| Objective: To screen the DNA differentially methylation genes(DMPs)in patients with primary IgA nephropathy(IgAN)by methylation gene chip,and to provide a new theoretical basis for further exploration of DNA methylation on the pathogenesis and progression of IgAN.Method: DNA was extracted from peripheral blood B lymphocytes of 3 patients with IgAN and 3 healthy controls.The methylations level of genome-wide DNA were detected by Nimble Gen 3 x 720 K methylation gene chip to find the DMPs.Combined with the genes of the genome-wide association studies(GWAS)of IgAN,protein interaction network(PPI)and gene function(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway analysis were performed to find the genes that may be related to the pathogenesis and progression of IgAN.At the same time,fresh peripheral blood was collected from 51 IgAN patients and 49 healthy controls,and the mRNA expressions of selected DMPs in peripheral blood lymphocytes were detected by RT-q PCR(real-time quantitative PCR).30 of the above IgAN patients and 30 healthy controls were selected,peripheral blood B lymphocytes were sorted by immunomagnetic beads,and pyrosequencing assays were performed to verify the methylation levels of DMPs.And then the correlations between methylation levels and mRNA expressions were analyzed to study the relationship between methylation and gene expression.Finally,the clinical baseline data as well as pathological data were collected during renal biopsy in IgAN patients to analyze the relationship between mRNA expression levels and clinical and pathological data.Results: 1.Methylation gene chip detected 2062 differentially methylation positions(DMPs)and 1839 differentially methylation genes(DMGs).Among them,there were 1170 hypermethylated genes,708 hypomethylated genes.2.GO/KEGG enrichment analysis and protein interaction network analysis screened four differentially methylation genes,CLEC10 A,ACCS,TNFRSF17,and CFHR1.3.RT-q PCR results showed that the mRNA expression level of TNFRSF17 in peripheral blood lymphocytes was increased in the IgAN group compared with the control group(P=0.03).4.Combined with the analysis of clinical data of patients,in peripheral blood lymphocytes of IgAN patients,the mRNA expression level of CLEC10 A was negatively correlated with hemoglobin(P=0.02,r=-0.33);the mRNA expression level of TNFRSF17 was negatively correlated with high-density lipoprotein(P=0.04,r=-0.33);the mRNA expression level of CFHR1 was negatively correlated with glomerular filtration rate(P=0.03,r=-0.31)and positively correlated with Ig G(P<0.01,r=0.40);the mRNA expression level of CFHR1 tended to be positively correlated with serum creatinine level(P=0.06,r=0.27).5.Combined with the analysis of pathological data of patients,the mRNA expression level of CLEC10 A in group E1 was increased compared with group E0(P<0.05);the expression level of CFHR1 mRNA in group S1 was increased compared with group S0(P=0.02);compared with M0,E0,and C0,TNFRSF17 tended to increase in M1,E1,and C1~2,but the difference was not statistically significant.6.In IgAN,the methylation level of TNFRSF17 Cp G1 locus was higher(P=0.02),and the methylation level of TNFRSF17 Cp G1 locus was positively correlated with the relative expression level of TNFRSF17 mRNA(P<0.05,r=0.39).7.Combined with clinical data analysis,the methylation level of TNFRSF17 Cp G1 site was negatively correlated with 24-hour urinary protein quantification;blood cholesterol level;low-density lipoprotein(P=0.02,r=-0.45;P=0.05,r=-0.46;P=0.03,r=-0.48).Conclusion:1.TNFRSF17 gene is hypermethylated in peripheral blood B lymphocytes of IgAN patients,and its mRNA expression level is increased in peripheral blood lymphocytes,and the methylation level is positively correlated with the mRNA expression level,suggesting that the abnormal methylation level of TNFRSF17 may be involved in the pathogenesis of IgAN.2.The expression level of CFHR1 mRNA in peripheral blood lymphocytes of IgAN patients is negatively correlated with glomerular filtration rate and tend to be positively correlated with serum creatinine levels,suggesting that CFHR1 may be associated with disease progression in IgAN. |
