Thiopurine Methyltransferase Genotype Research Articles

Implementation of TPMT testing

The activity of the enzyme thiopurine methyltransferase (TPMT) is regulated by a common genetic polymorphism. One in 300 individuals lack enzyme activity and 11% are heterozygous for a variant low activity allele and have an intermediate activity. The thiopurine drugs azathioprine, mercaptopurine and thioguanine are substrates for TPMT; these drugs exhibit well documented myelosuppressive effects on haematopoietic cells and have a track record of idiosyncratic drug reactions. The development of severe bone marrow toxicity, in patients taking standard doses of thiopurine drugs, is associated with TPMT deficiency whilst the TPMT heterozygote is at an increased risk of developing myelosuppression. Factors influencing TPMT enzyme activity, as measured in the surrogate red blood cell, are discussed in this review to enable an appreciation of why concordance between TPMT genotype and phenotype is not 100%. This is particularly important for lower/intermediate TPMT activities to avoid misclassification of TPMT status. TPMT testing is now widely available in routine service laboratories. The British National Formulary suggests TPMT testing before starting thiopurine drugs. Dermatologists were quick to adopt routine TPMT testing whilst gastroenterologists do not specifically recommend TPMT screening. TPMT testing is mandatory prior to the use of mercaptopurine in childhood leukaemia. Thiopurine drug dose and other treatment related influences on cell counts explain some of the differing recommendations between clinical specialities. TPMT testing is cost-effective and the major role is in the identification of the TPMT deficient individual prior to the start of thiopurine drugs.

OP006 Personalized thiopurine dosing based on TPMT genotyping reduces leucopenia occurrence and results in cost-savings in IBD patients. Results from a randomized trial in the Netherlands

Background: More than 20% of inflammatory bowel diseases (IBD) patients discontinue thiopurine therapy due to severe adverse drug reactions among which leucopenia is one of the most serious. Thiopurine S-methyltransferase (TPMT) pharmacogenetics has been proven effective for optimizing azathioprine/mercaptopurine safety. Nevertheless, TPMT screening is used in clinical practice on a very limited scale. The aim of our study was to assess whether pre-treatment TPMT genotype-based dosing reduces the occurrence of leucopenia and whether this strategy is cost-effective. Methods: We performed a randomized trial in thiopurine naive IBD patients starting on thiopurine treatment [the Dutch “Thiopurine response Optimization by Pharmacogenetic testing in Inflammatory bowel disease Clinics” (TOPIC) study (ClinicalTrials.gov: NCT00521950)]. Patients were randomly assigned to pre-treatment screening for three common variants in TPMT (TPMT∗2, ∗3A and ∗3C) or standard thiopurine treatment. Patients heterozygous for a TPMT variant received 50% of the standard thiopurine dose, patients homozygous for the tested variants 0-10%. We compared pre-treatment genotyped patients with patients receiving standard dose for the occurrence of leucopenia (leucocyte count

Monitoring thiopurine metabolites in korean pediatric patients with inflammatory bowel disease.

PurposeThis study aimed to assess the role of thiopurine S-methyltransferase (TPMT) and 6-thioguanine nucleotide (6-TGN) as predictors of clinical response and side effects to azathioprine (AZA), and estimate the optimal AZA dose in Korean pediatric inflammatory bowel disease (IBD) patients.Materials and MethodsOne hundred and nine pediatric IBD patients in whom AZA treatment was required were enrolled. Thiopurine metabolites were monitored since September 2010. Among them, 83 patients who had prescribed AZA for at least 3 months prior to September 2010 were enrolled and followed until October 2011 to evaluate optimal AZA dose, adverse effects and disease activity before and after thiopurine metabolite monitoring.ResultsThe result of the TPMT genotype was that 102 patients were *1/*1 (wild type), four were *1/*3C, one was *1/*6, one was *1/*16 (heterozygote) and one was *3C/*3C (homozygote). Adverse effects happened in 31 patients pre-metabolite monitoring and in only nine patients post-metabolite monitoring. AZA dose was 1.4±0.31 mg/kg/day before monitoring and 1.1±0.46 mg/kg/day after monitoring (p<0.001). However, there were no statistical differences in disease activity during metabolite monitoring period (p=0.34). Adverse effects noticeably decreased although reduction of the AZA dose since monitoring.ConclusionTPMT genotype and thiopurine metabolite monitoring could be helpful to examine TPMT genotypes before administering AZA and to measure 6-TGN concentrations during prescribing AZA in IBD patients.

