Abstract
OBJECTIVE: This exploratory, hypothesis-generating study aimed to investigate methylation differences in 16 angiogenesis- and fibrosis-related genes across diabetic retinopathy (DR) stages and proliferative diabetic retinopathy (PDR) subtypes (Florid and Gliotic), and to identify potential epigenetic biomarkers for disease progression. METHODS: DNA methylation levels were analyzed in 38 diabetic patients stratified into three groups: Florid PDR (n=16), Gliotic PDR (n=13), and controls (no DR/non-proliferative DR, n=9). Targeted genes included MMP9, EPO, AKR1B1, EGLN1, HIF1A, ICAM1, KDR, UCP1, SOD2, SERPINF1, PGF, RXRG, TGFB1, FLT1, FGF2, and VEGFA. Methylation profiling was performed via MethylTarget sequencing. RESULTS: In the Florid PDR group, AKR1B1 (P = 0.039) and MMP9 (P = 0.023) exhibited hypomethylation compared to controls. The Gliotic PDR group showed hypomethylation of EPO (P = 0.025), KDR (P = 0.023), MMP9 (P = 0.014), and UCP1 (P = 0.048) compared to controls. At the whole-promoter level, none of the 16 target genes showed statistically significant methylation differences between the Florid and Gliotic PDR groups. However, exploratory analysis of gene segments and individual CpG sites revealed nominal differences (e.g., in EGLN1) that warrant further investigation. Notably, EGLN1 displayed hypomethylation at three CpG sites and two gene segments in Gliotic PDR (P<0.05). CONCLUSIONS: Distinct hypomethylation profiles in AKR1B1, MMP9, EPO, KDR, and UCP1 were associated with PDR progression compared to no DR/non-proliferative controls. While no statistically significant differences were observed at the whole-promoter level for the 16 target genes between Florid and Gliotic PDR subtypes, segmental and CpG-level variations (particularly in EGLN1) suggest potential epigenetic heterogeneity. These preliminary findings highlight the need for further validation to assess their role as biomarkers or therapeutic targets.