescribing for reducing AZA-related adverse events. Key data include evidence in favor of TPMT testing in addition to the current practice of routine monitoring for reducing the number of AZA-related episodes of myelosuppression, averting deaths from neutropenic sepsis and improving health-related quality of life. Further data are needed for determining the cost-effectiveness of routine TPMT testing.European Journal of Human Genetics (2009) 17, 991–998; doi:10.1038/ejhg.2009.10; published online 18 February 2009 [ABSTRACT FROM AUTHOR]

Background and Aims: Azathioprine (AZA) is associated with a high frequency of adverse reactions. We examined polymorphism of the thiopurine S-methyltransferase (TPMT) gene to determine whether the TPMT genotype would be a predictive marker for the development of adverse reactions to AZA. Methods: The frequency of TPMT mutations was investigated in 147 Japanese inflammatory bowel disease (IBD) patients retrospectively. In these subjects, the presence of four mutant alleles (TPMT*2, *3B, *3C and *8) was determined by direct sequencing. The incidence of adverse reactions among patients carrying wild-type TPMT was investigated. The blood level of 6-thioguanine nucleotide (6-TGN) was measured and analyzed in 47 patients with wild-type TPMT. The results were analyzed in relation to the concomitant use of aminosalicylates (ASA). Results: Of the 147 patients, 144 (98.0%) were wild-type for TPMT (TPMT*1/*1) and three (2.0%) carried a mutant TPMT allele (TPMT*1/*3C). The incidence of adverse reactions was 33.3% (38/114) in the wild-type group. Leukopenia (WBC ≤ 3000/µL) was seen in 15.8% of the patients with wild-type TPMT. 6-TGN levels varied among 47 patients with wild-type TPMT. The blood levels of 6-TGN were significantly higher in the patients receiving concomitant ASA treatment compared with those not receiving concomitant ASA treatment ( P = 0.0033). Conclusion: The frequency of TPMT gene mutations is low among Japanese IBD patients. The incidence of adverse reactions to AZA was high, even in patients carrying wild-type TPMT. It is concluded that determination of TPMT genotype may not be useful in Japanese IBD patients to predict adverse reactions to AZA. [ABSTRACT FROM AUTHOR]

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • More than 27 mutations in the TPMT gene causing decreased TPMT activity have been reported in various races. • Decreased TPMT activity is associated with increased myelosuppression from azathioprine or thioguanine treatment. • TPMT*3C is the most common genetic variant found in Asian populations. WHAT THIS STUDY ADDS • TPMT*26 (208F→L) is a novel mutation, previously not reported, which is associated with reduced TPMT activity in three siblings in an Asian Chinese family. • This mutation is associated with increased sensitivity to azathioprine clinically. AIMS Azathioprine, mercaptopurine and thioguanine are commonly used to treat autoimmune disorders, leukaemia and solid organ transplantation. However, azathiopurine and its metabolites can also cause adverse reactions such as myelosuppression. These manifestations may be attributed to polymorphisms or mutations in the thiopurine methyltransferase (TPMT) gene that might result in low TPMT enzyme activity. Our aim was to investigate if azathioprine-related myelosuppression is associated with TPMT polymorphism, which in turn affects its enzyme activity. METHODS A 61-year-old Chinese man with severe atopic eczema developed moderate myelosuppression with standard doses of azathioprine. His TPMT activity was measured using radiochemical assay. Genotyping of TPMT *3C, *3A and *6 were screened using polymerase chain reaction-restriction fragment length polymorphism. Novel mutation was detected by sequencing. Family studies of his three other siblings were performed. RESULTS After 4 weeks of azathioprine treatment, the patient's white blood cells and absolute neutrophil count dropped by 40–45%. He was then taken off azathioprine, and blood counts returned to normal. TPMT activity test showed intermediate levels of 9.1 nmol h−1 ml−1 peripheral red blood cells (pRBC). Resequencing of the TPMT gene revealed a missense mutation Phe→Leu at 208 aa position in exon 9 (ss105107120). Two of his three siblings were heterozygous for 208F→L, which accounts for the decreased enzyme activity (brother 8.9 nmol h−1 ml−1 pRBC, sister 8.8 nmol h−1 ml−1 pRBC). The remaining sibling had wild-type allele with normal enzyme activity. Screening of 100 normal healthy Chinese subjects did not reveal any individual with this mutation. CONCLUSION We report a novel mutation TPMT*26 (208F→L) associated with a decrease in TPMT enzyme activity. [ABSTRACT FROM AUTHOR]

