Peripheral Whole Blood FOXP3TSDR Methylation Analysis:A Potential Marker In Severity Assessment Of Autoimmune Diseases And Chronic Microbial Infections

Objectives:In this study, we use HRM-PCR (High resolution melt-polymerase chain reaction) to determine the FOXP3TSDR (Treg-specific demethylated region) methylation status in autoimmune diseases and chronic cutaneous microbial infections. We aim to see how FOXP3TSDR methylation status affects natural regulatory T cell (Treg) levels in these conditions with altered but opposing immunological status. We also aim to explore the relationship between FOXP3TSDR methylation status and disease activity in systemic lupus erythematosus (SLE).Methods:Genomic DNA was isolated from200μl of peripheral venous whole blood of65patients with inactive and active SLE,30patients with psoriasis,30patients with chronic cutaneous microbial infections (acne, onychomycosis, genital herpes, and herpes simplex), and23healthy controls. Bisulfite conversion of unmethylated cytosines was done prior to the HRM-analysis. HRM-PCR was done on a rotor gene6000cycler to determine the FOXP3TSDR melting temperatures.Results:We found FOXP3TSDR to have the highest mean melting temperature (highly methylated) in active SLE patients compared to all the other groups (79.00±0.28vs all other groups, p<0.001). The psoriasis group also had a significantly high mean melting temperature when compared with the inactive SLE group (78.62±0.20vs78.49±0.29, p<0.05) and healthy controls (78.62±0.20vs78.44±0.25, p<0.01). There was no significant difference in melting temperature between inactive SLE and healthy controls (78.49±0.29vs78.44±0.25, p=0.464). Disease activity in SLE was positively correlated with melting temperature (methylation). On the other hand, patients with chronic microbial infections had significantly lower FOXP3TSDR mean melting temperature (demethylated) when compared with healthy controls (78.28±0.21vs78.44±0.25, p<0.05).Conclusion:The use of HRM-PCR to detect FOXP3TSDR methylation status is a reliable and easy way to predict nTreg levels in peripheral blood in different disease conditions. Analysis of FOXP3TSDR methylation status can be a useful tool in severity assessment and follow up of autoimmune diseases and chronic microbial infections. It may prove to be more sensitive and/or specific for active SLE than other disease conditions.