| Thiopurine .S-methyltransferase (TPMT) is a cytoplasmic enzyme that preferentially catalyzes the S-methylation of aromatic and heterocyclic sulphydryl compounds, such as 6-mercaptopurine (6-MP), Azathioprine (AZA) and 6-thioguanine (6-TG). 6-MP and 6-TG are thiopurine drugs, which are used in organ transplant recipients, autoimmune diseases and as anti-cancer drugs. 6-MP and 6-TG are the prodrugs, which require activation before they can exert their cytotoxic effects. These drugs form the same active metabolites, the thioguanine nucleotides (TGNs). Low TPMT activity may lead to a high level of intracellular TGNs and a potential risk of myelosuppression. TPMT activity is inherited as an autosomal codominant trait. The frequency distribution of TPMT activity in Caucasian population studies is trimodal: 0.3% subjects have undetectable activity, 11% have intermediate activity and 89% inherit high enzyme activity. The functional gene for human TPMT is located on chromosome band 6p22.3 and includes 10 exons and 9 introns. Up to now, 11 mutant alleles have been clearly identified as responsible for low or intermediate TPMT activity. Four deficient alleles, TPMT2, TPMT3A, TPMT3B and TPMT3C were detected in over 80%-95% of different ethnic groups with low or intermediate TPMT activity. In the current study, we would focus on the most common TPMT deficient alleles.AZA is the prodrug of MP, it is frequently used for immunosuppression after renal transplantation because of cheaper and lower toxicity than cyclosporin . But subjects for low TPMT activity run a hige risk of severe myelosuppression or death after treatment with standard doses of AZA. It is very important to investigate TPMT polymorphisms?for the patients treated with AZA after renal transplantation.Therefore, we studied TPMT polymorphisms in Han Chinese subjects in Henan province and the influence of hemodialysis on TPMT activity in uremic patients who were on the waiting list for renal transplantation, and intended to provide a stronger scientific basis to optimize drug therapy on the basis of each patient's genetic constitution.Methods1. TPMT GenotypingVenous blood samples were obtained from 140 unrelated healthy Han subjects in Henan. The proportion of men and women was 96/44, their mean age was 22.1 ?.2 years. They do not have the history including transfusion, significant medical illness, injury and taken any drugs during 4 months. 30 uremia patients(14 females, 16 males), their mean age was 32?.65 years. The proportion of men and women was 16/14, The genome DNA were extracted by classic SDS-proteinase K-hydroxybenzene-chloroform methods from blood. We used PCR-based methods to detect the most common functional mutations of TPMT gene as described. Briefly, an allele-specific PCR (ASPCR) procedure was performed to detect TPMT * 1 and TPMT * 2. PCR-RFLP(restriction fragment length polymorphism) was used for analysis of TPMT 3A. TPMT3B and TPMT3C.2. TPMT phenotyping2.1. Blood sample30 unrelated healthy Han subjects in Henan were recruited from a total of 140 subjects. 28 of them were homozygous for the TPMT1, the others were TPMT*3C. 30 uremia patients all were TPMT1. Venous blood samples (5ml each) were obtained from 30 healthy subjects and uremia patients before and after a 4-h period of hemodialysis. 5ml of whole blood collected in lithium heparinized tube was centrifuged at 2800g for 10 min at 4癈, the plasma and buffy coat were discarded. The red blood cells (RBC) were washed two times and centrifuged again. The number of RBC was counted and prepared to a final dilution of 41 09 cells/ml. RBC were lysed with cold distilled water (4 ml for 1 ml of solution) and centrifuged (2800xg, 20 min, 4癈). The samples were kept frozen at -20 癈 until the day of analysis.2.2. TPMT activity assayA new-HPLC assay was used to measure erythrocyte TPMT activity. TPMT activity is expressed in nanomoles of 6-MMP formed per milliliter of packed red blood cells per hour (nmol/h/PRBC).After slow thawing, 900ul lysate of RBC was chelated by adding C...
|
PDF Full Text Request