The main cause of azathioprine (AZA)/6-mercaptopurine (6MP)-induced adverse reactions is a reduction in the activities of the metabolizing enzymes thiopurine S-methyltransferase (TPMT) and inosine triphosphate pyrophosphohydrolase (ITPA). Adverse reactions develop at a high frequency in Japanese patients at half the dose required for European and American patients; however, the association with TPMT and ITPA gene polymorphisms in Japanese has not been fully investigated. Gene mutations of TPMT and ITPA, the major AZA/6-MP -metabolizing enzymes, were investigated retrospectively in 16 Japanese patients with inflammatory bowel disease (IBD) in whom AZA/6MP treatment induced adverse reactions. The TPMT gene was found to have a wild-type sequence in all patients, but in the ITPA gene a mutation, 94C>A, was detected at a rate of 50% (8/16), with 83.3% (5/6) occurring in patients with acute bone marrow suppression and 75% (3/4) in those with agranulocytosis. The 94C>A allele frequency was 10 of 32 (0.313; 95% CI, 0.180–0.486). Adverse reactions developed earlier in patients with the 94C>A mutation. However, in half the patients, no gene polymorphism was noted. It is suggested that the ITPA gene mutation is closely related to the adverse reactions of AZA/6-MP in Japanese patients, and screening for the mutant allele is useful for predicting the most serious adverse reactions, agranulocytosis and acute bone marrow suppression. [ABSTRACT FROM AUTHOR]

Thiopurine S-methyltransferase (TPMT)∗3A is degraded much more rapidly than is the ‘wild-type’ enzyme through a ubiquitin-proteasome-dependent process. It also forms aggresomes, suggesting a possible dynamic balance between degradation and aggregation. We set out to identify genes encoding proteins participating in these processes.Green fluorescent protein tagged TPMT∗3A was expressed in a Saccharomyces cerevisiae gene deletion library, and flow cytometry was used to screen for cells with high fluorescence intensity, indicating the loss of a gene essential for TPMT∗3A degradation.Twenty-four yeast genes were identified in functional categories that included ubiquitin-dependent protein degradation, vesicle trafficking, and vacuolar degradation. The presence of genes encoding proteins involved in vesicular transport and vacuolar degradation suggested a possible role in TPMT∗3A degradation for autophagy - a process not previously identified as a pharmacogenomic mechanism. In support of that hypothesis, TPMT∗3A aggregates increased dramatically in mutants for vacuolar protease and autophagy-related genes. Furthermore, TPMT∗3A expression in human cells induced autophagy, and small interfering RNA-mediated knockdown of ATG7, an autophagy-related human protein, enhanced TPMT∗3A aggregation but not that of TPMT∗3C or wild-type TPMT, indicating that autophagy contributes to TPMT∗3A degradation in mammalian cells. We also demonstrated that UBE2G2, the human homologue of the E2 ubiquitin-conjugating enzyme identified during the yeast genetic screen, was involved in TPMT∗3A degradation in human cells.These results indicate that autophagy should be considered among mechanisms responsible for the effects of pharmacogenetically significant polymorphisms that alter encoded amino acids. [ABSTRACT FROM AUTHOR]

Thiopurine S-methyltransferase (TPMT) is an enzyme responsible for the detoxification of the widely used thiopurine drugs. TPMT is genetically polymorphic and is associated with large interindividual variations in thiopurine drug toxicity and therapeutic efficacy. In this study, we performed an in-vitro analysis of TPMT variant alleles, namely, TPMT∗2, ∗3A, ∗3B, ∗3C, ∗5, ∗6, ∗7, ∗8, ∗9, ∗10, ∗11, ∗12, ∗13, ∗14, ∗16, ∗17, ∗18, ∗19, ∗20, ∗21, ∗22, ∗23, and ∗24.The wild-type TPMT proteins, TPMT.1 and 23 variants were heterologously expressed in COS-7 cells, and the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of 6-thioguanine S-methylation were determined.The expression levels of TPMT.2, TPMT.3A, TPMT.5, TPMT.12, TPMT.14, and TPMT.22 were considerably lower than that of TPMT.1 (P<0.005), and that of TPMT.18 was slightly reduced (P<0.05). The kinetic parameters of TPMT.3A, TPMT.3B, TPMT.5, TPMT.14, TPMT.18, TPMT.21, and TPMT.22 could not be accurately established because of no activity in 6-thioguanine S-methylation. The Vmax/Km values of TPMT.2, TPMT.7, TPMT.17, and TPMT.24 were displayed less than 10% of the wild-type.This functional analysis with respect to TPMT variants could provide useful information for individualization of thiopurine drugs therapy. [ABSTRACT FROM AUTHOR]

