Thiopurine Methyltransferase Genotype Research Articles

Thiopurine methyltransferase genotype and activity cannot predict outcomes of azathioprine maintenance therapy for antineutrophil cytoplasmic antibody associated vasculitis: A retrospective cohort study.

ObjectiveAzathioprine is a widely used immunosuppressive drug. Genetic polymorphisms and activity of the enzyme thiopurine methyltransferase (TPMT) have been associated with azathioprine efficacy and toxicity in several populations. We investigated whether these associations also exist for ANCA associated vasculitis (AAV) patients, who receive azathioprine maintenance therapy after remission induction with cyclophosphamide.Methods207 AAV patients treated with cyclophosphamide induction and azathioprine maintenance therapy were included and followed for 60 months. TPMT genotype and tertiles of TPMT activity were compared to relapse free survival and occurrence of adverse events, particularly leukopenia. Multivariable regression was performed to account for confounders.ResultsIn univariable analysis, relapse free survival was not significantly associated with TPMT genotype (P = 0.41) or TPMT activity (P = 0.07), although it tended to be longer in lower tertiles of TPMT activity. There was no significant association of TPMT genotype and activity with occurrence of any adverse event. In multiple regression, leukocyte counts at the end of cyclophosphamide induction were related to risk of leukopenia during azathioprine therapy [P<0.001; OR 0.54 (95% CI 0.43–0.68)] and risk of relapse during follow-up [P = 0.001; HR 1.17 (95% CI 1.07–1.29)] irrespective of TMPT genotype or activity.ConclusionTPMT genotype and activity were not independent predictors of relapse, and could not predict leukopenia or other adverse effects from azathioprine. Leukocyte counts after cyclophosphamide induction were related to both outcomes, implying a greater influence of cyclophosphamide response compared to azathioprine and TPMT in AAV patients.

Preemptive NUDT15 genotyping: redefining the management of patients with thiopurine-induced toxicity.

Thiopurine methyltransferase (TPMT) gene variants have achieved limited success in predicting the outcome of thiopurine therapy, which shows wide inter-individual variations. The literature indicates a strong association between the NUDT15 gene variant and thiopurine-induced toxicity in Asian patients. The present study intends to explore the role of the NUDT15 variant (C415T) in Indian patients on thiopurine therapy. NUDT15 and TPMT genotyping were performed using amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) and the restriction fragment length polymorphism (RFLP) technique. Of 370 samples received for TPMT testing, 206 samples were available for NUDT15 genotyping. The NUDT15 risk allele frequency was 10.7%, with the frequency of wild, heterozygous and mutant genotypes being 80.6%, 17.5% and 1.9%, respectively. TPMT variants were seen in 13 of 370 (3.5%) patients, whereas the NUDT15 variant was seen in 40 of 206 (19.4%) patients. Thiopurine-induced toxicity information was available for 101 patients, among whom 10 developed leukopenia and all harbored the NUDT15 variant (p<0.0001). NUDT15 was clinically more relevant than TPMT in terms of sensitivity and specificity, as well as with a statistically significant difference in thiopurine dose requirement for patients with the NUDT15 variant. A preemptive NUDT15 genotyping approach can therefore help identify high-risk patients (NUDT15 C415T positive) who could benefit from thiopurine dose reduction, thereby preventing fatal thiopurine-induced toxicity.

Role of TPMT and ITPA variants in mercaptopurine disposition.

