Longitudinal Profiling of DNA Methylation Reveals Age‐Varying CpG Sites and Novel Insights Into Aging Heterogeneity

Age-varying DNA methylation sites reflect increasing interindividual epigenetic divergence during aging, offering insights into health heterogeneity and potential for personalized interventions. Leveraging longitudinal DNA methylation data (3 waves over 5 years) from 135 relatively healthy Chinese older adults in the Rugao Longitudinal Ageing Study, we systematically characterized dynamic DNA methylation changes with age via mixed-effects modeling, identifying 125,353 age-associated (i.e., sites showing significant shifts in average methylation levels with age) and 3145 age-varying CpG sites (i.e., sites showing significant interindividual variability in methylation trajectories with age). Functional analysis revealed distinct enrichment profiles: age-associated CpG sites were enriched in nervous system development, cell signaling, and disease-related pathways, whereas age-varying CpG sites were enriched in cell adhesion, synaptic organization, and organ morphogenesis pathways. Notably, both categories showed significant enrichment in nervous system-related pathways, such as regulation of nervous system development and neuronal cell body. Established epigenetic clocks (e.g., HannumAge) were significantly enriched for age-associated CpG sites but not for age-varying sites. Furthermore, we quantified the pace of aging across eight major organ systems and identified 925 significant associations between organ-specific pace of aging and longitudinal methylation change rates at age-varying CpG sites. Pathway enrichment analysis revealed organ system-relevant biological functions-CpG sites associated with a given organ system were often enriched in pathways relevant to that system's function-with additional evidence of cross-system enrichment. Together, our findings elucidate the role of methylation variability in multi-organ systems aging and its potential for revealing mechanisms of aging heterogeneity and guiding precision monitoring and interventions.