Abstract: Thiopurine S-methyltransferase (TPMT) metabolizes cytotoxic thiopurine drugs used in the treatment of leukemia and inflammatory bowel disease. TPMT is a major pharmacogenomic target with 23 alleles identified to date. Several of these alleles cause rapid protein degradation and/or aggregation, making it extremely difficult to study the structural impact of the TPMT polymorphisms experimentally. We, therefore, have performed multiple molecular dynamics simulations of the four most common alleles [TPMT*2 (A80P), *3A (A154T/Y240C), *3B (A154T) and *3C (Y240C)] to investigate the molecular mechanism of TPMT inactivation at an atomic level. The A80P polymorphism in TPMT*2 disrupts helix α3 bordering the active site, which breaks several salt-bridge interactions and opens up a large cleft in the protein. The A154T polymorphism is located within the co-substrate binding site. The larger threonine alters the packing of substrate-binding residues (P68, L69, Y166), increasing the solvent exposure of the polymorphic site. This packing rearrangement may account for the complete lack of activity in the A154T mutant. The Y240C polymorphism is located in β-strand 9, distant from the active site. Side-chain contacts between residue 240 and helix α8 are lost in TPMT*3C. Residues 154 and 240 in TPMT*3A are connected through a hydrogen-bonding network. The dual polymorphisms result in a flattened, slightly distorted protein structure and an increase in the thiopurine-binding site solvent accessibility. The two variants that undergo the most rapid degradation in vivo, TPMT*2 and *3A, are also the most deformed in the simulations. [Copyright &y& Elsevier]

Introduction: 6-MP is a drug used at each stage of acute lymphoblastic leukemia ALL treatment in children. Polymorphism of TPMT gene encoding thiopurine S-methyltransferase has been associated with 6-MP therapy outcome. The study was aimed at determination of TPMT allele, i.e. TPMT*2 (G238C), TPMT*3A (G460A+A719G) and TPMT*3C (A719G) distribution in Polish ALL children, and their association with 6-MP side effects. The study group consisted of 132 children (82 boys and 50 girls aged 17-346 months) diagnosed with ALL, and 397 healthy subjects serving as controls. The TPMT polymorphism was assessed by PCR-RFLP and PCR-ASA techniques. Results: The TPMT*3A allele was found in 10 ALL patients (9 - TPMT*1/*3A and 1 - TPMT*3A/*3A). The TPMT*2 (G238C), TPMT*3B (G460A) and TPMT*3C (A719G) alleles were not found in the analyzed group. The frequency of TPMT*3A (G460A+A719G) allele was 4.2% (11/264 alleles), while the TPMT*1 wild-type allele was found with the frequency of 95.8% (253/264 alleles). No significant differences in the genotype and allele frequency were observed between the ALL and control groups. The 6-MP dose reduction rate during maintenance therapy was observed more frequently in ALL carriers of TPMT*3A allele (p=0.047; DTF =0.07). Moreover, the dose of 6-MP was reduced significantly more frequently (approximately 4-fold) due to leucopenia accompanied by respiratory tract infection (p=0.01; DTF=0.02). Conclusion: The results indicate that TPMT genotype influenced treatment outcome in ALL patients, and thus may be useful in optimizing 6-MP chemotherapy. [ABSTRACT FROM AUTHOR]

Azathioprine and 6-mercaptopurine have been used for many years in the treatment of inflammatory bowel disease. Approximately 0.3% of the population are homozygous for variant alleles associated with extremely low thiopurine S-methyltransferase enzyme activity. We describe the case of a young patient with ulcerative colitis, homozygous for TPMT*3A alleles, who suffered fatal azathioprine-induced myelotoxicity after standard dosing with azathioprine. Screening for decreased activity of TPMT in patients prior to azathioprine treatment is advised to minimize the risk of drug-induced toxicity. [ABSTRACT FROM AUTHOR]

