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Purpose  Progression of diabetic retinopathy is related to the duration and severity of hyperglycemia, and after 25 years of diabetes, 90% of patients show some signs of retinopathy. Despite initiation of many retinal molecular/biochemical abnormalities, including mitochondrial damage and epigenetic modifications, the disease remains asympotomatic in the initial stages. Our goal is to examine the utility of DNA methylation as a possible biomarker of diabetic retinopathy.    Methods  Genomic DNA (gDNA) was isolated from the buffy coat, isolated from blood of diabetic patients with proliferative (PDR) or no retinopathy (No-DR), and nondiabetic subjects (CONT). Methylation of mitochondrial DNA (mtDNA), especially its  D-Loop  (the site of mtDNA transcription/replication), was quantified by methylated DNA immunoprecipitation and methyl-specific PCR techniques. Results were confirmed in purified mtDNA. The specific  D-Loop  region with the highest DNA methylation was identified using five overlapping primers, and DNMT1 binding was quantified by chromatin immunoprecipitation. Promoter DNA methylation of DNA mismatch repair ( MLH1 ) and superoxide scavenging ( SOD2 ) enzymes were also quantified.    Results  Compared to CONT,  D-Loop  methylation was higher in PDR and No-DR groups, and the  D-Loop  region responsible for encoding the majority of the mtDNA-encoded genes had significantly higher methylation in the PDR group versus No-DR. Similarly, compared to No-DR, the PDR group also had hypermethylated  MHL1  and  SOD2  promoters.    Conclusions  Blood from PDR patients have higher DNA methylation, than seen in diabetic patients without retinopathy. Thus, DNA methylation can be used as a possible biomarker of diabetic retinopathy.    Translational Relevance  DNA methylation status in the blood of diabetic patients could serve as a potential noninvasive biomarker of retinopathy, and also an important readout parameter for testing longitudinal outcome of novel therapeutics for this blinding disease.

Epigenetic Modifications in Peripheral Blood as Potential Noninvasive Biomarker of Diabetic Retinopathy