Abstract
PurposeProgression 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.MethodsGenomic 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.ResultsCompared 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.ConclusionsBlood 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 RelevanceDNA 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.
Highlights
The International Diabetes Federation estimates approximately 425 million adults had diabetes in 2017, and with the prevalence of this disease rising at an alarming rate, by 2040 this number could be 642 million.[1]
Peripheral blood from diabetic patients has higher displacement loop (D-Loop) damage compared to nondiabetic controls, and the damage is significantly higher in patients with proliferative retinopathy compared to patients without retinopathy.[4]
Consistent with the results from the genomic DNA (gDNA), compared to CONT group 5mC levels at the D-Loop were significantly higher in the purified mitochondrial DNA (mtDNA) in both PDR and NoDR groups, and among these two diabetic groups, the PDR group had approximatley three-fold higher 5mC levels compared to the No-DR group (Fig. 1c)
Summary
The International Diabetes Federation estimates approximately 425 million adults had diabetes in 2017, and with the prevalence of this disease rising at an alarming rate, by 2040 this number could be 642 million.[1]. Proliferative stage in a relatively short period.[2,6] Severity of hyperglycemia is considered the key alterable risk factor, with the duration of diabetes intimately related to the development and progression of retinopathy. Maintenance of tight glycemic control impedes the development of diabetic retinopathy, in many patients, especially those with type 2 diabetes, hyperglycemia may remain undiagnosed for a long period of time.[3,7,8,9] the majority of patients do not, or cannot, maintain tight glycemic control for long durations, and their retina continues to be exposed to a hyperglycemic insult. Identifying noninvasive biomarkers to foretell the development of diabetic retinopathy and/or access the outcome of therapeutic intervention is critical
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