Thiopurine-methyltransferase variants in inflammatory bowel disease: Prevalence and toxicity in Brazilian patients

To analyze the prevalence of thiopurine-methyltransferase (TPMT) genotypes and their association with drug toxicity in inflammatory bowel disease (IBD) patients from southeastern Brazil. A total of 219 consecutive patients with IBD, of which 146 had Crohn's disease and 73 had ulcerative colitis, regularly seen at the outpatient unit of the Division of Gastroenterology at the University Hospital Pedro Ernesto of the State University of Rio de Janeiro, a tertiary referral center, were enrolled in this study from February 2009 to January 2011. We analyzed the presence of major TPMT genetic variants (TPMT 2, 3A, 3C) in IBD patients by means of a specific allele and RFLP-PCR. Genomic DNA was isolated from peripheral blood leukocytes by proteinase-K/Sodium Dodecyl Sulfate digestion and phenol-chloroform extraction. TPMT 2 (C238G), TPMT 3A (G460A/A719G), and TPMT 3C (A719G) genotypes were detected by real-time polymerase chain reaction followed by direct sequencing with specific primers. Clinical data were systematically recorded, and correlated with the genotype results. The distribution of the selected TPMT gene polymorphism TPMT 2 (C238G), TPMT 3A (G460A/A719G), and TPMT 3C (A719G) genotypes was 3.6%, 5.4%, and 7.7% of the patients, respectively. Among the side effects recorded from patients taking azathioprine, 14 patients presented with pancreatitis and/or an elevation of pancreatic enzymes, while 6 patients had liver toxicity, and 2 patients exhibited myelosuppression/neutropenia. TPMT polymorphisms were detected in 37/219 patients (8 heterozygous for 2, 11 heterozygous for 3A, and 18 heterozygous for 3C). No homozygotic polymorphisms were found. Despite the prevalence of the TPMT 3C genotype, no differences among the genotype frequencies were significant. Although no association was detected regarding myelotoxicity or hepatotoxicity, a trend towards the elevation of pancreatic enzymes was observed for TPMT 2 and TPMT 3C genotypes. The prevalence of TPMT genotypes was high among Brazilian patients. Variants genes 2 and 3C may be associated with azathioprine pancreatic toxicity in a IBD southeastern Brazilian population.

TPMT gene polymorphism detection by conventional PCR in pediatric acute lymphoblastic leukemia and its toxic effect

Introduction The purine analog mercaptopurine is a key medication for the successful treatment of childhood acute lymphoblastic leukemia, particularly for the consolidation and continuation therapies. Thiopurine S-methyltransferase (TPMT) catalyzes the inactivation of mercaptopurine. TPMT single-nucleotide polymorphisms can prospectively identify patients at higher risk for mercaptopurine toxicity. Patients and methods The TPMT genotype was determined by in-house conventional PCR followed by digestion of the product with restriction enzymes, MwoI FastDigest and AccI FastDigest. The study was carried out on a total of 80 participants: 40 pediatric patients with standard risk B-cell acute lymphoblastic leukemia and 40 age-matched and sex-matched healthy controls. Mercaptopurine was given to the patients in consolidation phase with oral dose of 75 mg/m 2 daily for 4 weeks. Toxicity of the drug was assessed at the end of this phase by complete blood profile and liver function tests. Results In the patients group, 97.5% were of the wild-type homozygous TPMT*1/*1 genotype and 2.5% were of the heterozygous TPMT*1/*3A genotype. In the control group, we identified 90% with the TPMT*1/*1 genotype, 7.5% with the TPMT*1/*3A genotype, and 2.5% with the TPMT*1/*3C genotype. Among the wild-type *1/*1 genotype patients in the patient group, 32.5% of patients suffered from either hepatoxicity and/or myelosuppression. Conclusion The homozygous wild-type TPMT*1/*1 genotype was the most frequent genotype in both cases and controls. TPMT*1/*3A was the most prevalent mutant genotype in this study. Although some patients had wild-type allele genotyping, they developed signs of toxicity.

