Thiopurine Methyltransferase Genotype Research Articles

Association between metabolic enzyme genotype of azathioprine and drug tolerance in patients with rheumatic diseases

To discuss the relationship between the genotype of thiopurine methyltransferase (TPMT) and azathioprine tolerance in the patients with rheumatic diseases. Four common mutation alleles of TPMT in 200 patients with rheumatic diseases [TPMT*2 (G238C), TPMT*3A (A719G/G460A), TPMT*3B (G460A), TPMT*3C (A719G)] were detected by allele specific polymerase chain reaction (AS-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-PFLP). 194 patients who had used azathioprine finished the 3 months' follow-up. In the 200 patients with rheumatic diseases, 4 cases of heterozygote of TPMT*3C were detected, but no mutation of TPMT*2, TPMT*3B or TPMT*3A was found. The genotypic frequency of wild-type homozygote was 98%, and that of heterozygote (TPMT*1/TPMT*3C) was 2%. Mutation allele frequency in these patients was 1%. Average of TPMT activity of the 4 cases of heterozygote was (2.4 +/- 1.2) U/ml red blood cells, significantly lower than that of the 196 cases of homozygote which was (12.2 +/- 6.8) U/ml RBC. In the 194 patients who had used azathioprine, bone marrow suppression occurred in 18 patients, 2 of which suffered severe crisis of hematopoietic system, and 6 of which were complicated with impaired liver function. In the 4 patients with heterozygote, 3 had used azathioprine, and bone marrow suppression occurred within 1 month of using the drug, including 2 cases of severe crisis of hematopoietic system. Patients with mutation alleles of TPMT are intolerant to azathioprine, and likely to have severe crisis of hematopoietic system. To detect the TPMT genotype before using azathioprine is significant to improve the therapeutic safety.

Current use of pharmacogenetic testing: a national survey of thiopurine methyltransferase testing prior to azathioprine prescription

Azathioprine is an immunosuppressant prescribed for the treatment of inflammatory conditions and after organ transplantation. Risk of neutropaenia has limited the effective use of azathioprine (AZA) and driven requirements for careful monitoring and blood tests. Thiopurine methyltransferase (TPMT) is a genetically moderated key enzyme involved in the metabolism of AZA that can be used to stratify individuals into different levels of risk of developing neutropaenia. Two techniques can be used to measure TPMT status: enzyme-level testing (phenotype testing) and DNA based testing (genotype testing). To identify the current uptake of TPMT enzyme-level testing, TPMT genotype testing, and, the role of guidelines; to inform the prescribing and monitoring of AZA. A survey was mailed to a consultant dermatologist, gastroenterologist, and rheumatologist at every NHS Hospital Trust in England. The survey comprised mainly closed questions exploring: use of AZA and monitoring; use of TPMT enzyme-level testing and genotype testing; and, the role of guidelines to guide prescribing practice. A 70% (n=287) response rate was obtained. The majority of respondents reported prescribing AZA (99%, n=283). Prescribing and monitoring patterns differed between individual respondents and between the three disciplines. TPMT enzyme-level testing was reportedly used by 67% (n=189) of respondents, but this differed by discipline (dermatologists 94%, gastroenterologists 60%, rheumatologists 47%). In 91% of cases enzyme-level testing was carried out prior to prescribing AZA. Genotype testing is not typically available to NHS clinicians but 15 clinicians (six dermatologists, six gastroenterologists, three rheumatologists) reported using it. Most consultants (82%) reported using guidelines to inform their AZA prescribing and monitoring (dermatologists 81%, gastroenterologists 75%, rheumatologists 94%). Two-thirds of the consultants surveyed in England are using TPMT enzyme-level testing, prior to AZA treatment. Uptake differs between specialities. High uptake of TPMT enzyme-level testing by dermatologists, compared with gastroenterologists and rheumatologists, may reflect national guidelines advocating its use prior to AZA. Uptake of enzyme-level testing may alter in other specialties as other guidelines are developed.

