Abnormal DNA Methylation In T Cells From Patients With Connective Tissue Diseases

Objective:To investigate whether alterations in DNA methylation occur in T cells of patients with connective tissue disease.Methods:(1) PBMC (peripheral blood mononuclear cells) cells were isolated from the peripheral venous blood of SLE,SSc,DM patients and healthy donors by density gradient centrifugation. CD4+ T cells were isolated using micro beads and protocols provided by the manufacturer.(2) Genomic DNA and total RNA were isolated with DNA/RNA kits.(3) Global methylation patterns was assayed using the Methylamp? Global DNA Methylation Quantification Kit.(4) mRNA levels of DNA methyltransferases (DNMTs) and methylated CpG binding proteins (MBDs) were measured by real-time quantitative polymerase chain reaction.Results:(1) Global DNA methylation level was significantly reduced in CD4+ T cells from SLE and SSc patients relative to controls (P=0.013; P=0.039, respectively).(2) DNMT1 and MBD4 mRNA expression were significantly lower in CD4+ T cells from SLE patients than that in controls (P=0.001; P=0.008, respectively), MBD2 and MeCP2 mRNA levels were significantly higher in CD4+ T cells from SLE patients compared to controls (P=0.008, P=0.027, respectively). DNMT1, MBD3 and MBD4 mRNA expression were significantly decreased in CD4+ T cells from SSc patients than that in controls(P=0.001, P=0.002, P=0.021, respectively) ; In DM patients CD4+ T cells, MBD2 and MeCP2 mRNA levels were significantly higher compared to controls (P=0.046, P=0.000 respectively).(3) The DNMT1 mRNA expression is positively correlated with global DNA methylation level in SLE patients CD4+T cells (r=0.746, P=0.013) . In SSc CD4+ T cells, the MBD4 mRNA expression is positively correlated with global DNA methylation level (r=0.804, P=0.005) .Conclusion: These data suggests that DNA methylation appears abnormal in T cells in SLE and SSc.