To explore the levels of thioguanine incorporated into DNA (DNA-TG), and erythrocyte levels of 6-thioguanine nucleotides (Ery-TGN) and methylated metabolites (Ery-MeMP) during 6-mercaptopurine (6MP)/Methotrexate (MTX) therapy of childhood acute lymphoblastic leukemia (ALL) and the relation to inosine triphosphatase (ITPA) and thiopurine methyltransferase (TPMT) gene variants. Blood samples were drawn during 6MP/MTX maintenance therapy from 132 children treated for ALL at Rigshospitalet, Copenhagen. The samples were analysed for thiopurine metabolites and compared to TPMT (rs1800460 and rs1142345) and ITPA (rs1127354) genotypes. Median DNA-TG (mDNA-TG) levels were higher in TPMT and ITPA low-activity patients as compared to wildtype patients (TPMTLA 549 vs. 364 fmol/µg DNA, p = 0.007, ITPALA 465 vs. 387 fmol/µg DNA, p = 0.04). mDNA-TG levels were positively correlated to median Ery-TGN (mEry-TGN)(rs = 0.37, p = 0.001), but plateaued at higher mEry-TGN levels. DNA-TG indices (mDNA-TG/mEry-TGN) were 42% higher in TPMTWT patients as compared to TPMTLA patients but no difference in DNA-TG indices was observed between ITPAWT and ITPALA patients (median 1.7 vs. 1.6 fmol/µg DNA/ nmol/mmol Hb, p = 0.81). DNA-TG indices increased with median Ery-MeMP (mEry-MeMP) levels (rs = 0.25, p = 0.001). TPMT and ITPA genotypes significantly influence the metabolism of 6MP. DNA-TG may prove to be a more relevant pharmacokinetic parameter for monitoring 6MP treatment intensity than cytosolic metabolites. Prospective trials are needed to evaluate the usefulness of DNA-TGN for individual dose adjustments in childhood ALL maintenance therapy.

Genetic Polymorphism of Thiopurine S-methyltransferase in Children with Acute Lymphoblastic Leukemia in Jordan

Background and Aims:It has been demonstrated that homozygote and heterozygote mutant allele carriers for thiopurine S-methyltransferase (TPMT) are at high risk of developing myelosuppression after receiving standard doses of 6-mercaptopurine (6-MP). The aim of this study was to determine the frequency of TPMT deficient alleles in children with acute lymphoblastic leukemia (ALL) in Jordan and to compare it with other ethnic groups.Methods:We included 52 ALL childhood cases from King Hussein Cancer Research Center in Jordan. Genotyping of the rs1800460, rs1800462, and rs1142345 SNPs was performed by polymerase chain reaction (PCR) followed by sequencing. Comparisons were made with historical data for controls and for both volunteers and cases from other middle-eastern countries.Results:Mutant TPMT alleles were present in 3.8% (2/52) of patients. Allelic frequencies were 1.0% for both TPMT*B and TPMT*C. None of the patients were heterozygous or homozygous for TPMT*3A or TPMT *2. We did not find statistically significant differences in the distribution of mutant alleles between Jordan and other middle-eastern countries for both healthy volunteers or ALL patients.Conclusions:The overall frequency of TPMT mutant alleles was low and did not exhibit differences compared to other middle-eastern countries, including Jordanian studies assessing TPMT mutant alleles in healthy volunteers. The current results question the value of TPMT genotyping in the Jordanian population.

High frequency of mutant thiopurine S-methyltransferase genotypes in Mexican patients with systemic lupus erythematosus and rheumatoid arthritis.

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.

Treatment-related sinusoidal obstruction syndrome in children with de novo acute lymphoblastic leukemia during intensification.

Sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease, has been described following treatment of acute lymphoblastic leukemia (ALL) with the anti-metabolite 6-thioguanine (6-TG). Previous studies incorporating daily 6-TG into maintenance chemotherapy demonstrated a high incidence of SOS, typically presenting after prolonged exposures to 6-TG. 6-TG continues to be used as a single, 14-day burst during intensification; however, SOS associated with brief courses of 6-TG is poorly described. We aim to describe this rare though clinically significant phenomenon. Children with 6-TG-related SOS were retrospectively identified from 680 de novo patients with ALL at Texas Children's Cancer Center over 8 years. Clinical characteristics and outcomes are described. Ten (1.5%) patients were identified with SOS. No predominant sex, ethnicity, or race was noted. SOS was diagnosed 16.5 (6-42) days from starting 6-TG. Isolated thrombocytopenia (IT) was noted in 9/10 patients and presented a median of 5 days prior to SOS. Refractoriness to platelet transfusions was noted in 8/10 patients, presenting a median of 2 days prior to SOS. Most patients were otherwise clinically stable outpatients upon presenting with IT or transfusion refractoriness. Fever was noted in 7/10 patients at diagnosis and 6/10 had documented or suspected infection within 14 days of SOS. Two patients died, while eight fully recovered. Intermediate thiopurine methyltransferase genotype was noted in 5/8 patients with data available. SOS following short courses of 6-TG in DI is clinically distinct from SOS following prolonged courses of 6-TG in maintenance, particularly in its early presentation and outcomes.

