Genes implicated in thiopurine-induced toxicity: Comparing TPMT enzyme activity with clinical phenotype and exome data in a paediatric IBD cohort
Tracy CoelhoGaia AndreolettiJames J. AshtonAkshay BatraNadeem AfzalYifang GaoAnthony P. WilliamsR. Mark BeattieSarah Ennis
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Abstract The aim of our study was to assess the utility of next generation sequencing (NGS) for predicting toxicity and clinical response to thiopurine drugs in paediatric patients with inflammatory bowel disease. Exome data for 100 patients were assessed against biochemically measured TPMT enzyme activity, clinical response and adverse effects. The TPMT gene and a panel of 15 other genes implicated in thiopurine toxicity were analysed using a gene based statistical test (SKAT-O test). Nine patients out of 100 (Crohn’s disease- 67, ulcerative colitis- 23 and IBDU-10) had known TPMT mutations associated with deficient enzyme activity. A novel and a highly pathogenic TPMT variant not detectable through standard genotyping, was identified through NGS in an individual intolerant to thiopurines. Of the 14 patients intolerant to thiopurines, NGS identified deleterious TPMT variants in 5 individuals whereas the biochemical test identified 8 individuals as intolerant (sensitivity 35.7% and 57.14%; specificity 93.75% and 50% respectively). SKAT-O test identified a significant association between MOCOS gene and TPMT activity (p = 0.0015), not previously reported. Although NGS has the ability to detect rare or novel variants not otherwise identified through standard genotyping, it demonstrates no clear advantage over the biochemical test in predicting toxicity in our modest cohort.Keywords:
Thiopurine methyltransferase
Exome
ITPA
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.
Thiopurine methyltransferase
ITPA
Multiplex
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OBJECTIVE To study the relationship between genetic polymorphism of thiopurine methyltransferase(TPMT) and inosine triphosphate pyrophosphatase (ITPA) with adverse drug reactions in 6-mercaptopurine in maintenance treatment of childhood acute lymphoblastic leukemia.METHODS Method of the allele specific PCR(ASPCR) and PCR-restriction fragment length polymorphism(PCR-RFLP) analysis the polymorphism of thiopurine methyltransferase(TPMT) and inosine triphosphate pyrophosphatase(ITPA),HPLC was used for the determination of TPMT and ITPA activity,reference to the adverse reactions of SFDA standards was taken as the ref erence for evaluation of the adverse reactions of 6-mercaptopurine treatment in children with acute lymphoblastic leukemia.RESULTS Risk of bone marrow suppression of TPMT * 3C is higher than wild genome.It was not found that the gene TPMT and ITPA polymorphisms linked with liver damage and gastrointestinal reactions.CONCLUSION Dosage adjustment in TPMT gene mutation can avoid the occurrence of bone marrow suppression.The relationship between TPMT and ITPA polymorphism and 6-mercaptopurine still need to integrate drug interactions with other drug metabolism enzymes in further study of genetic polymorphism.
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Objective To systematically investigate the relationships between thiopurine S-methyltransferase(TPMT)and inosine triphosphate pyrophosphatase(ITPA)polymorphisms and azathioprine-related adverse drug reactions in patients with kidney transplantation.Methods Erythrocyte TPMT and ITPA activity of 155 patients with kidney transplantation under AZA therapy was determined by high performance liquid chromatography(HPLC).The frequency of 4 common TPMT mutant alleles,TPMT2,3A,3B and 3C,was determined by allele-specific PCR and PCR-restriction fragment length polymorphism(PCR-PFLP)analysis.Meanwhile,the frequency of ITPA 94CA and IVS2+21AC was determined by real-time fluorescence PCR and PCR-PFLP.Results Among 155 patients in this study,120 did not experience any adverse reactions under standard dosage of AZA and they were classified as control group.The mean TPMT activity of control group was(37.26±11.60)U and the mean ITPA activity was(97.9±34.7)U.Thirty-five patients stopped azathioprine medication or were on reduced dose due to azathioprine-related side effects,of whom twelve developed hematotoxicity with mean TPMT activity of(22.92±10.67)U,much lower than control group(P0.05).Only two patients with TPMT3C heterozygous alleles were found in these twelve patients.AZA induced hematotoxicity was related to the lower TPMT activity.Eighteen patients developed hepatotoxicity and there was no statistical difference between the mean TPMT or ITPA activity and the control mean(P0.05).Only two cases with TPMT3C and four cases with ITPA 94CA heterozygous alleles were found.No significant associations between TPMT or ITPA genetic polymorphisms and AZA-related hepatotoxicity could be detected.In five patients with gastrointestinal disturbance(including one patient who developed hepatotoxicity simultaneously),two patients with deficient ITPA activity were homozygote for 94CA and the other three patients with low ITPA activity had 94CA heterozygous alleles.The patient who experienced flu-like symptoms showed deficient ITPA activity and he was the remaining homozygote for 94CA.Patients with ITPA 94CA homozygous alleles are at high risk to develop AZA-related gastrointestinal disturbance and flu-like symptoms.Conclusion Before commencing AZA treatment,it is important to measure erythrocyte TPMT activity in renal transplant recipients in order to prevent AZA-related hematotoxicity.Pretherapeutic screening of patients for erythrocyte ITPA activity and genetic polymorphisms should be useful to avoid AZA-related gastrointestinal disturbance and flu-like symptoms for safer and more tolerable AZA treatment.
