The Singnificance Of The Expression Of The TPMT In Childhood Acut Lymphoblastic Leukemia

Objective: By studying children with acute lymphoblastic leukemia and control group children mercaptopurine methyltransferase activity to explore the distribution of children in TPMT activity and its variation; Monitoring 6-TGNs concentrations of peripheral blood erythrocyte, in order to explore the relationship between TPMT genetic polymorphism and drug concentration;Assessing the correlation between drug concentration and drug efficacy as well as toxicity. By detecting TPMT gene polymorphism in children with ALL and in vivo active metabolite concentrations, combined with follow-up observation of clinical toxicity occurred, to analysis of the test of the significance of individual chemotherapy in ALL children.Methods: 1.HPLC method was carried out to determine TPMT activity(n=100), which activity at diagnosis and after taking Thiopurine drugs. At the same time determination of TPMT activity in healthy children(n=100), these children come from the health care clinic, TPMT activity is equal to unit time per unit mass of hemoglobin in the amount of the generated 6-MTG.. 2. Using online Primer3 software design primers, PCR products were purified by Shrimp alkaline enzyme and exonucleaseⅠ, detecting by ABI3130 Sequencer.To sequence TPMT gene of 10 exons and promoter region,which in patients with clinical events(n=30),Sequencing data analysis by software named Polyphred. 3. According to the program previously reported, A modified HPLC method to monitor the peripheral red blood cell concentrations of 6-TGNs,which abandons the time-consuming extraction and toxic substances in the extraction process, Instead, the application of 70% perchloric acid was used to precipitate proteins and then Then at 100℃to nucleotide hydrolysis, making it more conducive to the monitoring of clinical samples. Determination concentrations of 6-TGNs of consolidation therapy (CAM)(n=37), maintenance treatment (n=51)of ALL children, respectively, in order to analysis of correlation between drug concentration, TPMT activity and toxicity. Results:1,The average TPMT activity of Han children (31.72±10.31) umol.L-16-MTG / gHb.h, by the Kolmogorov-Smirnov test and the W test statistical analysis was normal (P =0.064).ALL and control group of children, no significant difference in distribution of TPMT activity ((31.38±10.20) vs (30.70±9.67), P = 0.577); No gender differences in TPMT activity (P = 0.45).2,When children of ALL take thiopurine, TPMT activity of the peripheral RBC changed, there are higher TPMT activity after treatment than before treatment((38.20±11.40)vs(31.72±10.31)), the difference was statistically significan(tP<0.01); TPMT activity before treatment and the percentage of growth was significant correlation (r=-0.804;P<0.01).3,ALL patients(n=100) were followed up of toxicity of the treatment process, there are significant differences in TPMT activity of the bone marrow suppression group and the group without bone marrow suppression ((20.96±7.24)v(s33.67±9.30),P<0.05), TPMT activity in severe liver toxicity group were similar to without liver toxicity group((30.30±7.37)vs(31.65±10.81),P=0.597).4,There are the five known SNP site and a newly discovered SNP sites were found While sequencing the DNA of TPMT gene(n=30) , 2/5 alleles mutations in exon were synonymous mutations (474T> C) and missense mutation (719A> G),respectively. Newly discovered SNP sites located in intron 7 (581-87C> T), has been submitted to GenBank dbSNP, in order to apply for the NCBI number. At the vicinity of the promoter region of -100, there are variation of the number of tandem repeats polymorphism, repeat count * V3-* V6, which consists of 17-18 base composition .5,During the TPMT gene sequencing about ALL patients with clinical events(n=30), only 3 children are heterozygosity mutations of 719A> G (TPMT*3C). We believe that, TPMT * 3C may be the only mutant alleles in Chinese Han people ,which can decreased activity of TPMT .6,VNTRs in the promoter regions, which binding transcription factor,can affect the transcription of TPMT,. However, VNTRs polymorphism in Han children did not cause differences in TPMT activity (P = 0.186), This phenomenon may be related to sample size, there may be ethnic differences, In need of large sample size studies to confirm this.7,6-TGNs concentration in peripheral red blood cells range of 15.7-1575.6 pmoL / 8×108RBC (median 259.4; average of 471.9); The correlation between pRBC 6-TGNs concentration at the first 10-12 days of consolidation therapy (CAM) and neutrophil count at 8th days after the end of treatment (ie after taking thiopurine 22th days) was negative (n=30,r =- 0.867, P= 0.00);Conclusion:1, TPMT activity of Han children was normal, the activity no significant difference between ALL children and healthy children, and no significant difference in defferent gender.2, When children of ALL take thiopurine, TPMT activity was significantly higher than diagnosis ,the part of the growth was negatively correlated with the initial visit, the reasons causing this phenomenon needs further study.3,We believe that TPMT*3C may be the only mutant alleles in Chinese Han people ,which can decreased activity of TPMT, VNTR polymorphism on the impact of research requires further study in large scale sample.4,Determination of TPMT phenotype - genotype could predict drug response to treatment, prevent from adverse events during chemotherapy.5,The correlation between pRBC 6-TGNs concentration and neutrophil count was significantly negative, at the same time ,there have a good relationship between the duration of neutropenia and 6-TGNs concentration ,which can be used as an early indicator of dose adjustment.6,TPMT activity determination, genotyping, combined with the determination of the concentration of 6-TGNs in pRBC could guide individual treatment.