Thiopurine Methyltransferase Deficiency Research Articles

Thiopurine S-methyl Transferase (TPMT) Enzyme Level in Healthy Sudanese Population

Background: Thiopurine drugs have limited use due to their toxicity, related to the enzyme thiopurine S-methyl transferase (TPMT) activity, which varies between individuals. This is the first study in Sudan, which aimed to assess the TPMT phenotypic status of healthy Sudanese volunteers. Methods: A total of 177 healthy volunteers from Sudan were included in the study. TPMT enzymatic activities were measured using the ELISA serum protocol. We used SPSS to analyze the data and determined enzyme level categories and normal range with Z scores and quartile tests. The Sudan Medical Specialization Board (SMSB) Ethical Committee approved the study. Results: There were 117 males and 60 females among the volunteers, with ages ranging from 16 to 70 years and a mean age ± SD of 28.0 ±1 0.2, median = 24. Most candidates were from the Afro-Asiatic linguistic group (64.5%), followed by Nilo-Saharan (18.6%) and Niger-Kordofanian (16.9%). The TPMT enzyme level ranged between 0.17 and 9.5 ng/ml, with a mean of 2.26 ± 0.75 ng/ml. The quartile classification included very low enzyme (<0.76 ng/ml) seen in 4 candidates (2.3%), intermediate low (0.76-1.4 ng/ml) seen in 34 (19.2%), the normal range (1.5 – 3.75 ng/ml) seen in 119 (67.2%), and high enzyme activity (>3.76 ng/ml) seen in 20 (11.3%). No significant correlations between age, sex, and ethnic groups were recorded. Conclusion: The normal TPMT enzyme activity is between 1.5 and 3.76 ng/ml. A higher prevalence of TPMT deficiency was recorded and compared with international studies. Pretreatment screening using serum ELISA test for TPMT enzyme activity should be used to predict the risk of toxicity.

Population-Specific Distribution of TPMT Deficiency Variants and Ancestry Proportions in Ecuadorian Ethnic Groups: Towards Personalized Medicine.

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

Implications of Tioguanine Dosing in IBD Patients with a TPMT Deficiency.

Tioguanine is metabolised by fewer enzymatic steps compared to azathioprine and mercaptopurine, without generating 6-methylmercaptopurine ribonucleotides. However, thiopurine S-methyl transferase (TPMT) plays a role in early toxicity in all thiopurines. We aimed to describe the hazards and opportunities of tioguanine use in inflammatory bowel disease (IBD) patients with aberrant TPMT metabolism and propose preventative measures to safely prescribe tioguanine in these patients. In this retrospective cohort study, all determined TPMT genotypes (2016-2021) were evaluated for aberrant metabolism (i.e., intermediate and poor TPMT metabolisers). Subsequently, all IBD patients on tioguanine with aberrant TPMT genotypes were evaluated for tioguanine dosages, adverse drug events, lab abnormalities, treatment duration and effectiveness. TPMT genotypes were determined in 485 patients, of whom, 50 (10.3%) and 4 patients (0.8%) were intermediate and poor metabolisers, respectively. Of these patients, 12 intermediate and 4 poor TPMT metabolisers had been prescribed tioguanine in varying doses. In one poor TPMT metaboliser, tioguanine 10 mg/day induced delayed pancytopenia. In general, reduced tioguanine dosages of 5 mg/day for intermediate TPMT metabolisers, and 10 mg two-weekly for poor TPMT metabolisers, resulted in a safe, long-term treatment strategy. Diminished or absent TPMT enzyme activity was related with a pharmacokinetic shift of tioguanine metabolism which is associated with relatively late-occurring myelotoxicity in patients on standard tioguanine dose. However, in strongly reduced dose regimens with strict therapeutic drug and safety monitoring, tioguanine treatment remained a safe and effective option in IBD patients with dysfunctional TPMT.