Genetic variation in thiopurine S-methyltransferase (TPMT) is a major factors for wide variation in the metabolism and safety of thiopurine drugs. We investigated the frequency of functional gene polymorphisms in 396 patients with inflammatory bowel disease and 300 healthy subjects. Frequencies of functionally deficient alleles TPMT*2, TPMT*3A, TPMT*3B, and TPMT*3B in the patient group were 0.1%, 4.3%, 0.1%, and 0.4%, respectively, and were similar to those of healthy subjects in the Czech population. Our results provide necessary information for pharmacoeconomic studies in the Czech Republic. [ABSTRACT FROM AUTHOR]

Background and objective:  The activity of thiopurine S-methyltransferase (TPMT), a key enzyme in the metabolism of purine analogues, displays wide inter-subject variability partly due to a genetic polymorphism. Previous studies have suggested adjusting purine analogues dosing according to TPMT activity but measurements are costly and time-consuming. It is still unclear, especially under treatment, whether the simpler TPMT genotyping reliably predicts enzyme activity. Our aim was to study the possible correlation of TPMT genotype with phenotype. Methods:  We determined the genotypic status and TMPT activity, at diagnosis and after 6 months of maintenance therapy, of 118 children with acute lymphoblastic leukaemia (ALL). Results and Discussion:  Eighty-nine per cent of the children had a homozygous wild-type genotype (group 1), 11% had one or two mutant allele(s) (group 2). At both time points, TPMT activity (U/mL peripheral red blood cell) was significantly higher in group 1 than in group 2 ( P < 0·001) but inter-group levels overlapped considerably. There was considerable heterogeneity in the percentage increase in TPMT activity after therapy, and little correlation between metabolites ratio [6-methylmercaptopurine derivative/6-thioguanine nucleotides (6-TGN)] and TPMT activity at the end of 6 months’ maintenance treatment. These results show that TPMT activity cannot be used as an accurate tool for 6-mercaptopurine monitoring. Conclusion:  Genotyping at diagnosis identifies patients with a homozygous mutant TPMT and may prevent severe and life-threatening toxicity. ALL treatment monitoring should preferentially be based on repeated determinations of intracellular active metabolites (6-TGN) and methylated metabolites. [ABSTRACT FROM AUTHOR]

Genetic data on the thiopurine S-methyltransferase (TPMT) polymorphism were obtained in population samples from Cabinda and Mozambique (located in the western and eastern coasts of sub-Saharan Africa, respectively). The overall frequency of TPMT-deficient alleles was 5.6% in Mozambique and 6.3% in Cabinda. Accordingly, one out of the 103 individuals from Cabinda tested had a genotype associated with TPMT deficiency, yielding a frequency that is threefold higher than heretofore reported in any population. In addition, in both Cabinda or Mozambique, TPMT*8 accounted for a significant proportion of non-functional alleles (nearly 40% in Cabinda). Since the substitution defining TPMT*8 seems to be highly specific of sub-Saharan Africa populations and given the fact it has not been integrated into the set of single nucleotide polymorphisms routinely tested for TPMT, a re-design of molecular screenings should be considered in the future in order to avoid serious underestimates of TPMT deficiency when the enzymatic profiles in populations are unknown. [ABSTRACT FROM AUTHOR]