Association of thiopurine methyltransferase status with azathioprine side effects in Chinese patients with systemic lupus erythematosus

Azathioprine (AZA) is indicated for the treatment of systemic lupus erythematosus (SLE). Thiopurine methyltransferase (TPMT) is the rate-limiting enzyme in the steps of AZA metabolization. Heritable deficiency of TPMT enzyme activity and polymorphisms may lead to leukopenia. This study aims to detect TPMT polymorphisms and TPMT enzyme activity in Chinese SLE patients and to describe the association between TPMT genotypes and adverse effects of AZA. One hundred and twenty-six SLE patients with present or previous thiopurine therapy were identified from a local database. Adverse effects were documented. No TPMT*2, TPMT*3A, or TPMT*3B mutant alleles were detected. TPMT*3C was detected in four patients (3.17%). The heterozygotes had significantly lower mean TPMT activity as compared to the homozygotes (2.38 ± 1.24 vs. 12.56 ± 7.02U/mL, P < 0.001). Twenty-seven cases (21.42%) exhibited adverse effects. All of the heterozygotes (4/4, 100%) developed severe leukopenia, and three cases (3/4, 75%) of whom exhibited alopecia simultaneously. The specificity of TPMT*3C for predicting leukopenia and alopecia was 100 and 99.17%, respectively, and the sensitivity was 28.57 and 60.00%, respectively. The mean value of TPMT activity with leukopenia (4.67 ± 3.01 vs. 13.2 ± 6.94U/mL RBC, P < 0.001) or alopecia (2.31 ± 1.16 vs. 12.65 ± 6.98U/mL RBC, P < 0.001) was significantly lower than those without. TPMT*3C was the most common mutant polymorphism found in the study group. TPMT activity is reduced in TPMT*3C mutant. AZA-induced leukopenia and alopecia were partly correlated to TPMT*3C heterozygotes and low TPMT activity. The results of this study suggest that the value of TPMT genotyping before AZA therapy was limited in Chinese SLE patients, considering the low sensitivity. Routine monitoring of TPMT activity before prescribing and continuous hematological monitoring dose were recommended.

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

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

High Intra-Individual Variability In Systemic Exposure To 6 Mercaptopurine (6MP) In Children With Acute Lymphoblastic Leukemia (ALL) Contributes To ALL Relapse: Results From a Children's Oncology Group (COG) Study (AALL03N1)

