Genome-wide mapping of sex-associated autosomal DNA methylation in major blood cell types

Sex differences shape human physiology and disease risk, yet their autosomal epigenetic basis remains incompletely understood. Using whole genome bisulfite sequencing data from iMETHYL database (∼100 adults) across three purified blood cell types (CD4⁺ T cells, monocytes, neutrophils), we performed comprehensive mapping of cell-type specific sex-associated DNA methylation. Using genome-wide Z-test and confidence-interval method, we identified thousands of autosomal differentially methylated sites (DMSs), the majority of which were cell type-specific. We found that neutrophils exhibited pronounced female-biased hypermethylation, whereas CD4⁺ T cells and monocytes showed more balanced patterns. DMSs were enriched within gene bodies and annotated to neuronal and adhesion functions, with T-cell activation uniquely associated in CD4⁺ T cells. Transcription factor binding site enrichment indicated hematopoietic regulators, suggesting that sex-associated methylation is tightly linked to early developmental processes within blood lineages. Differentially methylated regions overlapped genome-wide association study loci for lifetime smoking, multiple sclerosis, and psoriasis, indicating convergence between sex-associated epigenetic states and genetic susceptibility. Evolutionary analysis revealed limited conservation but identified a conserved intronic CpG within FIGN. This study provides the first genome-wide, cell type-resolved map of autosomal sex-associated DNA methylation in human blood and establishes a foundation for mechanistic and translational studies in sex-informed biology and medicine.