Associations Of Thiopurine S-methyltransferase And Inosine Triphosphate Pyrophosphatase Genetic Polymorphisms With Azathioprine-related Adverse Drug Reactions In Renal Transplant Recipients

Azathioprine (AZA) is a thiopurine prodrug commonly used in the chemotherapy of acute leukemia, for immunosuppression after solid-organ transplantation, and increasingly for immunomodulation in autoimmune disease. However, its use has been complicated by a high incidence of serious adverse drug reactions such as hematotoxicity, hepatotoxicity and gastrointestinal disturbance. The thiopurine S-methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) genetic polymorphisms have a significant clinical impact on the adverse reactions of AZA. Thus, investigation on associations of TPMT and ITPA genetic polymorphisms with AZA-related adverse reactions in renal transplant recipients conduces to individualize drug treatment and avoid severe adverse drug reactions.In the present study, 155 renal transplant recipients were recruited. Erythrocyte TPMT activity was measured in all samples by a new high-performance liquid chromatography (HPLC) procedure and genotype was determined for the TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C by allele specific polymerase chain reaction (ASPCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) method. Meanwhile, the influence to TPMT activity of drugs (ciclosporin, hydrocortisone and nifedipine, et al) which were usually used simultaneously with AZA was examined. In addition, another new HPLC method was used to measure erythrocyte ITPA activity in these patients and real-time fluorescence PCR and PCR-RFLP method were used to detect ITPA 94C>A and IVS2+21A>C polymorphisms. According to these results and the occurrence and type of adverse reactions to AZA, the relationships between TPMT and ITPA genetic polymorphisms and AZA-induced adverse reactions were systematically analyzed. The TPMT activity range of these 155 patients was 13.04~68.25U with a mean value of (36.00±12.32) U. TPMT activity showed a normal distribution. 30 patients (19.4%) had intermediat TPMT activity and no case with TPMT deficiency was found in all patients studied. All of 7 patients with TPMT*3C heterogyous alleles had intermediate TPMT activity. The allele frequency of the TPMT*3C was 2.26%. TPMT activity reduced in patients with TPMT*3C mutation. In vitro, ciclosporin, hydrocortisone, captopril and allopurinol had nearly no impacts on TPMT activity. Nifedipine highly inhibited TPMT activity, the mean IC50 value in intermediate TPMT activity group was (24.37±16.64)μg·mL-1 and in normal TPMT activity group was (11.81±10.03)μg·mL-1. The ITPA activity range of all patients was 0~191.2U with a mean value of (96.1±37.1) U. ITPA activity showed a bimodal distribution. The 3 patients who showed zero ITPA activity were ITPA activity deficiency and they were homozygote for 94C>A. All 31 patients who were heterozygote for 94C>A polymorphism showed low ITPA activity (<70U). The allele frequency of the 94C>A was 11.9%. The IVS2+21A>C mutation in the patients of this study was not found. It indicats that the ITPA 94C>A is probably a common polymorphism in the patients with lower ITPA activity.Among 155 patients in this study, 120 cases 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. 35 cases had stopped azathioprine medication or were on reduced dose due to azathioprine-related side effects. 12 patients developed hematotoxicity. The mean TPMT activity of them was (22.92±10.67) U, much lower than control group (t-test, P<0.05). Only 2 patients with TPMT*3C heterogyous allelles were found in these 12 patients. AZA induced hematotoxicity is related to the lower TPMT activity. 18 patients developed hepatotoxicity and there was no statistical difference between the mean TPMT or ITPA activity and the control mean (t-test, P>0.05). Only 2 cases with TPMT*3C and 4 cases with ITPA 94C>A heterogyous alleles were found. No significant associations between TPMT or ITPA genetic polymorphisms and AZA-related hepatotoxicity could be detected. In 5 patients with gastrointestinal disturbance (inclinding 1 patients who developed hepatotoxicity simultaneously), 2 patients with deficient ITPA activity were homozygote for 94C>A and other 3 patients with low ITPA activity had 94C>A heterozygous alleles. The patient who experienced flu-like symptoms showed deficient ITPA activity and he was the remaining homozygote for 94C>A. Patients with ITPA 94C>A homozygous alleles are at high risk to develop AZA-related gastrointestinal disturbance and flu-like symptoms.In 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.