Abstract Background Prolonged (∼2y) exposure to 6MP is associated with durable remissions in children with ALL. 6MP exerts cytotoxicity through conversion to thioguanine nucleotide (TGN) that is incorporated into DNA. Erythrocyte TGN concentration correlates with cytotoxicity. Systemic exposure to 6MP is influenced by: i) prescribed dose (dose intensity [DI]), ii) patient (pt) adherence, and iii) inherited capacity to metabolize 6MP by thiopurine methyltransferase (TPMT). Previous studies have shown the individual impact of these variables, but the contribution of each of these variable measured simultaneously in determining relapse risk is unknown – and was explored in the current study. Methods: We enrolled 744 pts in 1st remission and receiving oral 6MP (75 mg/m2/d) during maintenance. Monthly TGNs (∼6/ pt) yielded 3,953 measurements. 6MP DI (dose prescribed/ protocol dose) was ascertained for 113,682 days. TPMT genotyping classified pts as TPMT wild-type homozygotes (WT, 93.2%), heterozygotes (het, 6.7%) and homozygotes for deficient variants (def, 0.1%). Coefficient of variation (CV) was used to determine intra-individual variability in TGN (IQR=17.6-40.3), DI (3.2-28.2) and ANC (29.0-59.7). Using TGN CV% &lt;30 vs. ≥30, DI CV% &lt;20 vs. ≥20, and ANC CV% &lt;50 vs. ≥50, pts were classified as having stable TGN (s30TGN) vs. variable TGN (v30TGN), stable DI (s20DI) vs. variable DI (v20DI), and stable ANC (s50ANC) vs. variable ANC (v50ANC), respectively. 459 of these pts contributed 75,720 pt-days of electronically-monitored (MEMS) adherence data. Adherence rate was defined as days 6MP MEMS bottle was opened/days 6MP was prescribed. Results: Median age at diagnosis was 5y (1-19); 68% were males; 43% had high-risk disease. TGN levels, 6MP DI, and cumulative incidence (CI) of relapse are summarized in Table. 59 relapses occurred after a median follow-up of 6y (CI of relapse: 8.8% at 5y). TGN levels were higher (300 vs. 140 pmol/8*10^8 erythrocytes, p&lt;0.0001), 6MP DI was lower (0.65 vs. 0.86, p=0.0002) and risk of relapse tended to be lower (5y CI: 2.5% vs. 9.2%, p=0.1) among TPMT het/def vs. WT. Subsequent analyses were restricted to TPMT WT pts. Determinants of ALL relapse: Multivariable Cox regression analysis (adjusted for clinical factors) identified only adherence rate &lt;95% to be associated with relapse (HR=2.5, p=0.03); neither low TGN (&lt;140 [median]: HR=2.0, p=0.1), nor low DI (&lt;0.86 [median]; HR=1.3, p=0.5) increased relapse risk. However, pts with v30TGN had lower TGN levels (126.3 vs. 149.3, p&lt;0.0001), lower 6MP DI (0.86 vs. 0.90, p=0.01), lower adherence rates (85.4% vs. 92.7%, p=0.0002) (Table) and higher 5y CI of relapse (12.4% vs. 5.3%, p=0.02) than pts with s30TGN (Fig 1A). Multivariable Cox regression confirmed the increased relapse risk associated with v30TGN (HR=2.2, p=0.04). Similarly, pts with v20DI were at a higher relapse risk than pts with s20DI (5y CI: 13.4% vs. 6.7%, p=0.008, Fig 1B); multivariable analysis confirmed this association with v20DI (HR=1.8, p=0.04). Determinants of high intra-individual variability in TGN: Multivariable logistic regression analysis demonstrated that non-adherence (OR=2.1, p=0.0005) and high intra-individual variability in 6MP DI (v20DI: OR=5.5, p&lt;0.0001) were independently associated with v30TGN. The variability in 6MP DI, in turn, was due to high intra-individual variability in ANC (v50ANC: OR=2.7, p&lt;0.0001; using multivariable logistic regression analysis). Conclusions: Absolute TGN levels do not help prognosticate relapse risk; instead, high intra-individual variability in TGN levels, determined by both intra-individual variability in 6MP DI and lack of adherence to 6MP contribute to relapse risk in this population. These findings reinforce the need to minimize fluctuations in 6MP prescribed dose, as well as use measures to enhance adherence. Disclosures: Evans: St. Jude: In accordance with institutional policy, St. Jude allocates a portion of the income it receives from licensing inventions and tangible research materials to those researchers responsible for creating this intellectual property. Patents &amp; Royalties, Under this policy, I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics., Under this policy, I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Other. Relling:St. Jude: In accordance with institutional policy, St. Jude allocates a portion of the income it receives from licensing inventions and tangible research materials to those researchers responsible for creating this intellectual property. Patents &amp; Royalties, Under this policy, I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics., Under this policy, I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Other.

Prevalence of TPMT polymorphism in Indian patients requiring immunomodulator therapy and its clinical significance

Thiopurine methyltransferase (TPMT) enzyme plays a key role in the metabolism of azathioprine/6-mercaptopurine (6-MP). Mutations in the enzyme lead to generation of excess thioguanine, which causes suppression of various cell lineages, especially neutrophils. Data on the prevalence of TPMT polymorphism are available from Western and some Asian countries; such data from India are sparse. The aim of this research is to study the prevalence of TPMT mutation in Indian patients requiring immunomodulator therapy and its relation to the development of neutropenia on azathioprine therapy. In this retrospective study, data of all patients who underwent TPMT genotyping by PCR-RFLP and allele-specific PCR prior to immunomodulator therapy were analyzed. The frequency of on-treatment development of neutropenia (total neutrophil count <1,500 per cubic millimeters) was noted. Data were available on 126 patients (mean age, 42 [SD 13.6] years; 73 men and 53 women). The disease indications included ulcerative colitis (61), Crohn's disease (43), indeterminate colitis (1), autoimmune hepatitis (16), and others (5). TPMT genotype was wild in 120 patients (95.23 %) and heterozygous in 6 patients (4.77 %); no patient had homozygous mutation. Seven of 87 patients (6.8 %) who received azathioprine developed neutropenia; blood counts normalized on cessation of the drug in all. The incidence of neutropenia in patients with wild type was 6/84 (7.14 %) and with heterozygous type 1/3 (33 %) (p = 0.5764). Nearly 5 % of this population of patients requiring immunomodulator therapy was heterozygous carriers of the TPMT gene. Neutropenia was equally common in patients without and with the mutation.

