5-Hydroxymethylcytosine Signatures in Circulating Cell-Free DNA as Diagnostic Biomarkers for Late-Onset Alzheimer's Disease

5-Hydroxymethylcytosine (5hmC) is an epigenetic DNA modification that is highly abundant in nervous system. It has been reported that 5hmC is significant associated with Alzheimer's disease (AD). Changes in 5hmC signatures can be detected in circulating cell-free DNA (cfDNA), which has shown potential as a non-invasive liquid biopsy material. However, there is no research about genome-wide profiling of 5hmC in cfDNA and its potential for the diagnosis of AD to date. We carried out a case-control study and used a highly sensitive and selective high-throughput sequencing of chemical labels to detect the genome-wide profiles of 5hmC in human cfDNA and identified differentially hydroxymethylated regions (DhMRs) in AD patients and the control. We detected a significant difference of 5hmC enrichment in gene bodies which were linked to multiple AD pathogenesis-associated signaling pathways in AD patients compared with cognitively normal controls. AD patients can be well distinguished from cognitively normal controls by differentially hydroxymethylated regions (DhMRs) in cfDNA. Specially, we found 7 distinct genes (RABEP1, CPNE4, DNAJC15, REEP3, ROR1, CAMK1D, and RBFOX1) had prediction diagnostic potential based on their significant correlations with MMSE and MoCA scores. The present results suggest that 5hmC markers derived from plasma cfDNA can be served as an effective, minimally invasive biomarkers for clinical auxiliary diagnosis of late-onset AD.Funding: This work was supported by the National Natural Science Foundation of China (NSFC, No. 81472983), Natural Science Foundation of Shandong Province (ZR2020QH294, ZR2015HM024, 2019GSF108066), and Qingdao Postdoctoral Research Grant (RZ2000002906).Declaration of Interest: None to declare. Ethical Approval: The study was approved by The Ethics Committee of Qilu Hospital of Shandong University, and registered at the Chinese Clinical Trial Registry (No. ChiCTR2100042537).