Thiopurine Therapy Risk Evaluation for Inflammatory Bowel Disease Patients Using TPMT Enzyme Expression Method

Introduction: Thiopurine drugs, what are widely used medications for the treatment of inflammatory bowel diseases (IBD), have remarcable adverse effects like myelosuppression, leading to life threatening complications (severe infection or bleeding). Thiopurine methyltransferase (TPMT) plays a significant role in the metabolism of thiopurine drugs. Low TPMT activity in body is associated with pathological thiopurine drug metabolisms, overproduction of cytotoxic metabolites and myelosuppression. Methods: A purpose of our study was to compare TPMT enzyme expression analysis in patients with ulcerative colitis (UC) and Crohn's disease (CD) who are using azathioprine therapy with patients who have not yet begun this therapy. A prospective pilot study included 20 patients (55% female n=11 and 45% male n=9) having an age ranging from 22 to 79 years. All participants where admitted to Pauls Stradins Clinical university hospital (Riga, Latvia) from January 2017 to May 2017. All included patients received standard treatment of 5-ASA, steroid or azathioprine; leucocyte level was no lower than 4 x 109/L. The expression of TPMT in each blood sample was determined using ELISA (MyBioSource, USA) method. Results: All patient was previously diagnosed with IBD and now was admitted to hospital because of IBD exacerbation. 70 % of patients (n=14) was diagnosed with UC, 30 (n=6) with CD. 75% (n=15) of patients had not previously received azathioprine. 15% (n=3) had received azathioprine therapy, but stopped using it because of negative side effects like dyspepsia, acute pancreatitis, rash or exacerbation symptoms. 10% (n=2) was still receiving azathioprine therapy. Patients TPMT expression was in the range of 1.4 to 50 U/mL. All respondents were divided into the three TPMT enzyme activity groups. Activity of TPMT was low (< 5.5 U/mL) in 10% (n=2), intermediate (5.6-15.5 U/mL) in 5% (n=1) and normal/high (>15.6 U/mL) ir 85% (n=17) of patients. Conclusion: The results of this study confirmed the important role of the TPMT enzyme activity in the therapeutic drug monitoring. 15% of examined patients had decreased TPMT activity. Further studies in combination with TPMT genotype analysis are necessary for additional evaluation of treatment risks.

Critical Considerations in Anticancer Drug Development and Dosing Strategies: The Past, Present, and Future.

Critical Considerations in Anticancer Drug Development and Dosing Strategies: The Past, Present, and Future.

Significance and application of individualized detection of thiopurine S-methyltransferase in patients with chronic actinic dermatitis