B-225 Assessment of the Genotype Frequency of Thiopurine Methyltransferase (TPMT) Deficiency in a Large Cohort of Patients With Immune Mediated Inflammatory Disease and Cancer

Abstract Background Thiopurine methyltransferase (TPMT) alleles (*1/*2/*3A/*3B/*3C) are routinely genotyped to determine the metabolizer status of patients that plan to undergo thiopurine therapies (6-Mercaptopurine and Azathioprine). Screening of these alleles allows for personalized patient dosing and reduces the risk of severe or even fatal myelosuppression. The objective of this study was to determine the genotype frequency of TPMT deficiency in a large clinical cohort of patients with immune mediated inflammatory diseases and cancer. Methods All genotyping was performed in a reference clinical laboratory. Patient specimens were tested using genomic DNA isolated from whole blood. Clinical patient diagnoses were physician provided (international classification of disease, ICD-10). The genomic DNA was tested for the presence of three single nucleotide polymorphisms (SNPs) in the TPMT gene coding region and known to associate with reduced TPMT activity (rs1800462-Exon 5, rs1800460-Exon 7 and rs1142345-Exon 10). These polymorphisms determine the presence of *2/*3A/*3B/*3C alleles. Testing occurred from 10/26/2000 to 9/9/2022. Three different testing technologies were used during the 22 years of testing (PCR-RFLP from 2000 to 2003, Taqman™ chemistries from 2004 to 2015, and Luminex™ xMAP™ bead-based since 2016). Results A total of 219 129 patients were tested (mean age: 38 yrs, 56% female). As presented in the Table the genotype frequency for the wild type *1/*1 was 90.8% (95%CI: 90.7–90.9%), it was 9.0% (95%CI: 8.8–9.1%) for the heterozygous and 0.22% (95%CI: 0.020–0.024%) for homozygous variant, with similar frequency across disease states. The TPMT *1/*3B genotype frequency was very rare (<0.1%, n = 5) and the *3B/*3C homozygous variant (which cannot be distinguished from the *1/*3A) was calculated to have a frequency of 1:7 706 230 (95%CI: 1:7 043 228–1:8 369 232) and lower than initial estimates. Conclusion We estimated the frequency of TPMT genotypes in the largest cohort reported to date. The occurrence of the *3B/*3C genotype may be rarer than initially estimated.

Thiopurine Methyltransferase Intermediate Metabolizer Status and Thiopurine-Associated Toxicity During Maintenance Therapy in Childhood Acute Lymphoblastic Leukemia.

Mercaptopurine is a cornerstone of maintenance chemotherapy in childhood acute lymphoblastic leukemia (ALL). Its cytotoxic effects are mediated by 6-thioguanine nucleotides (TGNs) incorporation into lymphocyte DNA. Thiopurine methyltransferase (TPMT) inactivates mercaptopurine, and deficiency resulting from genetic variants increases TGN exposure and hematopoietic toxicity. Although mercaptopurine-dose reduction reduces toxicity risk without compromising relapse rate in patients with TPMT deficiency, dosing recommendations for those with moderately reduced activity (intermediate metabolizer (IM)) are less clear and their clinical impacts have yet to be established. This cohort study assessed the effect of TPMT IM status on mercaptopurine-associated toxicity and TGN blood exposure in pediatric patients with ALL initiated on standard dose mercaptopurine. Of 88 patients studied (mean age 4.8 years), 10 (11.4%) were TPMT IM, and all had undergone ≥ 3 cycles of maintenance therapy (80% completed). A larger proportion of TPMT IM than normal metabolizers (NM) had febrile neutropenia (FN) during the first two cycles of maintenance, reaching significance in the second cycle (57% vs. 15%, respectively; odds ratio=7.33, P < 0.05). Compared to NM, FN events occurred more frequently and with prolonged duration in IM in cycles 1 and 2 (adjusted P < 0.05). IM had a 2.46-fold increased hazard ratio for FN, and about twofold higher TGN level than NM (P < 0.05). Myelotoxicity was more common in IM than NM (86% vs. 42%, respectively) during cycle 2 (odds ratio=8.2, P < 0.05). TPMT IM initiated at a standard mercaptopurine dose are at greater risk for FN during early cycles of maintenance therapy, thus our findings support genotype-guided dose adjustment to reduce toxicity.