Assessment of thiopurine methyltransferase enzyme activity is superior to genotype in predicting myelosuppression following azathioprine therapy in patients with inflammatory bowel disease

Myelosuppression occurs in 2-7% of inflammatory bowel disease (IBD) patients treated with azathioprine, and can be associated with reduced activity of thiopurine methyltransferase (TPMT) in some patients. It has been proposed that pretreatment assessment of TPMT status reduces the incidence of toxicity and is cost-effective. To determine if screening for TPMT status predicts side-effects to azathioprine in patients with IBD and to ascertain whether screening by TPMT enzyme activity or genotype is superior. Sequential IBD patients were identified and azathioprine tolerance recorded. Blood was collected for measurement of TPMT activity and TPMT*3C, TPMT*3A and TPMT*2 genotypes. Of 130 patients, 25% stopped azathioprine because of toxicity. Four patients experienced severe myelosuppression (WCC < 2). Eleven of 17 patients with reduced TPMT activity were heterozygotes, including one patient with marked TPMT deficiency who experienced severe myelosuppression. There was no association between intermediate TPMT deficiency and any side-effect. Moderate reduction of TPMT activity in heterozygotes was not associated with toxicity, but very low TPMT activity caused severe myelosuppression in one patient. This would have been predicted by measuring TPMT activity but not by genotyping. Measurement of TPMT activity may therefore be superior to genotype in predicting severe myelosuppression.

Glutathione-S-transferase genotypes and the adverse effects of azathioprine in young patients with inflammatory bowel disease

Adverse drug reactions to azathioprine, the prodrug of 6-mercaptopurine, occur in 15%-38% of patients and the majority are not explained by thiopurine-S-methyltransferase (TPMT) deficiency. Azathioprine is known to induce glutathione depletion and consumption of glutathione is greater in cells with high glutathione-S-transferase (GST) activity compared with those with low activity; moreover, some reports indicate that GST might play a direct role in the reaction of glutathione with azathioprine. The association between polymorphisms of GST-M1, GST-P1, GST-T1, and TPMT genes and the adverse effects of azathioprine was therefore investigated. Seventy patients with inflammatory bowel disease (IBD), treated with azathioprine, were enrolled and clinical data were retrospectively determined. TPMT and GST genotyping were performed by polymerase chain reaction (PCR) assays on DNA extracted from blood samples. Fifteen patients developed adverse effects (21.4%); there was a significant underrepresentation of the GST-M1 null genotype among patients developing adverse drug reactions to azathioprine (odds ratio [OR] = 0.18, 95% confidence interval [CI] = 0.037-0.72, P = 0.0072) compared with patients who did not develop adverse effects. Patients heterozygous for TPMT mutations presented a marginally significant increased probability of developing adverse effects (OR = 6.38, 95% CI = 0.66-84.1, P = 0.062). Moreover, among the 55 patients who did not develop adverse effects, there was a significant underrepresentation of the GST-M1 null genotype among patients who displayed lymphopenia as compared with those that did not display this effect of azathioprine (OR = 0.15, 95% CI = 0.013-1.08, P = 0.032). Patients with IBD with a wildtype GST-M1 genotype present increased probability of developing adverse effects and increased incidence of lymphopenia during azathioprine treatment.

Thiopurine Methyltransferase Genotype Distribution in Irish Patients with Crohn's and Ulcerative Colitis: A Frequency Study

Aims: The aim of this study was to determine the frequency of TMPT polymorphisms in a population of Crohns and Ulcerative Colitis patients and whether or not this was different to the frequency reported in other Caucasian populations. Source material This was a blinded randomized study. The DNA used in this study was selected from a biobank of specimens used to previously to identify genetic associations with Crohns and Ulcerative Colitis.

Thiopurine S‐methyltransferase gene polymorphism in Japanese patients with autoimmune liver diseases

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). 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. 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. 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.

Analysis of Thiopurine S-methyltransferase Polymorphism in the Population of Serbia and Montenegro and Mercaptopurine Therapy Tolerance in Childhood Acute Lymphoblastic Leukemia

