THU0024 METHYLATION ANALYSIS OF VITAMIN D SIGNALING PATHWAY GENES IN RHEUMATOID ARTHRITIS PATIENTS

Abstract

Background:Vitamin D is known for its immunomodulatory and epigenome interacting effects. Vitamin D deficiency is frequently observed in rheumatoid arthritis (RA) patients compared to healthy controls, is also named as a potential risk factor in RA ethiopatogenesis and may alter DNA methylation of certain genes [1,2]. Still, causality of vitamin D deficiency in RA patients needs to be elucidated.Objectives:The aim of the study was to evaluate relationship between DNA methylation status of vitamin D related genes (VDR,CYP24A1,CYP2R1), miRNA-155 expression, vitamin D level and its association with RA.Methods:CpG islands in promoter region of theVDR,CYP24A1,CYP2R1genes were chosen for DNA methylation analysis by means of pyrosequencing. DNA from blood mononuclear cells of 31 RA patients and 31 age and sex matched healthy controls was assessed for methylation pattern after informed consent was obtained in Vilnius university Hospital Santaros klinikos Centre of Rheumatology. For miRNA analysis quantitative reverse transcription PCR was used. Chemiluminescent microplate immunoassay was used to asses 25(OH)D serum levels.Results:25(OH)D concentrations varied from deficiency (<50 nmol/l), insufficiency (50-75 nmol/l) to normal range (≥75-100 nmol/l) in RA (mean 47.49 nmol/l; SD ± 27.93) and healthy controls (mean 57.38 nmol/l; SD ± 29.93)).CYP24A1methylation level was significantly higher in comparison toVDR(p<0.0001) andCYP2R1(p<0.0001) genes in both groups.CYP24A1hypermethylation was also observed in older subjects (p=0.012). The study demonstrated a significant positive correlation between vitamin D concentration andVDR,CYP2R1genes methylation intensity (r2=0.31, p=0.014; r2=0.25, p=0.042, respectively). However, gene methylation frequency and methylation intensity showed no significant difference between RA patients and healthy controls (VDR– 2.4vs2.6 %,CYP24A1– 16.6vs15.3 %,CYP2R1– 2.6vs2.6 %) (p>0.05). To note, miRNA-155 expression negatively correlated withCYP24A1methylation intensity (r2=-0.43, p=0.009).Conclusion:Our study identified significant associations between theVDRandCYP2R1promoter methylation and vitamin D concentration. However, no significant differences in DNA methylation pattern between RA patients and healthy controls were detected. MiR-155 expression was associated withCYP24A1methylation level, confirming its possible involvement in vitamin D metabolism. The data of our study suggests that epigenetic phenomena are significantly involved in vitamin D metabolism and may have an indirect effect on RA ethiopatogenesis.