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ITPA
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Abstract: Thiopurine-S-methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) are crucial enzymes involved in the metabolism of thiopurine drugs. Significant interethnic variation in the expression of TPMT and ITPA is caused by single nucleotide polymorphisms of genes encoding these proteins. The aim of this study was to describe the distribution of TPMT and ITPA polymorphisms in healthy Tunisian subjects and to establish the metabolizer status of thiopurine drugs in this population. A total of 309 healthy Tunisian subjects were recruited among blood donors of Fattouma Bourguiba Hospital of Monastir. A written informed consent was obtained from all subjects. Whole blood samples were collected from every subject in ethylenediaminetetraacetic acid tubes. TPMT (c.238 G > C, c.460 G > A and c.719A > G) and ITPA (c.94C > A and IVS2+21A > C) mutations were genotyped using polymerase chain reaction-restriction fragment length polymorphism. The observed frequencies of TPMT*3A and TPMT*3C alleles were both 0.8%. The phenotype distribution of TPMT was bimodal: 96.8% of subjects were extensive metabolizers and 3.2% were intermediate metabolizers. Genotyping of ITPA revealed frequencies of 9% and 3% for IVS2+21A > C and c.94C > A mutations, respectively. Accordingly, a trimodal phenotype distribution was found: 75.4% of the subjects were extensive metabolizers, 23.4% were intermediate metabolizers, and 1.2% wereslow metabolizers. Combination of TPMT and ITPA genotyping has revealed that a quarter of the Tunisian Population carries polymorphisms that reduce the metabolic activities of these enzymes.
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Pharmacogenetic studies in inflammatory bowel diseases (IBD) are mainly focused on genes involved in the metabolism of Azathioprine (AZA). Use of AZA is limited by its toxicity, which occurs in 20-30% of patients. Variants in the Thiopurine S-methyltransferase (TPMT) and Inosine triphosphate pyrophosphatase (ITPA) genes have been associated with AZA toxicity, but also can contribute to the lack of response. The aims of this study were to determine the contribution of TPMT and ITPA variants in the development of AZA-related toxicity and response.Variants associated with the decrease of enzyme activity in TPMT and ITPA genes were genotyped with the Snapshot system in 232 IBD patients treated with AZA, and correlated with the clinical response and development of adverse drug reactions in a retrospective case-control study.Genotypic analysis showed that there is a statistical significance between c.94C > A variant on ITPA gene with non response to AZA treatment (p=0.005) and arthralgia (OR 8.2353; 95%CI 1.752-38.87, p=0.0041), as well as between mutant TPMT alleles and myelosuppression (OR 7.5; 95%CI 1.4456-38.91, p=0.0304).There is a positive correlation between c.94C > A variant on ITPA with clinical response. Mutant alleles on TPMT and the variant c.94C > A on ITPA gene predict side effects induced by AZA in our population (myelosuppression and arthralgia).