Cost-effectiveness analysis of genotype screening and therapeutic drug monitoring in patients with inflammatory bowel disease treated with azathioprine therapy: a Chinese healthcare perspective using real-world data.

BackgroundThis study aimed to analyze the cost-effectiveness of combining screening for thiopurine methyl transferase (TPMT) and nucleotide triphosphate diphosphatase (NUDT15) defective alleles with therapeutic drug monitoring (TDM) in Chinese patients with inflammatory bowel disease (IBD) treated with azathioprine (AZA).MethodsWe evaluated the cost-effectiveness of combining screening for NUDT15 and TPMT deficiency with TDM in patients receiving AZA treatment over a 1-year horizon by developing a decision tree model. Real-world data and published literature were used to derive model inputs. The model’s primary outcomes included quality-adjusted life-years (QALYs) and incremental cost-effectiveness ratios (ICERs). One-way and probabilistic sensitivity analyses were used to address uncertainty.ResultsCompared to NUDT15 genotyping, the combined TPMT/NUDT15 genotyping strategy cost an additional $13.83, yielding an ICER of $3,929.54/QALY, which was under the willingness-to-pay level of $30,425 per QALY in China. Compared to strategies with singular TPMT genotyping or no genotyping, the combined TPMT/NUDT15 genotyping strategy gained 0.00406 and 0.00782 QALYs and reduced the cost by $25.15 and $99.06, respectively. Additionally, incorporating TDM of AZA was more effective and less expensive than strategies without TDM. One-way sensitivity analysis revealed the expense attached to severe myelotoxicity to be the factor with the greatest influence in the present research. The application of the combined genotype screening strategy with TDM of AZA treatment was found to have a 91.7% chance of being cost-effective.ConclusionsFor Chinese patients with IBD who receive an AZA regimen, a strategy involving combined NUDT15/TPMT genotype screening prior to treatment initiation and incorporating TDM for treatment management is cost-effective compared to strategies involving genotyping of NUDT15 or TPMT alone or genotyping without TDM.

Star Allele-Based Haplotyping versus Gene-Wise Variant Burden Scoring for Predicting 6-Mercaptopurine Intolerance in Pediatric Acute Lymphoblastic Leukemia Patients.

Nudix Hydrolase 15 (NUDT15) and Thiopurine S-Methyltransferase (TPMT) are strong genetic determinants of thiopurine toxicity in pediatric acute lymphoblastic leukemia (ALL) patients. Since patients with NUDT15 or TPMT deficiency suffer severe adverse drug reactions, star (*) allele-based haplotypes have been used to predict an optimal 6-mercaptopurine (6-MP) dosing. However, star allele haplotyping suffers from insufficient, inconsistent, and even conflicting designations with uncertain and/or unknown functional alleles. Gene-wise variant burden (GVB) scoring enables us to utilize next-generation sequencing (NGS) data to predict 6-MP intolerance in children with ALL. Whole exome sequencing was performed for 244 pediatric ALL patients under 6-MP treatments. We assigned star alleles with PharmGKB haplotype set translational table. GVB for NUDT15 and TPMT was computed by aggregating in silico deleteriousness scores of multiple coding variants for each gene. Poor last-cycle dose intensity percent (DIP < 25%) was considered as 6-MP intolerance, resulting therapeutic failure of ALL. DIPs showed significant differences ( p < 0.05) among NUDT15 poor (PM, n = 1), intermediate (IM, n = 48), and normal (NM, n = 195) metabolizers. TPMT exhibited no PM and only seven IMs. GVB showed significant differences among the different haplotype groups of both NUDT15 and TPMT ( p < 0.05). Kruskal–Wallis test for DIP values showed statistical significances for the seven different GVB score bins of NUDT15. GVBNUDT15 outperformed the star allele-based haplotypes in predicting patients with reduced last-cycle DIPs at all DIP threshold levels (i.e., 5%, 10%, 15%, and 25%). In NUDT15-and-TPMT combined interaction analyses, GVBNUDT15 , TPMT outperformed star alleles [area under the receiver operating curve (AUROC) = 0.677 vs. 0.645] in specificity (0.813 vs. 0.796), sensitivity (0.526 vs. 0.474), and positive (0.192 vs. 0.164) and negative (0.953 vs. 0.947) predictive values. Overall, GVB correctly classified five more patients (i.e., one into below and four into above 25% DIP groups) than did star allele haplotypes. GVB analysis demonstrated that 6-MP intolerance in pediatric ALL can be reliably predicted by aggregating NGS-based common, rare, and novel variants together without hampering the predictive power of the conventional haplotype analysis.

