Preliminary Study On Hereditary Susceptibility Of The Patients With Immuno-related Pancytopenia

Objective:1. To investigate the global DNA methylation and the expression of genes that regulate methylation in CD4+T cells of the patients with IRP and to explore the role of methylation in pathogenesis of IRP.2. To study the expression of CD70and the methylation level of CD70promoter in immuno-related pancytopenia (IRP) patients, and explore the role of CD70in the pathogenesis of IRP.3. To detect and investigate the association of SNP variation of4immuno-related genetic susceptibility in IRP in Chinese Han population.Methods:Part1.30diagnosed IRP patients and15healthy controls were enrolled in this study and PBMC (peripheral blood mononuclear cells) were isolated from the peripheral venous blood by density gradient centrifugation. CD4+T cells were isolated from PBMC using magnetic cell separation technique and DNA from CD4+T cells were extracted by DNAout kit while RNA frome CD4+T cells were extracted by TRIZol seperately. The global DNA methylation was tested with ELISA like method using the MethylampTM Global DNA Methylation Quantification Kit (Colorimetric) and mRNA levels of DNA methyltransferases (DNMTs) and methylated CPG binding proteins (MBDs) were measured by real-time quantitative polymerase chain reaction(RT-PCR) and then statistically analyzed.Part235diagnosed IRP patients and15healthy controls were enrolled in this study and PBMC were isolated from the peripheral venous blood by density gradient centrifugation. CD4+T cells were isolated from PBMC using magnetic cell separation technique and test the expression of CD70on CD4+T cells by flow cytomety (FCM). mRNA level of CD70in PBMC CD4+T cells were measured by RT-PCR. PBMC CD4+T cells were treated by methylation kit and then the methylation level of CD70promoter were detected by methylation specific PCR (MSP).Part3.213diagnosed IRP patients and112healthy controls were enrolled in this study. PBMC were isolated from the peripheral venous blood by density gradient centrifugation. Extracting mononuclear cells genomic DNA by DNAout kit. Amplify the DNA sequence near the undertest purpose loci with primers designed according to the GenBank database and send the amplified products to biotechnology company to investigate the SNP variation of purpose loci (rsl800871、rs2476601、rs9463772、 rs13277113) and do the statistical analysis.Results:Part1. Compared with normal controls, the level of global DNA methylation in peripheral blood (PB) CD4+T cells of IRP patients was significantly lower(p<0.05). The level of global DNA methylation in PB CD4+T cells of untreated IRP patients (3.525+2.046)%and remission patients (4.790±1.471)%were significantly lower than that of normal controls (10.101±0.3449)%respectively, while the result in remission group and untreated group had no significant difference (P=0.062). DNMT3b mRNA level of untreated IRP patients (1.332±0.785) was significantly lower than that of normal controls (2.077±1.059) in CD4+T cells (P<0.05). The mRNA expression of MBD2was significantly higher in CD4+T cells from untreated (2.999±1.601) and remission IRP patients (2.055±1.576) than that in controls (0.581±0.247)(P<0.05). The MBD4mRNA level was significantly higher in CD4+T cells from untreated IRP patients (2.736±2.719) compared to that in normal controls (1.167±1.006)(P<0.05). DNMT3b mRNA expression was positive related with CD4±T cell DNA methylation in IRP patients (r=0.569, p<0.05). The MBD2mRNA expression negatively related to the level of global DNA methylation in PB CD4+T cells in IRP patients and the ratio of Thl/Th2(r=-0.763,p<0.05; r=-0.625,p<0.05)Part2. The percentage of CD70expression on CD4+T cells of untreated IRP patients(7.46±1.51)%was significantly higher than that of recovered IRP patients(5.95±1.34)%and normal controls (1.83±0.6)%and the result of recovered IRP patients was significantly higher than that of normal controls (P<0.05). The relative expressions of CD70mRNA in CD4+T cells were [2.314(0.200-6.084)]、[1.021(0.135-3.434)].[0.353(0.008～2.258)] in three groups respectively. The differences among untreated IRP patients, recovered IRP patients and normal controls were significant (P<0.05). The CD70promoter methylation level in CD4+T cells of all IRP patients was significantly lower than that of normal controls (p<0.05) but there was no significant difference among untreated IRP patients、recovered IRP patients and healthy controls. The expression of CD70positively correlated to the ratio of CD5+B cells (r=0.533, p<0.001).Part3The PTPN22SNP (rs2476601) G allele was presented in the all samples, no single-nucleotide polymorphism (rs2476601) was detected. Compared with normal controls, the genotype and allele frequencies of IL-10SNP (rs1800871) didn’t have significant difference(p>0.05). The A allele of BLK rs13277113was a risk factor(OR1.616,95%CI=1.113-2.347, p=0.015) and the morbidity in the population with this risk factor was1.616times in normal controls. The frequency distribution of AA genotype was5.263times normal controls. This showed that the AA genotype was relevant to the pathogenesis of IRP. T allele frequencies of IL-17F rs9463772in IRP patients was significantly higher than that in normal controls (14.6%vs.8.9%, OR=1.737,95%CI=1.020～2.959, p=0.04). The result showed that T allele of IL-17F rs9463772was a risk factor, the morbidity in the population with this risk factor was1.737times in normal controls.Conclusions:1. The globe DNA methylation decreased in CD4+T cells and the expression of methylation related regulating genes was abnormal in IRP patients. The global methylation of CD4+T cells positively related to the DNMT3b mRNA expression but negatively related to the MBD2mRNA expression and the ratio of Thl/Th2. All above demonstrate that hypomethylation and abnormal methylation related regulation genes correlated with autoantibodies production and the immunological function status of IRP.2. Demethylation of regulatory sequence contributes to CD70over expression in IRP CD4+T cells. The overexpression of CD70positively correlated to the ratio of CD5+B cells, which can produce antibodies, suggest that CD70play important role in IRP pathogenesis.3. The IL-17F rs9463772and BLK rs13277113polymorphism were associated with the susceptibility of IRP patients but the polymorphisms of PTPN22rs2476601and IL-10rs1800871were not associated with IRP in the population of Han nationality of China.