Abstract: Background: Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzed the S-methylation of thiopurine drugs. TPMT activity exhibits an interindividual variability, mainly as a result of genetic polymorphism. Patients with intermediate or deficient TPMT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. We determined a cut-off concentration of the TPMT activity assay less than which genotyping of the TPMT gene should be performed. In addition, the influence of hemodialysis on TPMT activity in uremic patients was examined. Methods: In 248 healthy subjects and 30 uremic patients, PCR-based methods were used to analyze the most common functional mutations TPMT⁎2, ⁎3A, ⁎3B and ⁎3C. A HPLC assay was used to measure erythrocyte TPMT activity in the whole population. Results: Seven TPMT⁎3C heterozygotes were identified, while TPMT⁎2, ⁎3A and ⁎3B alleles were not detected in 248 healthy subjects. The frequency of TPMT⁎3C allele was 1.4% (7/496). The TPMT activity in healthy subjects was normally distributed, ranged from 6.09 to 28.65 nmol/h/ml pRBC with a mean of 16.03±4.16 nmol/h/ml pRBC. The cut-off for high TPMT activity and intermediate TPMT activity was 10.07 nmol/h/ml pRBC. There were 19 intermediate activity healthy subjects (7.7%) and 229 high activity healthy subjects (92.3%), and no TPMT deficiency subject was found. All of the 229 healthy subjects with high activity had no mutant alleles, while 7 of the 19 subjects with intermediate activity had a mutant allele. Phenotypes were in good agreement with genotypes for 95% of subjects. The uremic patients were all homozygous for the wild-type allele whose TPMT activity was activated significantly before hemodialysis compared with TPMT activity after hemodialysis. Conclusions: We defined the cut-off values for the TPMT phenotyping assay at 10.07 nmol/h/ml pRBC, less than which additional genotyping elucidates the individual risk for drug therapy. In uremic patients, TPMT activity is increased by some uremic factors, and dialysis shifted their TPMT activity close to that of a healthy control group. [Copyright &y& Elsevier]

Background and aim: Thiopurine S-methyltransferase (TPMT) genotypes or phenotypes may be a predictive factor for azathioprine-induced toxicities. We investigated the genotypic status of TPMT to evaluate the risk of azathioprine-related adverse effects in Japanese patients with different liver diseases, including autoimmune hepatitis (AIH). Methods: 49 patients with AIH, 67 with primary biliary cirrhosis (PBC), and 120 with hepatitis C virus (HCV) were examined. TPMT genotypes were determined by PCR-restriction fragment length polymorphism-based assays. Results: The distribution of TPMT genotypes was 90% TPMT*1/TPMT*1, 8% TPMT*1/TPMT*3C, and 2% TPMT*3C/TPMT*3C in AIH, and 94% TPMT*1/TPMT*1, 4.5% TPMT*1/TPMT*3C, and 1.5% TPMT*3C/TPMT*3C in PBC. All except 1 patient with HCV had the TPMT*1/TPMT*1 genotype. Severe myelosuppression occurred in two of nine patients with AIH who received azathioprine, one of whom was homozygous for TPMT*3C. Conclusions: TPMT*3C variants are more frequent in patients with AIH or PBC than in patients with viral hepatitis or healthy volunteers in Japan. Pharmacogenetic screening for TPMT polymorphisms before commencing azathioprine therapy may help to prevent severe hematotoxicity in patients with TPMT deficiency. [ABSTRACT FROM AUTHOR]

Thiopurine drugs are metabolized, in part, by S-methylation catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low or undetectable TPMT activity are at high risk of severe, potentially fatal hematopoietic toxicity when they are treated with standard doses of thiopurines. As human TPMT activity is controlled by a common genetic polymorphism, it is an excellent candidate for the clinical application of pharmacogenetics. Here, we report a new molecular approach developed to detect point mutations in the TPMT gene that cause the loss of TPMT activity. A fluorescently labeled amplified DNA is hybridized with oligonucleotide DNA probes immobilized in gel pads on a biochip. The specially designed TPMT biochip can recognize six point mutations in the TPMT gene and seven corresponding alleles associated with TPMT deficiency: TPMT*2; TPMT*3A, TPMT*3B, TPMT*3C, TPMT*3D, TPMT*7, and TPMT*8. The effectiveness of the protocol was tested by genotyping 58 samples of known genotype. The results showed 100% concordance between the biochip-based approach and the established PCR protocol. The genotyping procedure is fast, reliable and can be used for rapid screening of inactivating mutations in the TPMT gene. The study also provides the first data on the frequency of common TPMT variant alleles in the Russian population, based on a biochip analysis of 700 samples. TPMT gene mutations were identified in 44 subjects; genotype *1/*3A was most frequent. [ABSTRACT FROM AUTHOR]