Thiopurine S-methyltransferase (TPMT) is an enzyme that converts thiopurine drugs into inactive metabolites. It is now well established that interindividual variation in sensitivity to thiopurines can be the result of the presence of genetic polymorphisms in the TPMT gene. The aim of this study was to determine the frequency and type of TPMT polymorphisms in the population of Serbia and Montenegro and to assess its relevance in the management of childhood acute lymphoblastic leukemia (ALL). Blood samples from 100 healthy adults and 100 children with ALL were analyzed for common mutations in the TPMT gene using polymerase chain reaction-based assays. The results revealed that allelic frequencies were 0.2% for TPMT*2, 3.2% for TPMT*3A, and 0.5% for TPMT*3B. A rare TPMT*3B allele was detected in 2 families. No TPMT*3C allele was found. The general pattern of TPMT-variant allele distribution as well as their frequencies in the population of Serbia and Montenegro, is similar to those determined for other Slavic and Mediterranean populations. The ability to tolerate 6-mercaptopurine (6-MP) -based maintenance therapy was used as a surrogate marker of hematologic toxicity. In the study of 50 patients with childhood ALL treated according to the BFM-like protocol, it was found that even TPMT-heterozygous patients are at greater risk of thiopurine drug-related leukopenia (mean duration of period when children missed therapy as a result of leukopenia for TPMT-heterozygous patients was 11.3 weeks vs 3.4 weeks for wild-type genotype patients, P < 0.01). In another group of 50 patients, the TPMT genotype was determined prospectively. The therapy protocol was modified considering their TPMT genotype. Administering reduced 6-MP dosages in the initial phase of maintenance allowed TPMT-heterozygous patients to later receive full protocol doses of both 6-MP and nonthiopurine therapy without omitting therapy resulting from myelotoxicity. These results justify performing TPMT genotyping before initiating thiopurine therapy in all children with ALL to minimize consequent toxicity.

Thiopurines in Inflammatory Bowel Disease - The Role of Pharmacogenetics and Therapeutic Drug Monitoring

Pharmacogenetics represents the study of variability in drug response due to genetic variations. Inflammatory bowel disease (IBD, i.e. primarily Crohn's disease and ulcerative colitis) is characterized by a chronic or relapsing inflammation of the digestive tract. The thiopurines 6-mercaptopurine (6-MP) and azathioprine (AZA), an imidazol derivative and pro-drug of 6-MP, are widely used in IBD, particularly in Crohn's disease. The metabolism of thiopurines is complex and individually variable. Thiopurine methyltransferase (TPMT) is a key enzyme in this metabolism and exhibits a genetic variability due to a number of variant alleles coding for a defective enzyme. The formation of biologically active thioguanine nucleotides (TGN) and methylated metabolites may vary considerably due to the TPMT activity. Patients with decreased TPMT activity are at increased risk of developing severe side effects if treated with conventional thiopurine doses, due to the accumulation of toxic metabolites. Determination of the TPMT phenotype or genotype is often used to identify individuals with increased risk for adverse events. Twenty-one variant TPMT alleles have been described, of which three are more common than the others. An association between inosine triphosphate pyrophosphatase polymorphisms and adverse events during thiopurine treatment has also been proposed. In this review, the clinical value of TPMT status determination and pharmacological monitoring of thiopurine metabolites are discussed as well as the increased interest in the use of 6-thioguanine, a thiopurine with a less complex metabolism, as an alternative for patients who do not tolerate AZA or 6-MP. It can be concluded that TPMT determination before start of thiopurine therapy is of value to identify individuals with increased risk for adverse reactions due to genetic enzyme deficiency. However, large prospective studies are still needed to evaluate the true benefit of monitoring thiopurine metabolites during thiopurine treatment. © 2006 Bentham Science Publishers Ltd.

TPMT genotype and its clinical implication in renal transplant recipients with azathioprine treatment

Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. Patients with intermediate or deficient TPMT activity are at risk of toxicity after receiving standard doses of these drugs. This study determined the frequencies of TPMT alleles (TPMT*2, *3A, *3B and *3C) and explored the association between TPMT genetic polymorphism and the development of adverse drug reactions in Chinese renal transplant patients receiving azathioprine (AZA). TPMT genotypes were determined using polymerase chain reaction-based assays in 122 renal transplant patients and 210 healthy subjects. Biochemical and clinical data were retrospectively evaluated after renal transplantation. Of 122 patients, eight (allele frequency 3.28%) were heterozygous for TPMT*3C and no TPMT*2, *3A or *3B or homozygous TPMT*3C subjects were identified. The pattern and frequency of the main mutant TPMT alleles were similar in patients and healthy subjects. Four of five patients (80%) with haematopoietic toxicity were heterozygotes. TPMT heterozygosity was associated with significant reductions in haematological indices and a significant decrease in cyclosporine plasma concentrations in the first year after renal transplantation. No association between TPMT genotype and renal rejection was identified. Our results, together with those of others pointing in the same direction, suggest that genotyping the major TPMT variant alleles may be a valuable tool in preventing AZA toxicity and optimization of immunosuppressive therapy.