Modeling the Outcome of Systematic TPMT Genotyping or Phenotyping Before Azathioprine Prescription: A Cost-Effectiveness Analysis.

Thiopurine S-methyltransferase (TPMT) testing, either by genotyping or phenotyping, can reduce the incidence of adverse severe myelotoxicity episodes induced by azathioprine. The comparative cost-effectiveness of TPMT genotyping and phenotyping are not known. Our aim was to assess the cost-effectiveness of phenotyping-based dosing of TPMT activity, genotyping-based screening and no screening (reference) for patients treated with azathioprine. A decision tree was built to compare the conventional weight-based dosing strategy with phenotyping and with genotyping using a micro-simulation model of patients with inflammatory bowel disease from the perspective of the French health care system. The time horizon was set up as 1year. Only direct medical costs were used. Data used were obtained from previous reports, except for screening test and admission costs, which were from real cases. The main outcome was the cost-effectiveness ratios, with an effectiveness criterion of one averted severe myelotoxicity episode. The total expected cost of the no screening strategy was €409/patient, the total expected cost of the phenotyping strategy was €427/patient, and the total expected cost of the genotyping strategy was €476/patient. The incremental cost-effectiveness ratio was €2602/severe myelotoxicity averted in using the phenotyping strategy, and €11,244/severe myelotoxicity averted in the genotyping strategy compared to the no screening strategy. At prevalence rates of severe myelotoxicity > 1%, phenotyping dominated genotyping and conventional strategies. The phenotype-based strategy to screen for TPMT deficiency dominates (cheaper and more effective) the genotype-based screening strategy in France. Phenotype-based screening dominates no screening in populations with a prevalence of severe myelosuppression due to azathioprine of > 1%.

Significance of NUDT15 gene in individualized treatment with 6-mercaptopurine in children with acute lymphoblastic leukemia

As an important drug during maintenance treatment of acute lymphoblastic leukemia (ALL), 6-mercaptopurine (6-MP) has several side effects, including hepatotoxicity and bone marrow suppression. Since its tolerability varies from person to person, 6-MP treatment should be individualized. The deficiency of thiopurine methyltransferase (TPMT) enzyme activity is associated with 6-MP intolerance. There is a lower frequency of mutation in TPMT alleles among Asian patients. Recent studies have shown that in ALL patients with NUDT15 gene mutation, the maximum tolerated dose of 6-MP is lower than the conventional dose. The article reviews the significance of NUDT15 gene in individualized treatment with 6-MP in children with ALL.

Analytical and clinical validation of an LC-MS/MS method to measure thiopurine S-methyltransferase activity by quantifying d3-6-MMP