Objective To investigate correlations of thiopurine S-methyltransferase (TPMT) activity (phenotype) and genotype with adverse reactions after oral azathioprine treatment in patients with chronic actinic dermatitis, and to explore optimal azathioprine dose for the treatment of chronic actinic dermatitis. Methods Totally, 70 patients with chronic actinic dermatitis treated with oral azathioprine were enrolled into this study and served as patient group, of whom, 12 had adverse reactions to azathioprine. In addition, 50 health checkup examinees were enrolled as the control group. EDTA-anticoagulated blood samples were collected from the patients and controls, and erythrocytes lysates were prepared. High performance liquid chromatography (HPLC) was performed to evaluate the activity of TPMT. Genome-wide DNA was extracted from the blood samples, and TPMT genotypes were detected by allele-specific PCR and PCR-restriction fragment length polymorphism (RFLP) analysis. Results There was no significant difference in the TPMT activity between the patient group and control group ([25.80 ± 8.34]U/ml vs. [23.58 ± 5.70]U/ml, t= 0.782, P > 0.05) . However, the TPMT activity was significantly lower in patients with adverse reactions than those without ([18.86 ± 9.22]U/ml vs.[27.27 ± 6.72]U/ml, t= 3.437, P < 0.05) . TPMT genotypes were detected among 120 samples, and a heterozygous mutation TPMT*3C (A719G) was found in the TPMT gene in 7 samples, including 5 in the patient group and 2 in the control group. The frequencies of the mutant genotype and allele were 5.8% (7/120) and 2.9% respectively. No significant difference in the frequency of TPMT*3C mutation was observed between the patient group and control group (χ2= 0.108, P= 0.742) . The TPMT*3C mutation was found in 3 of the 12 patients with adverse reactions, as well as in 2 of 58 patients without adverse reactions. Additionally, the frequency of TPMT*3C mutation was significantly higher in patients with adverse reactions than in those without (χ2= 40.093, P < 0.05) . Conclusion Adverse reactions after oral azathioprine treatment are associated with TPMT mutations and decreased TPMT activity in patients with chronic actinic dermatitis, so appropriately adjusting the azathioprine dose in patients with low-activity and mutant-type TPMT can facilitate safe treatment. Key words: Dermatitis, photoallergic; Azathioprine; Drug toxicity; DNA mutational analysis; Thiopurine methyltransferase

Pharmacokinetics of two 6-mercaptopurine liquid formulations in children with acute lymphoblastic leukemia.

A liquid formulation of 6-mercaptopurine (6-MP) was recently approved by the Food and Drug Administration (Purixan®) based on bioavailability (BA) data from healthy adults. We examined the pharmacokinetics (PK) and BA of 6-MP in children with acute lymphoblastic leukemia (ALL) comparing a marketed tablet, two extemporaneously prepared liquid formulations, and data from the approved liquid formulation. Twenty-two children (6-17 years) participated in a randomized two-way, crossover study of two cohorts. Group 1 (n = 11; five males) received a 5 mg/ml liquid formulation and the marketed 50 mg 6-MP tablet on separate occasions, and Group 2 (n = 11; five males) received a 50 mg/ml liquid formulation and the marketed tablet. The usual prescribed 6-MP dose (25-115 mg/m2 ) was given after an 8-hr fast. Serial blood samples were collected over 8 hr postdose. Plasma 6-MP concentrations were determined using a good laboratory practice (GLP)-validated liquid chromatography-tandem mass spectrometry method. PK parameters were calculated using noncompartmental analysis and compared within and between cohorts, and thiopurine methyltransferase (TPMT) genotype was analyzed. No patient had a TPMT genotype reflective of a poor metabolizer phenotype. Comparison of PK parameters between 5 and 50 mg/ml treatments revealed significant differences (P <0.05) in AUCN (where AUC is area under the curve), CmaxN , and Tmax . Comparisons within each group revealed significant differences in AUC0-∞ and Tmax in the 5 mg/ml group. Pharmacokinetic profiles of 6-MP established in healthy adults with the approved liquid formulation may not reflect the PK profile in children with ALL. Formulation-specific differences in PK may significantly impact the dose-exposure profile in these children and must be considered.

Nucleoside diphosphate-linked moiety X-type motif 15 C415T variant as a predictor for thiopurine-induced toxicity in Indian patients.