The toxicity of thiopurine drugs has been correlated to the activity of thiopurine S-methyltransferase (TPMT), whose interindividual variation is a consequence of genetic polymorphisms. We have herein investigated the relevance of some genetic markers for the prediction of thiopurine-related toxicities and to determine the genotype to phenotype correlation in the Slovenian population. The most prevalent mutant allele in the Slovenian population is TPMT*3A (4.1%), followed by TPMT*3C (0.5) and TPMT*3B (0.3), while the TPMT*2 allele was not found in any of the examined samples. TPMT enzyme activity distribution in the subgroup sample was bimodal and as such correlated with genetic data. Using a cutoff value of 9.82 pmol/107 RBC per h, the genetic data correctly predicted TPMT enzyme activity in 91.6% of the examined individuals. Pharmacogenetic TPMT analyses have therefore proved to have significant clinical implications for prediction of individuals’ responses to treatment with thiopurine drugs in order to avoid possible life-threatening therapy-related toxicities. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Thiopurine S-methyltransferase (TPMT) is an enzyme involved in the normal metabolic inactivation of thiopurine drugs. Patients with intermediate or no TPMT activity are at risk of toxicity after receiving standard doses of thiopurine drugs and it was shown that inter-individual differences in response to these drugs is largely determined by genetic variation at the TPMT locus. Objective: This study was designed to investigate in the Sardinian population the frequency distribution of four of the most common variants accounting for TPMT deficiency and to conduct comparative analyses with other populations in order to obtain insights into the main factors that have shaped diversity at the TPMT locus in Sardinia. Methods: DNA was extracted in 259 Sardinians and the frequencies of allelic variants of TPMT were determined using polymerase chain reaction-restriction fragment length polymorphism technique. Results: Among the 259 Sardinians genotyped, 6·95% were found to be heterozygous for one of four TPMT variants screened; for each variant the frequency estimate was 1·74%, 0·58%, 0·39% and 0·77% for TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C respectively. Conclusions: Although Sardinia does not show reduced diversity at the TPMT locus, the spectrum of TPMT allele frequencies affords evidence of remarkable influence of genetic drift and founder effects throughout its population history. In the broad context of the European TPMT diversity, the Sardinians come out as outliers, an observation consistent with previous genetic inferences that Sardinia has features of a genetic isolate. [ABSTRACT FROM AUTHOR]

Abstract: Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) is the key enzyme in the metabolism of thiopurine drugs. Determination of TPMT activity has been used for the individualization of thiopurine dose. We developed HPLC-UV assay for the determination of TPMT activity in human erythrocytes using 6-mercaptopurine as a substrate. Various extraction and chromatographic conditions were compared. In-house developed extraction with acetonitrile provided the lowest limit of quantification. TPMT activity was determined in 99 previously genotyped healthy Estonians. TPMT activity was expressed as the formation of 6-methylmercaptopurine ng/ml/h and normalized either to haemoglobin, haematocrit, erythrocyte count or protein content. The receiver-operating characteristic curve analysis revealed similar accuracy values for TPMT activity in predicting heterozygous and wild type individuals for each method of calculation. In healthy Estonians, TPMT activity varied from 21.5 to 129.6ng/ml/h. For heterozygous individuals (n =18), TPMT activity was 48.1±11.7ng/ml/h. Wild type individuals (n =81) revealed significantly higher TPMT activity 79.3±20.7ng/ml/h (P <0.001). This sensitive HPLC assay for quantitative determination of TPMT activity could easily be used in clinical settings. Under constant experimental conditions for haemolysate preparation no normalization is required. [Copyright &y& Elsevier]

Abstract: Background: Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. Several mutations in the TPMT gene have been identified which correlate with a low activity phenotype. The molecular basis for TPMT deficiency is not well defined in minority Chinese. We investigated differences in the activity of TPMT and the frequencies of mutant TPMT alleles in 4 ethnic groups of Chinese. Method: The frequency of 4 common TPMT mutant alleles, TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C, were determined in healthy subjects from Han (n =312), Jing (n =103), Yao (n =126) and Uygur Chinese (n =160) by allele-specific PCR and PCR-restriction RFLP analysis. TPMT activity in erythrocytes was determined by HPLC. Results: There was no significant difference in the mean TPMT activity between all ethnic groups studied and no subject with TPMT deficiency was found in all populations studied. TPMT*3C was found in 2.2% of Han and 1.9% of Jing Chinese. TPMT*2, TPMT*3B and TPMT*3A alleles were not detected in any of the Han or Jing Chinese tested. In contrast, 3.7% of Uygur Chinese had TPMT*3C and TPMT*3A alleles. Neither allele was detected in Yao Chinese. The overall frequencies of variant TPMT allele in Uygur were higher than in Han or Jing Chinese. However, neither the overall frequency of mutant TPMT alleles nor the genotype frequencies were significantly different between Han, Jing, Yao and Uygur Chinese. Conclusions: The TPMT*3C was the most prevalent allele in Han, Jing and Uygur Chinese, while TPMT*3A is a rare allele in Uygur Chinese who belong to Caucasian. Ethnicity may be an important factor affecting the variability in response to thiopurine chemotherapy. [Copyright &y& Elsevier]

