Genome-wide Analysis Reflects Novel 5-Hydroxymethylcytosines Implicated in Diabetic Nephropathy and the Biomarker Potential.

Aim: Diabetic nephropathy (DN) has become the most common cause of end-stage renal disease (ESRD) in most countries. Elucidating novel epigenetic contributors to DN can not only enhance our understanding of this complex disorder, but also lay the foundation for developing more effective monitoring tools and preventive interventions in the future, thus contributing to our ultimate goal of improving patient care. Methods: The 5hmC-Seal, a highly selective, chemical labeling technique, was used to profile genome-wide 5-hydroxymethylcytosines (5hmC), a stable cytosine modification type marking gene activation, in circulating cell-free DNA (cfDNA) samples from a cohort of patients recruited at Zhongnan Hospital, including T2D patients with nephropathy (DN, n = 12), T2D patients with non-DN vascular complications (non-DN, n = 29), and T2D patients without any complication (controls, n = 14). Differentially analysis was performed to find DN-associated 5hmC features, followed by the exploration of biomarker potential of 5hmC in cfDNA for DN using a machine learning approach. Results: Genome-wide analyses of 5hmC in cfDNA detected 427 and 336 differential 5hmC modifications associated with DN, compared with non-DN individuals and controls, and suggested relevant pathways such as NOD-like receptor signaling pathway and tyrosine metabolism. Our exploration using a machine learning approach revealed an exploratory model comprised of ten 5hmC genes showing the possibility to distinguish DN from non-DN individuals or controls. Conclusion: Genome-wide analysis suggests the possibility of exploiting novel 5hmC in patient-derived cfDNA as a non-invasive tool for monitoring DN in high risk T2D patients in the future.