Thiopurine methyltransferase genotype–phenotype discordance and thiopurine active metabolite formation in childhood acute lymphoblastic leukaemia

In children with acute lymphoblastic leukaemia (ALL) bone marrow activity can influence red blood cell (RBC) kinetics, the surrogate tissue for thiopurine methyltransferase (TPMT) measurements. The aim of this study was to investigate TPMT phenotype-genotype concordance in ALL, and the influence of TPMT on thiopurine metabolite formation. We measured TPMT (activity, as units ml(-1) packed RBCs and genotype) at diagnosis (n = 1150) and TPMT and thioguanine nucleotide (TGN) and methylmercaptopurine nucleotide (MeMPN) metabolites (pmol/8 × 10(8) RBCs) during chemotherapy (n = 1131) in children randomized to thioguanine or mercaptopurine on the United Kingdom trial ALL97. Median TPMT activity at diagnosis (8.5 units) was significantly lower than during chemotherapy (13.8 units, median difference 5.1 units, 95% confidence interval (CI) 4.8, 5.4, P < 0.0001). At diagnosis genotype-phenotype was discordant. During chemotherapy the overall concordance was 92%, but this fell to 55% in the intermediate activity cohort (45% had wild-type genotypes). For both thiopurines TGN concentrations differed by TPMT status. For mercaptopurine, median TGNs were higher in TPMT heterozygous genotype (754 pmol) than wild-type (360 pmol) patients (median difference 406 pmol, 95% CI 332, 478, P < 0.0001), whilst median MeMPNs, products of the TPMT reaction, were higher in wild-type (10 650 pmol) than heterozygous patients (3868 pmol) (P < 0.0001). In TPMT intermediate activity patients with a wild-type genotype, TGN (median 366 pmol) and MeMPN (median 8590 pmol) concentrations were similar to those in wild-type, high activity patients. In childhood ALL, TPMT activity should not be used to predict heterozygosity particularly in blood samples obtained at disease diagnosis. Genotype is a better predictor of TGN accumulation during chemotherapy.

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

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

PTU-108 Azathioprine Pharmacogenetics in Autoimmune Hepatitis

IntroductionAzathioprine (AZA) is widely used to treat autoimmune hepatitis (AIH). However, 20% of patients are intolerant of AZA and a further 18% are unresponsive. AZA metabolism is complex and in...

Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia

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. 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. 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 %). 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.

Tu1165 Discordance Between Thiopurine Methyltransferase Phenotype and Genotype Has Potential Implications in the Initiation Dose of Thiopurine Therapy in Pediatric Patients

Tu1165 Discordance Between Thiopurine Methyltransferase Phenotype and Genotype Has Potential Implications in the Initiation Dose of Thiopurine Therapy in Pediatric Patients

Incorporation of 6‐Thioguanine Nucleotides into DNA During Maintenance Therapy of Childhood Acute Lymphoblastic Leukemia—The Influence of Thiopurine Methyltransferase Genotypes