Interindividual variation seen in the thiopurine metabolism is attributed to the genetic variant in thiopurine methyltransferase (TPMT) gene leading to myelosuppression. In Asians, the thiopurine-induced toxicity is not completely explained by TPMT variants. Literature indicates that a newer genetic variant in nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) gene is associated with thiopurine intolerance. We aimed to determine the risk allele frequency of NUDT15 genetic variant and its association with thiopurine-induced toxicity in Indian patients. In this pilot study, 69 patients on thiopurine therapy were analyzed. The frequencies of thiopurine-induced leukopenia were recorded. NUDT15 (C415T) and TPMT (*2, *3A, *3B, and *3C) genotyping was performed using amplification refractory mutation system-polymerase chain reaction and restriction fragment length polymorphism technique. Results were validated by DNA sequencing. The NUDT15 CC, CT, and TT genotypes were found to be 86.9%, 11.5%, and 1.5%, respectively, whereas TPMT genetic variants were absent. Of 60 patients without NUDT15 variant, none developed leukopenia, whereas of nine patients with NUDT15 variant, six developed leukopenia (P-value < 0.0001). The mean thiopurine dose of 1.01 and 0.73 mg/kg/day for patients with wild and mutant NUDT15 alleles, respectively, was statistically significant (P < 0.01). The sensitivity and specificity for NUDT15 variant were 100% and 95.2%, respectively. The NUDT15 risk allele frequency was 7.2%. There are 6/69 (8.7%) patients who developed leukopenia and harbored NUDT15 variant, thus showing a strong association for thiopurine-induced toxicity. Hence, NUDT15 genotyping may be considered before thiopurine therapy in Indian patients.

Polymorphic variation in TPMT is the principal determinant of TPMT phenotype: A meta-analysis of three genome-wide association studies.

Thiopurine-related hematotoxicity in pediatric acute lymphoblastic leukemia (ALL) and inflammatory bowel diseases has been linked to genetically defined variability in thiopurine S-methyltransferase (TPMT) activity. While gene testing of TPMT is being clinically implemented, it is unclear if additional genetic variation influences TPMT activity with consequences for thiopurine-related toxicity. To examine this possibility, we performed a genome-wide association study (GWAS) of red blood cell TPMT activity in 844 Estonian individuals and 245 pediatric ALL cases. Additionally, we correlated genome-wide genotypes to human hepatic TPMT activity in 123 samples. Only genetic variants mapping to chromosome 6, including the TPMT gene region, were significantly associated with TPMT activity (P < 5.0 × 10-8 ) in each of the three GWAS and a joint meta-analysis of 1,212 cases (top hit P = 1.2 × 10-72 ). This finding is consistent with TPMT genotype being the primary determinant of TPMT activity, reinforcing the rationale for genetic testing of TPMT alleles in routine clinical practice to individualize mercaptopurine dosage.

P154 Complete sequence-based NUDT15 and TPMT variants for predicting thiopurine-induced leukopenia in patients with Crohn's disease

Background: Thiopurine-induced leukopenia is a life-threatening complication in Asians. Other than previously known thiopurine S-methyltransferase (TPMT) mutation, the NUDT15 genetic variant was recently identified to have a strong association with thiopurine-induced leukopenia. We investigated the association of NUDT15, TPMT, metabolites of thiopurines on leukopenia in patients with Crohn's disease (CD). Methods: We investigated the data of 168 adult patients with CD undergoing thiopurine treatment. Clinical evaluation and laboratory examinations were performed every 1–3 months. We carried out a complete sequencing analysis for evaluating NUDT15 and TPMT variants and measured thiopurine metabolites levels. Results: Of 168 patients, 34 patients (20.2%) had NUDT15 variant alleles. NUDT15 heterozygous variants were found in 28 patients (16.7%) and NUDT15 homozygous variants were found in 6 patients (3.6%). Among the 168 patients, 35 (20.8%) patients experienced leukopenia during first year of thiopurine treatment. NUDT15 variant types were strongly associated with developing leukopenia [10.9% wild type as reference; 48.5% heterozygous variant genotype; 100% homozygous variant genotype, odds ratio (OR) 3.44 (95% CI, 1.21–9.78)]. Table 1. The risk of leukopenia according to NUDT15 and TPMT genotype However, TPMT genotype was not associated with developing leukopenia (OR 0.58, 95% CI 0.07–4.96). Patients with NUDT15 homozygous variant genotype developed severe early leukopenia with average 88.2% (range, 84–94%) reduction from baseline WBC count at 4 weeks. Figure 1. WBC count as a percentage of baseline at 4 weeks after thiopurines treatment. In subgroup of NUDT15 variant type, there was no significant difference of 6-TGN levels between patients with and without leukopenia [298.5 (205–407) vs. 280.8 (174–327) pmol/8×108 RBC, p=0.344]. Table 2. Thiopurine metabolites levels and thiopurine-induced leukopenia in NUDT15 variant genotype subgroup Conclusions: This is the first complete sequence-based analysis for NUDT15 variants in Asian populations. Our findings suggest that NUDT15 genotype should be detected rather than TPMT before initiating thiopurines. Thiopurines treatment should not be recommended to patients with NUDT15 homozygous variant genotype due to severe early leukopenia.