Thiopurine methyltransferase Genotype Is Not a Risk Factor for Secondary Malignant Neoplasias after Treatment for Childhood Acute Lymphoblastic Leukemia on Berlin-Frankfurt-Muenster Protocols.

The thiopurines mercaptopurine and thioguanine are important components of contemporary polychemotherapeutic treatment protocols for acute leukemias. Thiopurines are prodrugs that undergo intestinal and hepatic metabolism. Activation occurs via a multistep pathway to form thioguanine nucleotides, which are thought to be the major cytotoxic compound through triggering cell cycle arrest and apoptosis. This process is in competition with direct inactivation of thiopurines or their metabolites by thiopurine S-methyltransferase (TPMT). TPMT is a cytosolic enzyme that is ubiquitously expressed in the human body and catalyzes the S-methylation of thiopurines. The TPMT locus is subject to genetic polymorphism, with heterozygous individuals (5 to 11% of Caucasians) having intermediate TPMT activity, and homozygous individuals (0.3 to 0.5% of Caucasians) having low TPMT activity. At least 20 variant TPMT alleles (*2 to *18) have been described so far that confer decreased enzyme activities compared to the TPMT*1 wild-type allele. TPMT genotype is highly concordant with TPMT phenotype. With regard to long-term adverse effects, patients who have diminished TPMT activity were shown to be at increased risk of developing chemotherapy-induced acute myeloid leukemia and radiation-induced second brain tumors tumors after exposure towards mercaptopurine during therapy for childhood acute lymphoblastic leukemia (ALL). To investigate if such an association is generalizable to other entities of secondary malignant neoplasms (SMN) and different treatment approaches for ALL, we collected specimens of 72 patients who developed a SMN after ALL treatment on Berlin-Frankfurt-Muenster (BFM) protocols, analyzed their TPMT genotype and compared genotype frequencies to these in the general ALL patient population. The 72 patient cohort consisted of 49 hematological SMN (half of them received cranial irradiation), 12 brain tumors, and 11 other solid SMN. Neither in the entire patient group nor in subgroup analyses, differences in allele frequencies of TPMT variants conferring diminished enzyme activity were detectable when comparing SMN patients to the overall ALL patient population. Thus, low TPMT activity does not seem to play a major role in the etiology of SMN after treatment for childhood ALL according to BFM treatment strategies. Factors potentially helpful for the explanation of the previously described relationsship of TPMT activity with SMN will be presented.

Cost-Effectiveness of Pharmacogenomics in Clinical Practice: a Case Study of Thiopurine Methyltransferase Genotyping in Acute Lymphoblastic Leukemia in Europe

Only a few studies have addressed the cost-effectiveness of pharmacogenetics interventions in healthcare. Lack of health economics data on aspects of pharmacogenetics is perceived as one of the barriers hindering its implementation for improving drug safety. Thus, a recent Institute for Prospective Technological Studies (IPTS) study, entitled 'Pharmacogenetics and pharmacogenomics: state-of-the-art and potential socio-economic impact in the EU' included an explorative cost-effectiveness review for a pharmacogenetic treatment strategy compared with traditional medical practice. The selected case study examined the cost-effectiveness of thiopurine methyltransferase (TMPT) genotyping prior to thiopurine treatment in children with acute lymphoblastic leukemia (ALL). Information for the cost-effectiveness model parameters was collected from literature surveys and interviews with experts from four European countries (Germany, Ireland, the Netherlands and the UK). The model has established that TPMT testing in ALL patients has a favorable cost-effectiveness ratio. This conclusion was based on parameters collected for TPMT genotyping costs, estimates for frequency of TMPT deficiency, rates of thiopurine-mediated myelosuppression in TPMT-deficient individuals, and myelosuppression-related hospitalization costs in each of the four countries studied. The mean calculated cost per life-year gained by TPMT genotyping in ALL patients in the four study countries was euro 2100 (or euro 4800 after 3% discount) based on genotyping costs of euro 150 per patient. Cost per life-year gained is expected to further improve following the introduction of the wider use of TMPT genotyping and the availability of lower cost genotyping methods. Our analysis indicates that TPMT genotyping should be seriously considered as an integral part of healthcare prior to the initiation of therapy with thiopurine drugs.