The Journal of Clinical PharmacologyVolume 53, Issue 6 p. 670-674 Brief Report Incorporation of 6-Thioguanine Nucleotides into DNA During Maintenance Therapy of Childhood Acute Lymphoblastic Leukemia—The Influence of Thiopurine Methyltransferase Genotypes Maria S. Ebbesen, Maria S. Ebbesen Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, Copenhagen, DenmarkSearch for more papers by this authorJacob Nersting, Jacob Nersting Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, Copenhagen, DenmarkSearch for more papers by this authorJack H. Jacobsen, Jack H. Jacobsen Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, Copenhagen, DenmarkSearch for more papers by this authorThomas L. Frandsen, Thomas L. Frandsen Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, Copenhagen, DenmarkSearch for more papers by this authorKim Vettenranta, Kim Vettenranta Division of Hematology–Oncology, Department of Pediatrics, University of Tampere, Tampere, FinlandSearch for more papers by this authorJonas Abramsson, Jonas Abramsson Department of Pediatrics, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, SwedenSearch for more papers by this authorFinn Wesenberg, Finn Wesenberg Department of Pediatrics, Oslo University Hospital/Medical Faculty, University of Oslo, Oslo, NorwaySearch for more papers by this authorKjeld Schmiegelow, Corresponding Author Kjeld Schmiegelow Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, Copenhagen, Denmark The Faculty of Medicine, Institute of Gynecology, Obstetrics and Pediatrics, University of Copenhagen, Copenhagen, Denmark Corresponding Author: Kjeld Schmiegelow, Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, JMC-4072, Copenhagen, Denmark Email: kschmiegelow@rh.dkSearch for more papers by this author Maria S. Ebbesen, Maria S. Ebbesen Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, Copenhagen, DenmarkSearch for more papers by this authorJacob Nersting, Jacob Nersting Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, Copenhagen, DenmarkSearch for more papers by this authorJack H. Jacobsen, Jack H. Jacobsen Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, Copenhagen, DenmarkSearch for more papers by this authorThomas L. Frandsen, Thomas L. Frandsen Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, Copenhagen, DenmarkSearch for more papers by this authorKim Vettenranta, Kim Vettenranta Division of Hematology–Oncology, Department of Pediatrics, University of Tampere, Tampere, FinlandSearch for more papers by this authorJonas Abramsson, Jonas Abramsson Department of Pediatrics, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, SwedenSearch for more papers by this authorFinn Wesenberg, Finn Wesenberg Department of Pediatrics, Oslo University Hospital/Medical Faculty, University of Oslo, Oslo, NorwaySearch for more papers by this authorKjeld Schmiegelow, Corresponding Author Kjeld Schmiegelow Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, Copenhagen, Denmark The Faculty of Medicine, Institute of Gynecology, Obstetrics and Pediatrics, University of Copenhagen, Copenhagen, Denmark Corresponding Author: Kjeld Schmiegelow, Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, JMC-4072, Copenhagen, Denmark Email: kschmiegelow@rh.dkSearch for more papers by this author First published: 15 April 2013 https://doi.org/10.1002/jcph.81Citations: 28Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume53, Issue6June 2013Pages 670-674 RelatedInformation

Thiopurine methyltransferase genotyping in Palestinian childhood acute lymphoblastic leukemia patients.

BackgroundThe genetic polymorphism of thiopurine methyltransferase (TPMT) is well characterized in most populations. Four common polymorphic alleles are associated with impaired activity of the enzyme. These are TPMT*2 (238G>C), TPMT*3B (c.460G>A), TPMT*3A (c.460G>A and c.719A>G) and TPMT*3C (c.719A>G). The aim of the present study was to determine the frequency of TPMT polymorphisms and their association with the occurrence of adverse events, during 6-mercaptopurine therapy in pediatric acute lymphoblastic leukemic (ALL) patients in Gaza Strip.MethodsA total of 56 DNA samples from all pediatric ALL patients admitted to the pediatric hematology departments of Gaza strip hospitals were analyzed. Genomic DNA from peripheral blood leukocytes was isolated and the TPMT*2, TPMT*3B TPMT*3A and TPMT*3C allelic polymorphism was determined by PCR-RFLP and allele specific PCR technique.ResultsNo TPMT*2, *3B or *3C alleles were detected. Only one, out of 56 patients, was found heterozygous for the TPMT*3A allele. Thus, the frequency of TPMT*3A allele was calculated to be 0.89%. Fourteen patients of ALL were suffering from myelotoxicity during 6-MP therapy. From our results, no significant association could be established between clinical and laboratory data and/or the presence of the mutation in TPMT gene.ConclusionTPMT*3A was the only deficiency allele detected in our population with an allelic frequency of 0.89%. Other polymorphic alleles in TPMT gene, or factors other than TPMT polymorphisms may be responsible for the development of myelosuppression in cases that don’t carry the investigated TPMT alleles (*2, *3A, *3B and *3C). Therefore, more studies are recommended to study such factors.