Whole-Blood Thiopurine S-Methyltransferase Genotype and Phenotype Concordance in Iranian Kurdish Ulcerative Colitis (UC) Patients.

Thiopurine methyl transferase (TPMT), a drug-metabolizing enzyme, catalyzes methylation and consequently, the metabolism of thiopurine compounds used for treatment of inflammatory bowel disease (IBD). Individuals who are homozygous recessive or have extremely low TPMT activity need to avoid thiopurines because of concern for significant leukopenia. The aim of this research was to determine TPMT phenotypes and genotypes in IBD patients to predict the risk of thiopurine toxicity before treatment. The present case-control study consisted of 210 ulcerative colitis patients and 212 unrelated healthy controls from the population of western Iran. TPMT phenotype and genotype were determined by HPLC and allele specific PCR and PCR-RFLP, respectively. TPMT phenotyping and genotyping were compatible and demonstrated no frequency for deficient, 2.2% for low, and 97.8% for normal-activity which is different compared with the results of other studies. There was a significant negative correlation between TPMT activities as calculated based on nmol6MTG/gHb/h and the Hb levels in both UC (r = -0.54, p < 0.001) and control groups (r = -0.27, p < 0.001). Interestingly, a significant positive correlation between Hb levels and TPMT activities was seen when the enzyme activity was calculated in mU/L in both UC patients (r = 0.14, p = 0.05) and in control subjects (r = 0.43, p < 0.001). The overall concordance rate between TPMT phenotypes and genotypes of mutants to alleles (9 out of 422), based on receiver-operating characteristic (ROC) curve, yielded a sensitivity of 94.7% and specificity of 90% for mU/L and a sensitivity of 85.6% and specificity of 90% for nmol6MTG/gHb/h. The use of mU/L is more appropriate than nmol6MTG/gHb/h for expressing TPMT activity, and there is better correlation between genotypes and phenotypes of TPMT based on mU/L. The frequency of known mutant TPMT alleles in western Iran (Kurd population) is low suggesting low risk of thiopurine drug toxicity in IBD patients from this region.

Thiopurine S-Methyltransferase Polymorphisms in Korean Dermatologic Patients.

BackgroundThiopurine S-methyltransferase (TPMT) is an important enzyme in the metabolism of thiopurines including azathioprine (AZA), 6-mercaptopurine, and 6-thioguanine. TPMT genotyping is widely used for screening of AZA-related toxicity during routine clinical practice in Korea. However, the data of TPMT genotypes and its AZA-related toxicity have not been studied in the field of dermatology.ObjectiveThe aim of this study was to evaluate the genetic basis of TPMT polymorphism in Korean dermatologic patients and subsequently to investigate the relationship between mutant TPMT and adverse responses to AZA treatment.MethodsThis study was retrospective, single-center study. One hundred forty-nine Korean dermatologic patients who underwent TPMT screening test were included. Each patient's medical records, the result of TPMT screening test, dose and treatment period of AZA, and side effects, were reviewed. Laboratory tests were assessed at each visit in order to monitor adverse drug reactions. Leukopenia grading was used in accordance with the common terminology criteria for adverse events (CTCAE) ver. 4.03.ResultsBehçet's disease was the leading disorder among the patients. The frequency of TPMT mutation was 4.0% (6/149) among the participants in this study. Four of the six patients with genetic alterations were treated with a low-dose AZA regimen, but no AZA-related adverse events were observed.ConclusionOur results suggest that 1) TPMT polymorphisms in Korean dermatologic patients are similar to those previously reported in Asian patients with the most common mutant allele being TPMT*3C and 2) AZA can be used in the patients with these polymorphisms under a careful dosing regimen.