Utility of thiopurine methyltransferase genotyping and phenotyping, and measurement of azathioprine metabolites in the management of patients with autoimmune hepatitis

Azathioprine is a key drug in the management of autoimmune hepatitis (AIH), with effects mediated via conversion to 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP), the latter controlled by thiopurine methyltransferase (TPMT). Our aims were to evaluate the role of TPMT genotyping and phenotyping and to examine 6-TG and 6-MMP metabolite levels in patients with AIH. TPMT genotyping and phenotyping was performed on 86 patients with AIH, and metabolites evaluated in assessable patients. Eighty-six patients with AIH received azathioprine; 22 developed toxicity and 4/22 were heterozygous for TPMT alleles. Cirrhosis was more common amongst patients who developed toxicity (12/22 (54.5%) versus 19/64 (29.6%), P=0.043). Patients who required persistent prednisone at equivalent azathioprine doses had a higher mean fibrosis stage (P=0.044). TPMT activity, but not metabolites, was lower in patients with stage III/IV fibrosis versus stage I/II fibrosis (30+/-1.92 versus 35.2+/-1.93, P=0.044). Azathioprine dose significantly correlated with measured 6-TG levels (r=0.409, P<0.0001) and 6-MMP levels (r=0.387, P<0.001). Advanced fibrosis but not TPMT genotype or activity predicts azathioprine toxicity in AIH. Overlap in 6-TG and 6-MMP metabolite levels is noted whether or not steroid therapy is used to maintain remission.

No Induction of Thiopurine Methyltransferase During Thiopurine Treatment in Inflammatory Bowel Disease

The aim of this study was to follow, during standardized initiation of thiopurine treatment, thiopurine methyltransferase (TPMT) gene expression and enzyme activity and thiopurine metabolite concentrations, and to study the role of TPMT and ITPA 94C > A polymorphisms for the development of adverse drug reactions. Sixty patients with ulcerative colitis or Crohn's disease were included in this open and prospective multi-center study. Thiopurine naïve patients were prescribed azathioprine (AZA), patients previously intolerant to AZA received 6-mercaptopurine (6-MP). The patients followed a predetermined dose escalation schedule, reaching target dose at Week 3; 2.5 and 1.25 mg/kg body weight for AZA and 6-MP, respectively. The patients were followed every week during Weeks 1–8 from baseline and then every 4 weeks until 20 weeks. TPMT activity and thiopurine metabolites were determined in erythrocytes, TPMT and ITPA genotypes, and TPMT gene expression were determined in whole blood. One homozygous TPMT-deficient patient was excluded. Five non compliant patients were withdrawn during the first weeks. Twenty-seven patients completed the study per protocol; 27 patients were withdrawn because of adverse events. Sixty-seven percent of the withdrawn patients tolerated thiopurines at a lower dose at Week 20. There was no difference in baseline TPMT enzyme activity between individuals completing the study and those withdrawn for adverse events (p = 0.45). A significant decrease in TPMT gene expression (TPMT/huCYC ratio, p = 0.02) was found, however TPMT enzyme activity did not change. TPMT heterozygous individuals had a lower probability of remaining in the study on the predetermined dose (p = 0.039). The ITPA 94C > A polymorphism was not predictive of adverse events (p = 0.35).

