Analysis of Epigenetic Changes in Vitamin D Pathway Genes in Rheumatoid Arthritis Patients

Abstract

Background: Rheumatoid arthritis (RA) is an autoimmune inflammatory disease with complex etiopathogenesis launched by multiple risk factors, including epigenetic alterations. RA is possibly linked to vitamin D that is epigenetically active and may alter DNA methylation of certain genes. Therefore, the study aimed to evaluate the relationship between DNA methylation status of vitamin D signaling pathway genes (VDR, CYP24A1, CYP2R1), vitamin D level and associations with RA.Materials and Methods: Totally 76 participants (35 RA patients and 41 healthy controls) were enrolled from a case-control vitamin D and VDR gene polymorphisms study regarding age and vitamin D concentration. CpG islands in promoter regions of the VDR, CYP24A1, CYP2R1 genes were chosen for DNA methylation analysis by means of pyrosequencing. Chemiluminescent microplate immunoassay was used to assess 25(OH)D serum levels. RA clinical data, i.e. the disease activity score C-reactive protein 28 (DAS28 – CRP) as well as patient-reported outcome questionnaires were recorded.Results: The study showed similar methylation pattern in the promoter regions of vitamin D pathway genes in RA and control group with p>0.05 (VDR gene 2.39% vs. 2.48%, CYP24A1 gene 16.02% vs. 15.17% and CYP2R1 2.53% vs. 2.41%). CYP24A1 methylation intensity was significantly higher in compare to methylation intensity of VDR and CYP2R1 genes in both groups (p<0.0001). A tendency of higher vitamin D concentration in cases having methylated VDR (57.57±28.93 vs. 47.40±29.88 nmol/l), CYP24A1 (53.23±26.22 vs. 48.23±34.41 nmol/l) and CYP2R1 (60.41±30.73 vs. 44.54±27.63 nmol/l) genes and a positive correlation between VDR, CYP2R1 methylation intensity and vitamin D level in RA affected participants was revealed (p>0.05). A significantly higher CYP24A1 methylation intensity (p=0.0104) was detected in blood cells of vitamin D deficient (<50 nmol/l) RA patients vs. vitamin D deficient controls.Conclusions: Our data suggests some indirect associations between DNA methylation status of vitamin D pathway genes and vitamin D level in RA.

Methylation of the Vitamin D Receptor (VDR) gene in human disorders

“…The study identified a significant reduction of VDR methylation levels in RA patients compared to CTRs, suggesting that this signature may represent a potential disease biomarker [23]. In contrast with these results, a second epigenetic study revealed no significant differences in the methylation levels of 10 CpG sites within the VDR primary promoter between a group of 35 RA patients and 41 healthy subjects [19]. However, this controversial result could be attributed to both the small size of the study cohort and the small number of CpG sites analyzed.…”

“…The function of the vitamin D is mediated by its binding with VDR on the membrane of immune system cells such as dendritic cells, monocytes and B and T lymphocytes [17]. Consequently, the genetic variability and epigenetic modifications in the VDR could alter immune homeostasis, significantly impacting the onset and progression of autoimmune diseases, such as Rheumatoid Arthritis (RA), Multiple Sclerosis (MS) and Behcet's Disease (BD) (Table 1) [18][19][20][21]. RA is a progressive autoimmune disease characterized by chronic joint inflammation and pain, attributed to the influx of CD4+ TCs into the synovial lining and the increase of macrophage-like and fibroblastlike cells that produce degradative enzymes and proinflammatory cytokines [22].…”

Methylation of the Vitamin D Receptor Gene in Human Disorders

“…The study identified a significant reduction in the VDR methylation levels in RA patients compared to CTRs, suggesting that this signature may represent a potential disease biomarker [ 26 ]. In contrast with these results, a second epigenetic study revealed no significant differences in the methylation levels of 10 CpG sites within the VDR primary promoter between a group of 35 RA patients and 41 healthy subjects [ 22 ]. However, this controversial result could be attributed to both the small size of the study cohort and the small number of CpG sites analyzed.…”

“…The function of the 1,25(OH) 2 D 3 is mediated by its binding with the VDR on the membrane of immune system cells such as dendritic cells, monocytes, and B and T lymphocytes [ 20 ]. Consequently, the genetic variability and epigenetic modifications in the VDR could alter immune homeostasis, significantly impacting the onset and progression of autoimmune diseases, such as rheumatoid arthritis (RA), multiple sclerosis (MS), and Behcet’s disease (BD) [ 21 , 22 , 23 , 24 ]. RA is a progressive autoimmune disease characterized by chronic joint inflammation and pain, attributed to the influx of CD4+ TCs into the synovial lining and the increase in macrophage-like and fibroblast-like cells that produce degradative enzymes and proinflammatory cytokines [ 25 ].…”

Role of the Vitamin D Receptor (VDR) in the Pathogenesis of Osteoporosis: A Genetic, Epigenetic and Molecular Pilot Study

“…In addition, epigenetic signatures, including DNA methylation, seem to modulate VDR gene expression and appear intensely involved in bone diseases [ 26 , 27 ]. In this context, higher vitamin D concentration was shown to be associated with higher VDR promoter methylation in rheumatoid arthritis (RA) patients [ 28 ]. Interestingly, recent data suggest a link between Cdx2 genotype-specific VDR expression and differential VDR methylation, regardless of ethnicity [ 12 ], supporting the complex interaction between genetics, epigenetics and environment in OP pathogenesis.…”