The Expression Of DNA Methyltransferase MRNA In Children With Persistent And Chronic Immune Thrombocytopenia

BackgroundImmune thrombocytopenia(ITP)is the most common hemorrhagic disease among children. Based on the course of disease, ITP is divided into three categories:newly diagnosed ITP, persistent ITP and chronic ITP. Newly diagnosed ITP is easy to cure, while persistent and chronic ITP are more difficult to cure. The specific pathogenesis of ITP has not been fully clarified so far.It has been demonstrated that abnormal DNA methylation contributes to the occurrence and development of various diseases including autoimmune disease and tumor. Some researchers have found that adult ITP patients have DNA methylation which contributes to occurrence of ITP, while little study has been conducted about the relationship between DNA methylation and pathogenesis of pediatric persistent and chronic ITP.ObjectiveThis paper aims to study the relationship between DNA methylation and pathogenesis of pediatric persistent and chronic ITP by analyzing the DNA methyltransferase(DNMTs) mRNA expression in pediatric persistent and chronic ITP with RT-PCR.Methods1. Object of study. During the period of Jan 2014 to Jan 2015, hundreds of persistent and chronic ITP children accepted inpatient, outpatient or follow-up treatment in pediatrics of First Affiliated Hospital of Xinxiang Medical University.25 children are randomly selected form these ITP children, forming the experiment group and another 20 children identified as healthy through physical examination form the control group, according to ITP diagnosis and classification standards proposed by Subspecialty Group of Hematology, the Society of Pediatrics, Chinese Medical Association. Both experiment and control group don’t use folic acid, vitamin B6 and vitamin B12 before 4 weeks. No statistical difference of age and gender in both experiment and control group. Specimen collection:collect 2ml peripheral blood before breakfast with EDTA as anticoagulantion using bioclean operation.2. Detection of DNMTs mRNA expression. Separate peripheral blood lymphocyts, extract RNA using Trizol, and detect expression of DNMT1, DNMT3A, DNMT3B mRNA using RT-PCR.3. Statistical analysis. Conduct statistical analysis with SPSS 16.0. Measurement data are represented by mean±standard variation (-x±S); enumeration data is expressed as perce-ntage (%); difference of two sample’s mean is caculated using independent sample t-test method; enumeration data is verified with chi-square test method; correlation analysis of DNMTs mRNA expression and clinical data is conducted using Bivariate method, and P<0.05 indicates significant difference.Results1. The experiment group has a PLT of (36.2±19.6)×109/L, and control group (168.8±46.8)×109/L. It can be seen that experiment group has a significantly lower PLT than control group. The difference (t=-11.85, P=0.000) is statistically meaningful.2. The expression of DNMT1 mRNA is (0.17±0.05) and (0.27±0.10) for experiment and control group respectively. Experiment group has a much lower value than control group. The difference (t=-3.912, P=0.001) is statistically meaningful. The expression of DNMT3A mRNA is (0.20±0.10) and (0.32±0.11) for experiment and control group respectively. Experiment group has an obvious lower value than control group. The difference (t=-3.779,P=0.000) is statistically meaningful. The expression of DNMT3B mRNA is (0.16±0.11) and (0.31±0.11) for experiment and control group respectively. Experiment group has an obvious lower value than control group. The difference (t=-4.641, P=0.000) is statistically meaningful.3. The expression of DNMT1 and DNMT3B mRNA is positively correlated with r=0.433 and P=0.031; the expression of DNMT3A and DNMT3B mRNA is also positively correlated with r=0.721 and P=0.000; The expression of DNMT1 and DNMT3A mRNA has no correlation with r=0.277 and P=0.180.4. The expression of DNMTs mRNA is negatively correlated with age (DNMT1: r=-0.565, P=0.003; DNMT3A:r=-0.563, P=0.003; DNMT3B:r=-0.506, P=0.01).5. The expression of DNMT1 mRNA in male and female is (0.184±0.059) and (0.164±0.050) respectively, which indicates no obvious difference in different genders (t=0.948, P=0.353). The expression of DNMT3A mRNA in male and female is (0.187±0.082) and (0.219±0.115) respectively, which indicates no obvious difference in different genders (t=-0.78, P=0.443). The expression of DNMT3B mRNA in male and female is (0.123±0.106) and (0.182±0.116) respectively, which indicates no obvious difference in different genders (t=-1.29, P=0.210).Conclusions1. DNA hypomethylation, which may contribute to the pathogenesis, exists in pediatric persistent and chronic ITP.2. DNMTs mRNA may has synergistic effect in the process of pediatric persistent and chronic ITP.3. The expression of DNMTs mRNA has relationship with age probably.