Background: Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme involved in the metabolism of these thiopurine drugs. Methylation of thiopurine drugs by TPMT competes with the formation of their active 6-thioguanine nucleotide metabolite, thereby potentially modulating the therapeutic and toxic effects of these drugs. Objective: To analyze the thiopurine S-methyltransferase allelic frequencies in Taiwan aborigines and Taiwanese. Methods: We used polymerase chain reaction–restriction fragment length polymorphism method to determine the allelic frequencies of TPMT variants (TPMT*1–TPMT*8) in 409 Taiwan aborigines and 117 Taiwanese. Results and discussion: The results showed that the allelic frequencies of TPMT*1 were 99·88% and 98·72% for Taiwan aborigines and Taiwanese respectively. The allelic frequencies of TPMT*3C were 0·12% and 1·28% for Taiwan aborigines and Taiwanese respectively. No TPMT*2, 3A, 3B, 3D and 4–8 were found in these populations. Conclusion: Our results provide useful information for using thiopurine drugs in these populations. [ABSTRACT FROM AUTHOR]

Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. Genetic polymorphisms for TPMT are a major factor responsible for large individual variations in thiopurine toxicity and therapeutic effect. The present study investigated the functional effects of human TPMT variant alleles that alter the encoded amino acid sequence of the enzyme, TPMT∗2, ∗3A, ∗3B, ∗3C and ∗5 to ∗13. After expression in COS-1 cells and correction for transfection efficiency, allozymes encoded by these alleles displayed levels of activity that varied from virtually undetectable (∗3A,∗3B and ∗5) to 98% (∗7) of that observed for the wild-type allele. Although some allozymes had significant elevations in apparent Km values for 6-mercaptopurine and S-adenosyl-L-methionine (i.e. the two cosubstrates for the reaction), the level of enzyme protein was the major factor responsible for variation in activity. Quantitative Western blot analysis demonstrated that the level of enzyme protein correlated closely with level of activity for all allozymes except TPMT∗5. Furthermore, protein levels correlated with rates of TPMT degradation. TPMT amino acid sequences were then determined for 16 non-human mammalian species and those sequences (plus seven reported previously, including two nonmammalian vertebrate species) were used to determine amino acid sequence conservation. Most human TPMT variant allozymes had alterations of residues that were highly conserved during vertebrate evolution. Finally, a human TPMT homology structural model was created on the basis of a Pseudomonas structure (the only TPMT structure solved to this time), and the model was used to infer the functional consequences of variant allozyme amino acid sequence alterations. These studies indicate that a common mechanism responsible for alterations in the activity of variant TPMT allozymes involves alteration in the level of enzyme protein due, at least in part, to accelerated degradation. [ABSTRACT FROM AUTHOR]

Background: Thiopurine drugs are used as immunosuppressant or cytotoxic drugs. Thiopurine S-methyltransferase (TPMT) methylates and thereby modulates the therapeutic and toxic effects of these drugs. The activity of TPMT is affected by genetic polymorphism of TPMT alleles, and these alleles have not been studied in Tibetans and Bolivians. Objectives: To analyse the TPMT allelic frequencies in Tibetans and Bolivians. Methods: We developed an inexpensive method for collecting blood and extracting genomic DNA. Genomic DNA was extracted from blood spots of 50 Tibetans and 115 Bolivians. The frequencies of allelic variants of TPMT gene ( TPMT*1 to TPMT*8) were determined using polymerase chain reaction-restriction fragment length polymorphism technique. Results: The allelic frequencies of TPMT*1 were 99 and 93·48% for Tibetans and Bolivians, respectively. The corresponding allelic frequencies of TPMT*3A were 0 and 6·52% and those of TPMT*3C were 1·0 and 0%. No TPMT*2, 3B, 3D, 4–8 were found in these two populations. Conclusions: As with Caucasian populations, TPMT*3A is the most prevalent mutant allele in Bolivians. Our results may be of value in helping to guide the prescription of thiopurine drugs in these populations. [ABSTRACT FROM AUTHOR]