BackgroundIdentification of patients with thiopurine S-methyltransferase (TPMT) deficiency prior to thiopurine drug therapy has become routine clinical practice worldwide. To measure TPMT activity, traditional radiochemical assays have been replaced by chromatographic methods. MethodInspired by the increasing number of isotope labelled sources that may be of benefit for the TPMT assay, a new LC-MS/MS method for TPMT activity was developed and validated. Isotope labelled d3-S-adenosyl-l-methionine (d3-SAM) was selected for the enzymatic methylation of mercaptopurine during sample incubation; d3-6-methylmercaptopurine (d3-6-MMP) with d2-2, 8-hypoxanthine as the internal standard was quantified to ascertain individual TPMT activity. ResultsThe validation of the analytical part of this method showed good linearity (coefficient of determination 0.9999 in the range of 1–500 ng/mL) with the intra-and inter-day impression CV% between 7.6% and 9.1% and 3.7% and 9.2%, respectively. Recovery ranged from 94.9% to 112.3%. The specificity of the enzymatic reaction was validated by using 108 clinical check samples. After compared with traditional radiochemical assay and genotype results, all homozygous and heterozygous deficiency clinical checks fitted into the nominal groups, inter-batch and intra–batch impression CV% were between 2.3% and 9.7%. ConclusionWith the inclusion of isotope labelled substrate, interfering non-enzymatic methylation no longer results in potential false assignment of abnormal patients. Furthermore, the method can be applied to patients who have already been prescribed thiopurine drugs. This new LC-MS/MS is therefore a favourable clinical routine application to test TPMT activity, as it shows excellent performance in identifying patients with TPMT deficiency.

Azathioprine Therapy in a Pediatric TPMT-Deficient Patient—Still an Option

We describe the case of a pediatric patient on azathioprine therapy with previously undiagnosed homozygote thiopurine S-methyltransferase (TPMT) deficiency, resulting in myelotoxic thiopurine metabolite levels. The patient was successfully treated with a very low azathioprine dose of 50 mg once a week (4% of standard dose), guided by frequent thiopurine metabolite measurement and a close clinical surveillance. We demonstrate that azathioprine therapy still might be an effective and safe therapeutic option in pediatric thiopurine S-methyltransferase-deficient IBD patients.

AZATHIOPRINE INDUCED PANCYTOPENIA-A CASE REPORT

&lt;p&gt;Azathioprine (AZA), a prodrug of 6 mercaptopurine, is an immunosuppressant that can be used as adjunctive therapy with corticosteroids in the treatment of arteritic form of ischemic optic neuropathy. Although myelotoxicity is known to occur while using azathioprine, severe pancytopenia is rarely seen. Patients with thiopurine methyltransferase (TPMT) deficiency are at high risk of developing severe myelosuppression. A 63 year* old female with ischemic optic neuropathy was initiated treatment with oral methylprednisone. As two courses of oral steroids showed no significant improvement, oral azathioprine 25 mg twice daily was added and gradually increased to 50 mg twice daily with relief of symptoms. 6 mo later, she was admitted with acute stroke and lab reports showed low levels of total blood counts or pancytopenia. The patient was put on broad spectrum antibiotics; given injection of granulocyte colony stimulating factor 300 mcg subcutaneously and blood transfusion as correction till counts normalised. She improved over 14 d and on next follow-up counts were in normal range. Causality was assessed by Naranjo causality assessment scale and a probable relationship was obtained between azathioprine and pancytopenia with a score of 6. Variations in TPMT activity occurs due to genetic polymorphism. Physicians should be aware of the possibility of myelosuppression while prescribing azathioprine. Frequent blood count monitoring is the most convenient way to avoid this problem where testing for TPMT deficiency is not possible.&lt;/p&gt;

Early Assessment of Thiopurine Metabolites Identifies Patients at Risk of Thiopurine-induced Leukopenia in Inflammatory Bowel Disease.