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Pharmacogenomics
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Summary Background Thiopurines are widely used for the treatment of inflammatory bowel disease, but are associated with the development of side effects. It has been suggested that the enzyme inosine triphosphate pyrophosphatase (ITPA) plays a role in the digestion of thiopurines and that defective activity resulting from polymorphisms in the inosine triphosphate pyrophosphatase encoding genes may be associated with thiopurine‐induced side effects. Current studies are controversial regarding this hypothesis. Aim To perform a meta‐analysis and gain more insight into a possible correlation between thiopurine‐induced side effects and ITPA polymorphisms. Methods We explored Medline for articles on ITPA polymorphisms and thiopurine toxicity. Studies that compared ITPA polymorphism frequencies among thiopurine‐tolerant and ‐intolerant adult inflammatory bowel disease patients were included in this meta‐analysis. Results Nine published studies investigated associations between ITPA polymorphisms and thiopurine toxicity. Six studies (with 751 patients included) met our inclusion criteria and were processed in the meta‐analysis. This analysis demonstrates that the ITPA 94C→A polymorphism, is not significantly associated with any of the studied side effect parameters. Conclusions This meta‐analysis does not prove a correlation between the development of thiopurine toxicity and the ITPA 94C→A polymorphism. This implies that there is no clinical relevance to determine ITPA polymorphisms in thiopurine‐treated patients.
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Adverse reactions to thiopurines may be predisposed by thiopurine methyltransferase (TPMT) or inosine triphosphate pyrophosphatase (ITPA) gene mutations.We examined the frequencies of TPMT and ITPA gene polymorphisms in 812 Korean patients with inflammatory bowel diseases using denaturing high performance liquid chromatography and direct sequencing.The allele frequencies of TPMT*2, TPMT*3A, TPMT*3B, and TPMT*3C were 0, 0, 0, and 0.010 (17/1624), respectively. For the ITPA polymorphism, 173 subjects were heterozygous and 5 were homozygous for the 94C>A missense mutation (allele frequency of A, 0.113). Moreover, the 87T>C, IVS2+21A>C, and IVS2+53C>T polymorphisms were found in one patient each (1/1624), respectively. Of these, 87T>C and IVS2+53C>T were novel single nucleotide polymorphisms of the ITPA gene whose clinical significance should be further evaluated.Our data describe TPMT and ITPA gene mutation patterns among Koreans and provide a basis for screening studies to identify patients at high risk for myelotoxicity from thiopurine drugs.
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Azathioprine (aza) therapy is beneficial in the treatment of inflammatory bowel disease, but 10%-30% of patients cannot tolerate aza therapy because of adverse drug reactions. Thiopurine S-methyltransferase (TPMT) deficiency predisposes to myelotoxicity, but its association with other side effects is less clear. Inosine triphosphatase (ITPA) mutations are other pharmacogenetic polymorphisms possibly involved in thiopurine metabolism and tolerance.We analyzed data from a 6-month prospective study including 71 patients with Crohn disease undergoing first-time aza treatment with respect to aza intolerance. Patients were genotyped for common TPMT and ITPA mutations and had pretherapy TPMT activity measured.Early drop-out (within 2 weeks) from aza therapy was associated with ITPA 94C > A [P = 0.020; odds ratio (OR), 4.6; 95% confidence interval (95% CI), 1.2-17.4] and low TPMT activity [<10 nmol/(mL erythrocytes . h); P = 0.007; OR = 5.5; 95% CI, 1.6-19.2]. A high-risk group defined by ITPA 94C > A or TPMT <10 nmol/(mL erythrocytes . h) showed significant association with early drop-out (P = 0.001; OR = 11.3; 95% CI, 2.5-50.0) and all drop-outs (P = 0.002; OR = 4.8; 95% CI, 1.8-13.3). For only drop-outs attributable to aza-related side effects (n = 16), there was a significant association with ITPA 94C > A (P = 0.002; OR = 7.8; 95% CI, 2.1-29.1). Time-to-event analysis over the 24-week study period revealed a significant association (P = 0.031) between the time to drop-out and ITPA 94C > A mutant allele carrier status.Patients with ITPA 94C > A mutations or low TPMT activity constitute a pharmacogenetic high-risk group for drop-out from aza therapy. ITPA 94C>A appears to be a promising marker indicating predisposition to aza intolerance.
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