P324 Value of thiopurine methyltransferase genotyping for prevention of myelotoxicity in routine clinical practice in patients with inflammatory bowel disease treated with azathioprine

P324 Value of thiopurine methyltransferase genotyping for prevention of myelotoxicity in routine clinical practice in patients with inflammatory bowel disease treated with azathioprine

Clinical Pharmacogenetics Implementation Consortium Guidelines for Thiopurine Methyltransferase Genotype and Thiopurine Dosing: 2013 Update

The Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for Thiopurine Methyltransferase Genotype and Thiopurine Dosing was originally published in March 2011. We reviewed recent literature and concluded that although relevant new evidence has been generated, none of the evidence would change the primary dosing recommendations in the original guideline; therefore, the original publication remains clinically current. Up-to-date information on thiopurine methyltransferase (TPMT) gene alleles and nomenclature can be found at PharmGKB (http://www.pharmgkb.org). Clinical Pharmacology & Therapeutics (2013); 93 4, 324–325. doi:10.1038/clpt.2013.4

Development and Validation of a Rapid and Reliable Method for &lt;i&gt;TPMT&lt;/i&gt; Genotyping using real-time PCR

It is widely accepted that many medications exhibit inter-individual variability in their efficacy and toxicity due to polymorphisms in genes encoding drug-metabolising enzymes. One of the most often cited examples in this context is thiopurine S-methyltransferase (TPMT) polymorphism. TPMT is a phase 2 detoxification enzyme that catalyzes the S-methylation of thiopurine drugs such as thioguanine and 6-mercaptopurine. Approximately 11% of the Caucasian population carry a heterozygous deficiency of this enzyme causing intermediate enzyme activity, whereas 0.3% show a homozygous deficiency. In both cases, severe myelosuppression can develop upon treatment with thiopurines. These are commonly used in the treatment of leukemia. Therefore, genotyping of patients before treatment is absolutely necessary. Development of a fast and reliable real-time PCR application for TPMT genotyping would greatly improve thiopurine treatment regimens and allow the avoidance of adverse drug reactions. Blood was obtained from a Caucasian cohort of 143 individuals. After extraction of DNA, all samples were genotyped for TPMT polymorphisms *2, *3A, *3B, and *3C by real-time PCR as well as by PCR-RFLP as the reference method, in order to validate the new method. Four different genotypes were found in the population studied. Of the 143 individuals investigated, 1 was heterozygous for TPMT*2 (0.70%), 2 were heterozygous for TPMT*3B (1.40%), and 8 heterozygous for TPMT-*3C (5.60%). No homozygous genotype could be identified. In total, 7.7% of the individuals carried mutations. Results from the newly developed real-time PCR were 100% concordant with those obtained using standard PCR-RFLP analysis, leading to 100% sensitivity and specificity. The hands-on time is approximately one third of the time needed for standard PCR-RFLP methods. A new high-throughput genotyping method could be successfully established and optimised for the commonly found mutant alleles TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A), and TPMT*3C (A719G) via real-time PCR on the LightCycler (Roche) instrument and using the standard PCR-RFLP as reference method.

Successful Azathioprine Treatment with Metabolite Monitoring in a Pediatric Inflammatory Bowel Disease Patient Homozygous forTPMT*3C

Thiopurine S-methyltransferase (TPMT) methylates purine analogues, showing TPMT activity in inverse relation to concentrations of active metabolites such as 6-thioguanine nucleotide (6-TGN). With conventional dosing of thiopurines, patients with homozygous variant TPMT alleles consistently suffer from severe myelosuppression. Here, we report a patient with TPMT*3C/*3C who managed successfully with monitoring of thiopurine metabolites. The patient was an 18-year-old male diagnosed with Crohn's disease. The standard dose of azathioprine (AZA) (1.8 mg/kg/day) with mesalazine (55.6 mg/kg/day) was prescribed. Two weeks after starting AZA treatment, the patient developed leukopenia. The DNA sequence analysis of TPMT identified a homozygous missense variation (NM_000367.2: c.719A>G; p.Tyr240Cys), TPMT*3C/*3C. He was treated with adjusted doses of azathioprine (0.1-0.2 mg/kg/day) and his metabolites were closely monitored. Leukopenia did not reoccur during the follow-up period of 24 months. To our knowledge, this is the first case of a patient homozygous for TPMT*3C successfully treated with azathioprine in Korea. While a TPMT genotyping test may be helpful to determine a safe starting dose, it may not completely prevent myelosuppression. Monitoring metabolites as well as routine laboratory tests can contribute to assessing drug metabolism and optimizing drug dosing with minimized drug-induced toxicity.