Only a quarter of thiopurine-induced myelotoxicity in inflammatory bowel disease [IBD] patients is related to thiopurine S-methyltransferase deficiency. We determined the predictive value of 6-thioguanine nucleotide [6-TGN] and 6-methylmercaptopurine ribonucleotide [6-MMPR] concentrations 1 week after initiation [T1] for development of leukopenia during the first 8 weeks of thiopurine treatment. The study was performed in IBD patients starting thiopurine therapy as part of the Dutch randomized controlled TOPIC trial [ClinicalTrials.gov NCT00521950]. Blood samples for metabolite measurement were collected at T1. Leukopenia was defined by leukocyte counts of <3.0 × 109/L. For comparison, patients without leukopenia who completed the 8 weeks on the stable dose were selected from the first 272 patients of the TOPIC trial. Thirty-two patients with, and 162 patients without leukopenia were analysed. T1 threshold 6-TGN concentrations of 213 pmol/8 × 108 erythrocytes and 3525 pmol/8 × 108 erythrocytes for 6-MMPR were defined: patients exceeding these values were at increased leukopenia risk (odds ratio [OR] 6.2 [95% CI: 2.8-13.8] and 5.9 [95% CI: 2.7-13.3], respectively). Leukopenia rates were higher in patients treated with mercaptopurine, compared with azathioprine (OR 7.3 [95% CI: 3.1-17.0]), and concurrent anti-TNF therapy (OR 5.1 [95% CI: 1.6-16.4]). Logistic regression analysis of thiopurine type, threshold concentrations, and concurrent anti-tumour necrosis factor [TNF] therapy revealed that elevations of both T1 6-TGN and 6-MMPR resulted in the highest risk for leukopenia, followed by exceeding only the T1 6-MMPR or 6-TGN threshold concentration (area under the curve 0.84 [95% CI: 0.76-0.92]). In ~80% of patients, leukopenia could be explained by T1 6-TGN and/or 6-MMPR elevations. Validation of the predictive model is needed before implementing in clinical practice.

Pharmacogenomics for leukemia treatment.

The pharmacokinetics and pharmacodynamics of therapeutic drugs can greatly vary among individuals. For example, it is sometimes necessary to alter the treatment of childhood acute lymphoblastic leukemia from the standard protocol. Genetic variation is one important factor, which can exert a wide range of effects on sensitivities and responses to therapeutic agents. Thiopurine S-methyl transferase (TPMT) is a useful test for predicting 6-mercaptopurine (6-MP) sensitivity in Caucasians. However, it is not effective for predicting the 6-MP therapeutic responses of Japanese patients because the frequency of TPMT deficiency is lower in the Japanese population (approximately 1% versus approximately 10% in Caucasians). Recently, NUDT15 polymorphisms have been reported to be predictive factors contributing to responsiveness to thiopurine therapy in Asians. The associations between genetic variants and therapeutic responses have been reported in Western countries. However, questions remain about whether results studying other races are applicable to Japanese due to differences in genetic variant frequencies among races. To provide personalized therapy based on genetic factors, we need to ascertain the relationships between genetic variants and therapeutic responses in Japanese childhood acute lymphoblastic leukemia cases.

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.

PTH-062 Treating TPMT Deficient Patients with Thiopurines: A Tertiary IBD Centre Experience

