ABNORMAL METHYLATED DNA REGIONS INDICATE AN ATYPICAL RESPONSE OF THE CD4+ T CELLS IN IGA NEPHROPATHY PATIENTS

Introduction and Aims: Immunoglobulin A nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide among patients undergoing renal biopsy. The pathogenesis of this disease seems to have a strong genetic component but so far, no genetic variants or genes underlying these loci have been identified as causative or affecting the pathology. In this setting, also the DNA methylation could be an important factor influencing the pathology. Rapid advances in the field of epigenetics are now revealing a molecular basis for how heritable information other than DNA sequence can influence gene function. To assess possible changes in CpG methylation in IgA nephropathy, we analyzed the CpG methylation in a genome-wide manner on the DNA derived from CD4+ Cells from 6 IgAN samples and 6 healthy controls. Methods: To assess possible changes in CpG methylation in IgAN, we analyzed the CpG methylation in a genome-wide manner on the DNA derived from CD4+ Cells from 6 IgAN samples and 6 healthy subjects (HS). DNA methylation analysis was performed by the Illumina Human-Methylation450 BeadChip that interrogates DNA methylation at more than 485000 CpGs. All statistical analysis was performed using R and the RnBeads R package for comprehensive analysis of DNA methylation data. Identified CpG, differentially methylated, were further validated on ten different samples of IgAN and HS. Gene expression studies by Real-time PCR were performed on identified methylated genes/promoters to verify the correspondence between the methylation status and the gene expression. Results: We found 171 CpG sites differentially methylated with a Δbeta > 0.2 and p<0.05. 95 CpG sites were down-methylated and 76 were up-methylated. Differential methylation on the region level was computed across gene regions with sites in the promoter region, 5'UTR, first exon, gene body, and 3'UTR in order to provide the broadest, most comprehensive view of methylation state. Analysis of the regions revealed 161 tiling regions, 12 promoters, 12 genes and 5 CpG islands differentially methylated. In particular, 8 regions differentially methylated included 8 genes involved in the response and proliferation of CD4+ T cells.We confirmed by real-time PCR that 4 hyper-methylated genes were down-regulated and the 4 hypo-methylathed genes were up-regulated in IgAN patients respect to controls. Moreover, we found a region containing the transcription site of the precursor of miR-886 gene that was hyper-methylated.We showed that the miR-886 regulated some key genes of the T cell receptor signalling and of the T-helper response such as the TGF-β, TOLLIP and RUNX3. Transfection of the miRNA inhibitor on CD4+ immortalized cells confirmed the modulation of these gene target by miR-886. Conclusions: We found, for the first time, some specific region abnormally methylated in IgAN patients, some of which including genes involved in the T cell receptor (TCR) signalling and in the CD4+ T cell response and proliferation. These methylated regions led to the altered expression of genes of the TCR signal transduction, indicating an atypical response of the CD4+ T cells of IgAN patients.