Alterations of 5-hydroxymethylcytosines in circulating cell-free DNA reflect retinopathy in type 2 diabetes

Diabetic retinopathy (DR) is a common microvascular complication that may cause severe visual impairment and blindness in patients with type 2 diabetes mellitus (T2DM). Early detection of DR will expand the range of potential treatment options and enable better control of disease progression. Epigenetic dysregulation has been implicated in the pathogenesis of microvascular complications in patients with T2DM. We sought to explore the diagnostic value of 5-hydroxymethylcytosines (5hmC) in circulating cell-free DNA (cfDNA) for DR, taking advantage of a highly sensitive technique, the 5hmC-Seal. The genome-wide 5hmC profiles in cfDNA samples from 35 patients diagnosed with DR and 35 age-, gender-, diabetic duration-matched T2DM controls were obtained using the 5hmC-Seal, followed by a case-control analysis and external validation. The genomic distribution of 5hmC in cfDNA from patients with DR reflected potential gene regulatory relevance, showing co-localization with histone modification marks for active expression (e.g., H3K4me1). A three-gene signature (MESP1, LY6G6D, LINC01556) associated with DR was detected using the elastic net regularization on the multivariable logistic regression model, showing high accuracy to distinguish patients with DR from T2DM controls (AUC [area under curve] = 91.4%; 95% CI [confidence interval], 84.3– 98.5%), achieving a sensitivity of 88.6% and a specificity of 91.4%. In an external testing set, the 5hmC model detected 5 out of 6 DR patients and predicted 7 out of 8 non-DR patients with other microvascular complications. Circulating cfDNA from patients with DR contained 5hmC information that could be exploited for DR detection. As a novel non-invasive approach, the 5hmC-Seal holds the promise to be an integrated part of patient care and surveillance tool for T2DM patients.