Despite extensive research on 5-methylcytosine (5mC) in relation to smoking, there has been limited exploration into the interaction between smoking and 5-hydroxymethylcytosine (5hmC). In this study, total DNA methylation (5mC+5hmC), true DNA methylation (5mC) and hydroxymethylation (5hmC) levels were profiled utilizing conventional bisulphite (BS) and oxidative bisulphite (oxBS) treatment, measured with the Illumina Infinium Methylation EPIC BeadChip. An epigenome-wide association study (EWAS) of 5mC+5hmC methylation revealed a total of 38,575 differentially methylated positions (DMPs) and 2023 differentially methylated regions (DMRs) associated with current smoking, along with 82 DMPs and 76 DMRs associated with former smoking (FDR-adjusted p p p −5). The substantial number of recognized DMPs, including 5mC+5hmC (7069/38,575, 2/82), 5mC (0/33, 1/1), and 5hmC (2/8, 0/2), have not been previously reported. Our findings corroborated previously established methylation positions and revealed novel candidates linked to tobacco smoking. Moreover, the identification of hydroxymethylated CpG sites with suggestive links provides avenues for future research.