Thiopurine Methyltransferase Genotype Testing in Paediatric Patients in South Australia. A Retrospective Audit into Prescribing Practices.

Background: Thiopurines are used to treat a number of medical conditions including inflammatory bowel disease, acute lymphoblastic leukemia and severe eczema in children. Pre prescription identification of variant alleles helps reduce thiopurine related adverse events. The aim of the study was to explore the clinical utility of TPMT genotyping in a paediatric population in South Australia, specifically the uptake of testing and whether the results are guiding appropriate dosing of thiopurines in keeping with current established international guidelines. Methods: A retrospective audit was conducted reviewing all patients below the age of 18 years who underwent TPMT genotyping in South Australia during the 10-year period between January 2004 and January 2014. Data regarding demographics and prescribing practices was collected from the medical records of 260 paediatric patients. Results: Paediatric gastroenterologists requested 67% of the TPMT genotypes performed. Loss of function alleles were confirmed in almost 9% of cases. There were positive correlations between adverse events and whether the test was used correctly (p 0.011) and with subspecialty unit (p<0.001). Oncology recorded the largest percentage of adverse events 63.5% whilst only comprising 16.5% of the total dataset. Conclusion: The safest prescribing practice in all groups of patients is to ensure the TPMT gentyope is performed prior to administration and dosing is guided by the results and established guidelines.

Early prediction of thiopurine-induced hepatotoxicity in inflammatory bowel disease.

Hepatotoxicity, gastrointestinal complaints and general malaise are common limiting adverse reactions of azathioprine and mercaptopurine in IBD patients, often related to high steady-state 6-methylmercaptopurine ribonucleotide (6-MMPR) metabolite concentrations. To determine the predictive value of 6-MMPR concentrations 1 week after treatment initiation (T1) for the development of these adverse reactions, especially hepatotoxicity, during the first 20 weeks of treatment. The cohort study consisted of the first 270 IBD patients starting thiopurine treatment as part of the Dutch randomised-controlled trial evaluating pre-treatment thiopurine S-methyltransferase genotype testing (ClinicalTrials.gov NCT00521950). Blood samples for metabolite assessment were collected at T1. Hepatotoxicity was defined by alanine aminotransaminase elevations >2 times the upper normal limit or a ratio of alanine aminotransaminase/alkaline phosphatase ≥5. Forty-seven patients (17%) presented hepatotoxicity during the first 20 weeks of thiopurine treatment. A T1 6-MMPR threshold of 3615 pmol/8 × 108 erythrocytes was defined. Analysis of patients on stable thiopurine dose (n = 174) showed that those exceeding the 6-MMPR threshold were at increased risk of hepatotoxicity: OR = 3.8 (95% CI: 1.8-8.0). Age, male gender and BMI were significant determinants. A predictive algorithm was developed based on these determinants and the 6-MMPR threshold to assess hepatotoxicity risk [AUC = 0.83 (95% CI: 0.75-0.91)]. 6-MMPR concentrations above the threshold also correlated with gastrointestinal complaints: OR = 2.4 (95% CI: 1.4-4.3), and general malaise: OR = 2.0 (95% CI: 1.1-3.7). In more than 80% of patients, thiopurine-induced hepatotoxicity could be explained by elevated T1 6-MMPR concentrations and the independent risk factors age, gender and BMI, allowing personalised thiopurine treatment in IBD to prevent early failure.

NUDT15 polymorphisms are better than thiopurine S-methyltransferase as predictor of risk for thiopurine-induced leukopenia in Chinese patients with Crohn's disease.