Pharmacogenomics: methods and protocols

Pharmacogenetics is about how the genetic inheritance of a person affects his response to medicine, examining relationships between genes and drug metabolism, drug response and adverse effects. Despite a reasonably long history of this field, which took off in the 1950s, success stories in cancer pharmacogenetics were rather lacking, with the most quoted examples being thiopurine methyltransferase (TPMT) genotype affecting the metabolism of mercaptopurine, and association between the promoter polymorphism in UDP-glucuronosyltransferase 1A1 gene and irinotecan induced adverse drug reactions. In the last decade, the generation of a huge amount of genetic information by the Human Genome Project and the HapMap project, accompanied by the rapid improvement in genotyping technologies, have fuelled an increase in genetics studies on the aetiology of common traits. These same reasons also enable pharmacogenetic studies to move from mostly single gene based to genome-wide investigations. Reflecting this gain in scale, the term ‘pharmacogenomics' is now often used. Together with the development of targeted cancer therapy with drugs like Glivec (Imatinib), Iressa (Gefitinib) and Herceptin (Trastuzumab), pharmacogenomics is set to play a bigger role in both understanding cancer drug mechanisms and in clinical practice. Pharmacogenomics: methods and protocols, is one of the latest additions to the long running ‘Methods on Molecular Biology' series. This book aims to introduce the latest technology in complex genetics to its readers, presumably bench-based scientists who are interested in recreating the experimental system for pharmacogenomic studies in their laboratory. The opening chapter provides a historical overview of the field and is followed by 12 chapters of protocols. The last section deals with the management of pharmacogenomic information. The core of this book is the protocols, which are divided into two groups. The first group contains four chapters on functional analysis of gene variations, with three of them describing methodologies in investigating allele specific expression. The second part covers a range of genotyping techniques, from fluorescence-based microsatellite typing to SNP typing methods including pyrosequencing, Taqman and MALDI-TOF (Sequenom). Each of these chapters begins with an abstract and a brief introduction explaining the theoretical basis of the system, before launching into the detailed description of materials and methods. The protocols are concise and well written – in most cases enough instructions are provided for the reader to follow the experiment without the help of supplementary information. Their value, however, depends largely on the technique being described. The protocols are most useful for complicated assays like the Haplotype-specific Chromatin Immunoprecipitation, where multiple molecular biology techniques has to be carried out and the fine-tuning of experimental parameters vital to eventual success. Genotyping techniques, on the other hand, are often highly standardised, and instruction manuals can be obtained from the company manufacturing the genotyping equipment, making some of the protocols less interesting. What distinguishes this book from the standard protocols, however, is the Notes section of each chapter, where the ‘tricks' of the experimental system are discussed. The authors of this book are leading experts in complex genetics and pharmacogenetics, and their experience in applying particular techniques is the most valuable information for the readers. The tips they provided are very specific and often pinpoint both practical details (down to the particular type of flask to use) and issues in good experimental design (e.g. suggesting appropriate controls). By following them the readers can get a better grasp of the critical steps in the each procedure and hence have a higher chance of success when setting them up. Most of the protocols contain a short description of the expected output from the experiment, often consists of a figure or a screenshot. It would be beneficial to expand this section and include more information on troubleshooting. I suspect the majority of the readers of Pharmacogenomics: methods and protocols will only need a few of the protocols described. In the ideal world a book like this would be helpful when deciding on the particular technology at the beginning of a pharmacogenomic study. Practically though, the choice often depends on the availability of machinery and expertise in the laboratory rather than any other factor. So all in all, this book is a fine collection of protocols describing state of the art methods in functional and complex genetics that can be used in pharmacogenomics. While it might not become a most treasured item for individual scientists, it is certainly a good reference book to have in one's institute library.

5-Aminosalicylate therapy is associated with higher 6-thioguanine levels in adults and children with inflammatory bowel disease in remission on 6-mercaptopurine or azathioprine

Small uncontrolled trials have suggested that 5-aminosalicylate (5-ASA) medications increase 6-thioguanine nucleotide (6-TGn) levels in adults with Crohn's disease (CD) on azathioprine (AZA) or 6-mercaptopurine (6-MP), presumably through the inhibition of thiopurine methyltransferase (TPMT). We tested the theory that coadministration of 5-ASA agents with AZA/6-MP results in higher 6-TGn levels in a large cohort of children and adults with CD or ulcerative colitis (UC). A retrospective cohort study identified all children and adults treated for IBD with AZA/6-MP at 2 tertiary medical centers. Patients were included if their TPMT genotype was known and 6-TGn and 6-methymercaptopurine (6-MMP) levels had been obtained after 3 months of clinical remission at a stable dose of AZA/6-MP. 6-TGn and 6-MMP levels were compared between patients taking and those not taking 5-ASA medications through the use of linear regression models to identify and adjust for potentially confounding variables. Of the 126 patients included, 88 were taking 5-ASA medications. Patients on 5-ASA agents had higher mean 6-TGn levels after adjustment for confounding variables (Delta6-TGn, 47.6 +/- 21.8 pmol/8 x 10 red blood cells; P = 0.03). CD and TPMT heterozygosity was independently associated with higher 6-TGn levels (P = 0.01 and P = 0.03, respectively). 5-ASA exposure was not associated with a change in 6-MMP levels. We found that 5-ASA therapy is associated with higher 6-TGn levels in children and adults with IBD on 6-MP/AZA. TPMT inhibition may not explain this effect because 5-ASA exposure did not affect 6-MMP levels. The observed association of CD with higher 6-TGn levels is novel and needs to be verified in prospective studies.

