Background SLE is a complex autoimmune disease with heterogeneous manifestations and unpredictable outcomes. Early diagnosis is challenging due to non-specific symptoms, and current treatments only manage symptoms. Epigenetic alternations, including 5-Hydroxymethylome (5hmC) modifications, are important contributors to SLE pathogenesis. However, the 5hmC modification status in circulating cell-free DNA (cfDNA) of patients with SLE remains largely unexplored. We investigated the distribution of 5hmC in cfDNA of patients with SLE and healthy controls (HCs), and explored its potential as an SLE diagnosis marker.

Methods We used 5hmC-Seal to generate genome-wide 5hmC profiles of plasma cfDNA and bioinformatics analysis to screen differentially hydroxymethylated regions (DhMRs). In vitro mechanistic exploration was conducted to investigate the regulatory effect of CCCTC-binding factor (CTCF) in 5hmC candidate biomarkers.

Results We found distinct differences in genomic regions and 5hmC modification motif patterns between patients with SLE and HCs, varying with disease progression. Increased 5hmC modification enrichment was detected in SLE. Additionally, we screened 151 genes with hyper-5hmC, which are significantly involved in SLE-related processes, and 5hmC-modified BCL2, CD83, ETS1 and GZMB as SLE biomarkers. Our findings suggest that CTCF regulates 5hmC modification of these genes by recruiting TET (ten-eleven translocation) protein, and CTCF knockdown affected the protein expression of these genes in vitro.

Conclusions Our findings demonstrate the increased 5hmC distribution in plasma cfDNA in different disease activity in patients with SLE compared with HCs and relating DhMRs involved in SLE-associated pathways. Furthermore, we identified a panel of SLE relevant biomarkers, and these viewpoints could provide insight into the pathogenesis of SLE.