Methylation matters: A case control study on epigenetic alteration in diabetic retinopathy

Dear Editor, Diabetes mellitus continues to represent a major global health burden, affecting more than 500 million individuals worldwide. Diabetic retinopathy (DR) remains one of the leading causes of visual impairment among working-age adults.[1] Although chronic hyperglycemia is a well-recognized driver of microvascular damage, increasing evidence suggests that epigenetic mechanisms may play a key role in the development and progression of diabetic complications.[2] Among these mechanisms, DNA methylation has emerged as an important regulator of gene expression and may contribute to the phenomenon of metabolic memory.[3] In this context, we conducted a hospital-based case–control pilot study to evaluate the association between global DNA methylation and diabetic retinopathy in patients with type 2 diabetes mellitus. The study was conducted at the Institute of Ophthalmology in collaboration with the Rajiv Gandhi Centre for Diabetes and Endocrinology, J. N. Medical College, Aligarh Muslim University, between March 2023 and December 2024. Seventy patients with type 2 diabetes mellitus were enrolled after obtaining informed consent. Among them, 33 patients had diabetic retinopathy and 37 diabetic patients without retinopathy served as controls. All participants underwent a comprehensive ophthalmic examination including best-corrected visual acuity using ETDRS charts, slit-lamp bio microscopy, and dilated fundus examination with a +78 diopter lens and indirect ophthalmoscopy. Peripheral venous blood samples were collected for biochemical investigations and genomic DNA extraction. Global DNA methylation levels were quantified using a 5-methylcytosine DNA ELISA-based assay. Baseline demographic and metabolic characteristics were largely comparable between the two groups, although the duration of diabetes and glycated hemoglobin (HbA1c) levels were significantly higher among patients with diabetic retinopathy. The mean global DNA methylation level was significantly elevated in patients with DR compared with diabetic controls without DR (2.28 ± 1.46 vs. 1.45 ± 0.16; P < 0.001). Furthermore, DNA methylation levels demonstrated a progressive increase with increasing severity of retinopathy. The mean methylation levels were 1.55 ± 0.06% in mild nonproliferative DR, 1.62 ± 0.04% in moderate nonproliferative DR, 2.00 ± 0.76% in severe nonproliferative DR, and markedly higher at 3.74 ± 2.15% in proliferative diabetic retinopathy. This graded increase suggests a potential relationship between epigenetic alterations and disease severity. Receiver operating characteristic (ROC) curve analysis demonstrated good discriminatory ability of global DNA methylation in identifying diabetic retinopathy, with an area under the curve of 0.882 (95% CI: 0.80–0.96). Using an optimal cutoff value of >1.53%, the sensitivity and specificity for detecting DR were 93.9% and 78.4%, respectively. Multivariate logistic regression analysis showed that global DNA methylation remained significantly associated with DR after adjusting for potential confounding variables. The observed increase in global DNA methylation may reflect hyperglycemia-induced epigenetic modifications. Chronic oxidative stress, mitochondrial dysfunction, and activation of DNA methyltransferases have been proposed as mechanisms through which persistent hyperglycemia induces epigenetic changes that persist even after glycemic control. Such mechanisms may contribute to the metabolic memory phenomenon implicated in the progression of DR. Our findings are consistent with previous studies reporting altered DNA methylation patterns in diabetic complications.[4] However, most previous studies have employed techniques such as high-performance liquid chromatography or gene-specific methylation assays. In contrast, the ELISA-based method used in our study provides a relatively simple and cost-effective approach that may have potential for translational application in clinical research settings. The present study has certain limitations. The sample size was modest and the study population was hospital-based, which may limit generalizability. Additionally, only global DNA methylation levels were assessed, whereas gene-specific methylation changes may provide deeper mechanistic insights. Finally, the cross-sectional design precludes establishing a causal relationship between methylation alterations and DR. Despite these limitations, our findings highlight a potential association between increased global DNA methylation and DR in patients with type 2 diabetes mellitus. These results support the growing evidence that epigenetic mechanisms may contribute to the pathogenesis of diabetic microvascular complications. Larger longitudinal studies evaluating gene-specific methylation patterns are warranted to further clarify the role of epigenetic regulation in DR and its potential clinical implications. Authors contribution All the authors have read and agreed to the script, discussed the results and commented on the manuscript at all stages. All authors read and approved the final manuscript. Financial support and sponsorship: Nil. Conflicts of interest: There are no conflicts of interest.