Outcome in acute lymphoblastic leukemia: Influence of thiopurine methyltransferase genetic polymorphisms

Current treatment protocols for childhood acute lymphoblastic leukemia always include thiopurine drugs such as 6-mercaptopurine (6-MP), which is administrated daily during maintenance therapy. Once 6-MP is an inactive prodrug, it needs to be activated to thioguanine nucleotides (TGNs) to exert cytotoxicity. However, 6-MP can also be methylated by the polymorphic enzyme thiopurine S-methyltransferase (TPMT), thus reducing TGNs formation. In order to assess the influence of TPMT polymorphism in ALL, we have used PCR/HCSGE (Horizontal Conformation-Sensitive Gel Electrophoresis) based methods to characterise molecularly a sample of 110 children with ALL. Four distinct alleles associated with TPMT deficiency (TPMT*3A, *3C, *2 and *8) were found in heterozygous individuals representing 11.8% of the sample. We have also compared several parameters related with ALL outcome between subsamples of children homozygous or heterozygous for TPMT. In the heterozygous group, 6-MP dosages were lower and the number of whole interruptions during treatment or interruptions due to toxicity as well as the number of relapses was higher compared to the other group. Despite our results do not reach statistical significance, this study emphasizes the importance to introduce the prospective analysis of TPMT genotype, prior to any ALL treatment.

Genotyping of thiopurine methyltransferase in patients with acute leukemia using LightCycler PCR

Patients with acute lymphoblastic leukemia (ALL) are treated with thiopurines. Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurines and is subject to genetic polymorphism. The TPMT genotype was determined in 55 patients with ALL. Three patients were heterozygous for allelic variants of TPMT. We describe an assay for the genotyping of the TPMT polymorphism using real-time fluorescence polymerase chain reaction (PCR) to facilitate rapid processing of samples. This strategy is superior to standard PCR-restriction fragment length polymorphism genotyping methods and provides informative data on TPMT polymorphisms in patients prior to treatment with thiopurines.

Inosine Triphosphate Pyrophosphatase and Thiopurine S-Methyltransferase Genotypes Relationship to Azathioprine-Induced Myelosuppression

The use of azathioprine (AZA) in inflammatory bowel disease (IBD) patients is limited by toxicity, which occurs in up to 20% of treated patients. Mutations in the thiopurine S-methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) genes have been associated with the occurrence of AZA-related toxicity. The aim of our study was to determine the relative contribution of ITPA and TPMT mutations to the development of toxicity induced by AZA treatment in IBD patients. ITPA(94C>A, IVS2+21A>C) and TPMT (238G>C, 460G>A, and 719A>G) genotypes were assessed in 262 IBD patients (159 females, 103 males; 67 patients with ulcerative colitis, 195 patients with Crohn's disease) treated with AZA and were correlated with the development of leukopenia and hepatotoxicity. Leukopenia (leukocyte count, <3.0 x 10(9)/L) was observed in 4.6% of treated patients. The frequencies of mutant ITPA 94C>A and TPMT alleles were significantly higher in the leukopenic population compared with patients without leukopenia (16.7% and 5.4%, respectively, for ITPA 94C>A, and 20.8% and 4%, respectively, for TPMT). Moreover, the ITPA 94C>A and TPMT mutations predicted leukopenia: ITPA 94C>A odds ratio, 3.504; 95% confidence interval, 1.119-10.971 (P = .046); TPMT odds ratio, 6.316; 95% confidence interval, 2.141-18.634 (P = .004). Neither TPMT nor ITPA genotype predicted hepatotoxicity. ITPA 94C>A and TPMT polymorphisms are associated with AZA-related leukopenia in IBD patients.