Introduction Thiopurine S-methyltransferase (TPMT) activity is inherited, with around 1 in 300 Caucasian individuals being deficient. Patients with zero TPMT activity are at risk of severe myelosuppression if given standard doses of thiopurines (TP), therefore TP are rarely prescribed. Our unit has treated a small number of these patients with 5–10% of the standard dose. Our experience was evaluated. Methods All patients with TPMT deficiency (enzyme activity Results 23 TPMT deficient patients (8 from Gastroenterology) were identified from 7100 patients. 5 (22%) patients were treated with TP, all of whom had inflammatory bowel disease. Patient 1–32 yr old Caucasian female with ileocolonic Crohn’s disease (CD) referred from another centre (TPMT 1). She was prescribed 50 mg/day mercaptopurine (MP) (0.66 mg/kg) before her TPMT was checked. After 10 days she developed severe pancytopenia (Hb 74 g/L, WCC 1.8x10 9 /L, Plts 62x10 9 /L) requiring hospital admission. She made a full recovery after cessation of the drug and is now in remission on infliximab. Patient 2–56 yr old Caucasian male with ileal and perianal CD (TPMT 2). He was prescribed 2.5 mg/day azathioprine (Aza) (0.03 mg/kg) for 1 year with normal FBC and liver function tests. His mean TGNs were sub therapeutic at 161 pmol/mL (normal range 235–450 pmol/8x108 8 RBC). He continued to have active disease despite Aza and was switched to methotrexate (MTX). Patient 3–47 yr old black, African female with pan-ulcerative colitis (UC) (TPMT 9). She was prescribed 10 mg/day Aza (0.11 mg/kg), which was increased to 20 mg/day (0.22 mg/kg) after 2 months because of sub therapeutic TGNs (83 pmol/mL). After the dose increase she developed drug-induced hepatitis with ALT 378 IU/L. The Aza was switched to MTX and the hepatitis resolved. Patient 4–32 yr old Caucasian female with pan-UC (TPMT 0). She was prescribed 12.5 mg/alternate days MP (0.1 mg/kg/day) which was decreased to 12.5 mg twice a week (0.06 mg/kg/day) due to supratherapuetic TGNs (1816 pmol/mL). Her FBC and liver function tests were normal but she continued to have active disease and has recently started on anti-TNF therapy. Patient 5–49 yr old Caucasian male with pouchitis after a pan-proctocolectomy for UC (TPMT genotype *3 A/*3 C). He was prescribed Aza 5 mg/day (0.07 mg/kg) with normal FBC and slightly supra therapeutic TGNs (695 pmol/mL). He continued to have active disease after 6 months so Aza was stopped and starting MTX was advised. Conclusion In this small cohort of patients, treatment with thiopurines failed to achieve clinical remission, despite aiming for therapeutic TGNs in 3 patients. Patients prescribed ≤5% of the recommended dose tolerated the drug well but pancytopenia and drug induced hepatitis occurred in doses above that. Low doses of TP may be considered with careful blood monitoring, particularly FBC, however other treatments are likely to be more beneficial to induce and maintain remission of CD and moderate to severe UC. Disclosure of Interest None Declared

The Outlier in All of Us: Why Implementing Pharmacogenomics Could Matter for Everyone.

The field of pharmacogenomics originally emerged in the 1950s from observations that a few rare individuals had unexpected, severe reactions to drugs. As recently as just 6 years ago, prominent views on the subject had largely remained unchanged, with authors from the US Food and Drug Administration (FDA) citing the purpose of pharmacogenetics as "tailoring treatment for the outliers." It should not be surprising if this is the prevailing view--the best-studied pharmacogenomic drug examples are indeed just that, genetic explanations of extreme responses or susceptibilities among usually a very small fraction of the human population. Thiopurine methyltransferase (TPMT) deficiency as a cause of severe myelosuppression upon treatment with azathioprine or mercaptopurine is found as a heterozygous trait in only ∼ 10% of patients, and homozygous (deficiency) carriers are even more rare--occurring in fewer than 1 in 300 patients. Malignant hyperthermia resulting from inhaled anesthetics and succinylcholine is believed to have a genetic incidence of only about 1 in 2000 people.

Genotyping Reduces Leukopenia in TPMT-Deficient Patients

Patients with thiopurine methyl transferase (TPMT) deficiency, which in its heterozygous form affects about 10% of the population, are at increased

Update on thiopurine pharmacogenetics in inflammatory bowel disease.

Azathioprine and 6-mercaptopurine remain pivotal therapies for the maintenance of disease remission in patients with Crohn's disease and ulcerative colitis. While thiopurine S-methyltransferase deficiency was the first pharmacogenetic phenomenon to be recognized to influence thiopurine toxicity and reliably predict leukopenia, it does not predict other adverse effects, nor does it explain most cases of thiopurine resistance. In recent years, a number of other genetic polymorphisms have received increasing attention in the literature. In particular, SNPs in NUDT15 and in the class II HLA locus have been shown to predict thiopurine-related leukopenia and pancreatitis. The aim of this review is to provide a concise update of genetic variability which may influence patient response to azathioprine and 6-mercaptopurine.

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.