Combined epigenomic landscapes of 5mC, 5hmC, and 6mA modifications in papillary thyroid carcinogenesis.

Papillary thyroid carcinoma (PTC), though genetically characterized, lacks comprehensive epigenomic profiling. Here we present the first combined analysis of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and 6-methyladenine (6mA) in PTC pathogenesis. Using mDIP-seq on matched tumor and adjacent normal tissues from six treatment-naïve patients, we mapped genome-wide distributions of these modifications, revealing distinct spatial patterns and tumor-specific differentially methylated regions (DMRs) with modification-specific functional partitioning. Silhouette-guided feature selection refined 7,117 DMRs to a 187-locus diagnostic panel achieving robust tumor-normal discrimination, with chromosomes 16 and 19 as recurrent epigenetic hotspots (2.1-7.9-fold enrichment). Integration of TCGA data revealed mutation-dependent epigenetic-transcriptional associations: BRAF-mutant tumors showed selective upregulation of epigenetically dysregulated genes, whereas RAS-mutant tumors displayed distinct transcriptional patterns. Co-expression network analysis highlighted disrupted transcriptional coordination in tumors. This work establishes a multidimensional epigenetic framework for PTC with combinatorial diagnostic signatures and condition-specific associations between DNA modifications and driver mutations (BRAF vs. RAS).