Genetic Variation in the MTHFR Gene Influences Thiopurine Methyltransferase Activity

The immunosuppressive drug 6-mercaptopurine (6-MP) and its prodrug azathioprine are used in the treatment of inflammatory bowel disease and other disorders of immune regulation (1). Thiopurine methyltransferase (TPMT) inactivates 6-MP by methylation. The genetic variants TPMT *2 to *19 are associated with decreased TPMT activity (2), and TPMT*3A , * 3C , and *2 are the most common deficiency-associated variants (1). A heterozygous TPMT genotype (1 in 10 individuals from the general population) is associated with an increased risk of myelosuppression with standard-dose azathioprine therapy (3) and a favorable response to reduced-dose thiopurine therapy (1). Patients with complete TPMT deficiency (1 in 300 individuals from the general population) are at high risk for myelosuppression (4). The erythrocyte TPMT activity distribution is continuous, and concordance between genotype and phenotype in the carrier range varies, depending on where the cutoff is established between the ranges for carriers and noncarriers. We propose that genetic variation in folate metabolism influences TPMT activity and contributes to the lack of concordance between genotype and phenotype in the carrier range. TPMT irreversibly transfers a methyl group from S -adenosylmethionine (SAM) to 6-MP, forming 6-methylmercaptopurine (6-MeMP) and S -adenosylhomocysteine (SAH). The adenosyl moiety of SAH is subsequently cleaved, and homocysteine is remethylated to methionine. The methyl donor for this folate-dependent remethylation cycle is 5-methyltetrahydrofolate, which is formed from 5,10-methylenetetrahydrofolate (MTHF) in a reaction catalyzed by 5,10-MTHF reductase (MTHFR). The MTHFR 677C>T (A222V) thermolabile variant (5) and the 1298A>C (E429A) variant (6) are associated with decreased MTHFR activity. The homozygous MTHFR 677TT genotype occurs in 8%–10% of the population (7), shows 30%–50% of wild-type activity in lymphocytes (8)(9), and is associated with hyperhomocysteinemia (10), DNA hypomethylation (11), increased risk of neural tube defects(12), and decreased risk of some cancers (13)(14). The homozygous 1298CC …

Genetic Polymorphism of Thiopurine S-Methyltransferase in the Russian Population.

Thiopurine S-methyltransferase (TPMT) is a key enzyme in the catabolism of 6-mercaptopurine (6MP), a key component of therapy for childhood acute lymphoblastic and promyelocytic leukemias and lymphoblastic non-Hodgkin lymphoma. TPMT mutations reduce TPMT enzymatic activity; therefore, TPMT-deficient patients develop severe hematopoietic toxicity when treated with standard doses of 6MP (50–75 mg/m2/day). The frequency of TPMT alleles has not previously been evaluated in the Russian Federation. We determined the allele frequency and the pattern of mutant TPMT alleles in Russian children with hematologic malignancies (leukemia/lymphoma) and other diseases, and in healthy adults, in different areas of the Russian Federation. A microchip was designed to detect the main mutant alleles of human TPMT (TPMT*2, *3A, *3B, *3D, *7, and *8) and was used to assay 982 samples. Only 3 inactivating mutations of the TPMT gene were identified: 45 subjects (4.6%) had TPMT genotype *1/*3A, 8 (0.8%) had *1/*3C, and 2 (0.2%) had *1/*2. No homozygous genotypes and no mutant *3B, *3D, *7, or *8 alleles were found. The estimated frequency of the wild-type allele was 97.1% (95% CI, 93.8%–100%) and that of variant alleles was 2.9% (95% CI, 0%–6%). Most individuals with the *1/*3A genotype were of Slavic background, and genotype *1/*3C was found in a buryat (Mongolian race) and in residents of the North Caucasian and the Urals regions. These findings indicate ethnic differences in the frequency of nonfunctional TPMT alleles. The overall frequency of mutant TPMT genotypes in the Russian Federation is 5.6%, which is similar to that observed in the white populations of the United States and Europe.