Ethnicity is an important variable influencing drug response. Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurine drugs. Previous population studies have identified ethnic variations in both phenotype and genotype of TPMT, but limited information is available within Chinese population that comprises at least 56 ethnic groups. The current study was conducted to compare both phenotype and genotype of TPMT in healthy Han and Yao Chinese children. TPMT activity was measured in healthy Chinese children by a HPLC assay ( n = 213, 87 Han Chinese and 126 Yao Chinese). Allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) were used to determine the frequency of TPMT mutant alleles ( TPMT* 2, TPMT* 3 A, TPMT* 3B and TPMT* 3C) in these children. There was no significant difference in the mean TPMT activity between Han and Yao Chinese children. A unimodal distribution of TPMT activity in Chinese children was found and the mean TPMT activity was 13.32 ± 3.49 U ml−1 RBC. TPMT activity was not found to differ with gender, but tended to increase with age in Yao Chinese children. TPMT* 2, TPMT* 3B and TPMT* 3A were not detected, and only one TPMT* 3C heterozygote (Han child) was identified in 213 Chinese children. Erythrocyte TPMT activity of this TPMT* 3C heterozygote was 12.36 U ml−1 RBC. The frequency of the known mutant TPMT alleles was 0.2%[1/426] in Chinese children. The frequency distribution of RBC TPMT activity was unimodal. The frequency of the known mutant TPMT alleles in Chinese Children is low and TPMT* 3C appears to be the most prevalent among the tested mutant TPMT alleles in this population. [ABSTRACT FROM AUTHOR]

To determine the incidence of the thiopurine S-methyltransferase (TPMT) genetic polymorphism in the Thai population. Genomic DNAs were isolated from peripheral blood leucocytes of 200 healthy Thais. The frequencies of five allelic variants of the TPMT gene, TPMT* 2, * 3A, * 3B, * 3C and * 6 were determined using allele specific polymerase chain reaction (PCR) or PCR-Restriction fragment length polymorphism technique. Of the 200 Thai subjects participating in this study, 181 subjects (90.5%) were homozygous for TPMT* 1, 18 subjects (9.0%) were heterozygous for TPMT* 1/* 3C. Only one subject (0.5%) was homozygous for TPMT* 3C. The frequency of TPMT* 3C mutant allele was 0.050. Although the TPMT* 3C is the most prevalent mutant allele in Asian populations, the frequency of this defective allele is significantly higher in Thais than has been reported in other Asian populations. [ABSTRACT FROM AUTHOR]

A non-extraction high-performance liquid chromatographic (HPLC) method has been developed for the determination of 6-methylthioguanine (6-MTG), as part of the determination of thiopurine S-methyltransferase activity (TPMT) in erythrocytes. Erythrocyte lysate is added to a glass vial containing substrates and incubation buffer, which is then sealed for the rest of the analysis. Enzyme incubation, sample preparation, and analysis are then undertaken without further sample-handling steps. The need for a solvent extraction step has been overcome by heating the incubate to 85 °C to stop the enzyme reaction. The heat inactivation step precipitates protein which upon centrifugation forms a thin film in the bottom of the glass vial enabling the supernatant to be injected directly onto the HPLC system. The assay shows excellent precision and recovery with a within-batch imprecision giving a co-efficient of variation of 2.9% (mean=41.5 nmol 6-MTG/g Hb/h, n=10) and 5.1% (mean=12.6 nmol 6-MTG/g Hb/h, n=10). The between-batch imprecision gives a co-efficient of variation of 8.2% (mean=11.1 nmol 6-MTG/g Hb/h, n=11) and 7.3% (mean=41.0 nmol 6-MTG/g Hb/h, n=16). Determination of the TPMT activity in 120 people shows a range of enzyme activity of 11.3–63.8 nmol 6-MTG/g Hb/h with a mean and median activity of 34.8 and 34.2 nmol 6-MTG/g Hb/h, respectively. TPMT is increasingly used in clinical practice to ensure optimisation of treatment with thioguanine drugs. This direct HPLC method minimises sample-handling, reduces inherent imprecision, the possibility of laboratory error and with the potential for further automation, makes it ideal for use in a regional referral laboratory. [Copyright &y& Elsevier]