Thiopurine-induced leukopenia is the most common dangerous adverse event in Asians. NUDT15 R139C was recently proposed to be a promising biomarker for leukopenia with thiopurine therapy in Asians, but this has not been replicated in the Chinese population. To investigate the influence of NUDT15 R139C, thiopurine S-methyltransferase (TPMT), 6-TGN and 6-MMPR on thiopurine-induced leukopenia in Chinese patients with Crohn's disease. Clinical and epidemiological characteristics were reviewed from medical records. NUDT15 R139C and TPMT were genotyped. 6-TGN/6-MMPR concentrations were measured with high-performance liquid chromatography (HPLC). A total of 253 patients were included, 65 (25.7%) of whom experienced leukopenia. The median follow-up with thiopurine treatment was 38.0 weeks (range, 1-192 weeks). NUDT15 R139C was strongly associated with the incidence of leukopenia (70.2% mutation vs. 12.8% wild type; P=8.61×10-19 ; odds ratio, 10.80; 95% CI, 5.89-19.83). However, TPMT genotype was not found to be correlated with the incidence of leukopenia (P = 0.44). In subgroup of NUDT15 wild type, there was significant difference of 6TGN concentration between patients with and without leukopenia (413.0 (174.2-831.4) vs. 279.7 (77.3-666.9) pmol/8 × 108 RBC, P = 0.0055). In contrast, no association was found in patients with NUDT15 R139C variant alleles (P = 0.26). 6-MMPR was not correlated with leukopenia (P = 0.84). In Chinese patients, it is strongly recommended to detect NUDT15 genotype rather than TPMT before initiating thiopurine drugs. 6TGN concentration should be routinely monitored in CD patients with NUDT15 wild type. As for CT genotype, starting at low dose and careful monitoring for leukopenia and 6TGN levels is recommended.

Frequency of thiopurine methyltransferase mutation in patients of Mediterranean area with inflammatory bowel disease and autoimmune disorders

Background and aimsFew studies exist on the frequency of thiopurine methyltransferase (TPMT) mutation in patients from Southern Europe. We aimed to evaluate the frequency of TPMT mutation in a homogeneous Sicilian cohort of patients with inflammatory bowel disease (IBD), autoimmune and hematological disorders, the rate of thiopurine-related adverse events, and its association with the TPMT genotype. ResultsAmong 105 patients with IBD, 45 with autoimmune disease, and 34 with hematologic diseases, the homozygous TPMT variant genotype was found in one patient only (0.5%), while the heterozygous TPMT genotype was identified in 8 patients (4.3%). In patients with IBD, leukopenia was observed in ten patients: one had the homozygous TPMT genotype, one the heterozygous genotype, and the remaining eight the wild type genotype. ConclusionsThe frequency of TPMT mutation in a Mediterranean area was low. TPMT genotyping is not a sensitive tool for predicting thiopurine-induced leukopenia.

A Simple Method for TPMT and ITPA Genotyping Using Multiplex HRMA for Patients Treated with Thiopurine Drugs.

Thiopurine methyltransferase (TPMT) and inosine triphosphatase (ITPA) are crucial enzymes involved in the metabolism of thiopurine drugs: azathioprine and 6-mercaptopurine, used in the treatment of leukemia or inflammatory bowel diseases (IBD). The activity in these enzymes correlates with the genetic polymorphism of the TPMT and ITPA genes, respectively, which determines an individual reaction and dosing of thiopurines. Three main TPMT alleles: TPMT*2 (c.238G>C), TPMT*3A (c.460G>A, c.719A>G) and TPMT*3C (c.719A>G) account for 80–95 % of inherited TPMT deficiency in different populations in the world. In the ITPA gene, a c.94C>A mutation is significantly associated with an adverse thiopurine reaction. The aim of this study was to develop a quick and highly sensitive method for determining major TPMT and ITPA alleles. Here we present the molecular test for genotyping c.238G>C, c.460G>A, c.719A>G and c.94C>A changes based on multiplex high resolution melting analysis (HRMA). We analyzed DNA samples from 100 clinically diagnosed IBD patients treated with thiopurine drugs, and a known genotype in the positions 238, 460 and 719 of the TPMT gene as well as in position 94 of the ITPA gene. Our results obtained with multiplex HRMA indicated 100 % accuracy in comparison with data from restriction fragments length polymorphism (RFLP) and standard DNA sequencing. We conclude, that multiplex HRMA can be used as a quick, sensitive and efficient alternative diagnostic method compared to conventional techniques for the determination of TPMT*2,TPMT*3A and TPMT*3C alleles and c.94C>A change in the ITPA gene.