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

## TL;DR

This study tracks DNA methylation changes in older adults over time, revealing how methylation variability increases with age and contributes to differences in aging.

## Contribution

The study identifies age-varying CpG sites and links their methylation changes to organ-specific aging rates, offering new insights into aging heterogeneity.

## Key findings

- 3145 age-varying CpG sites showed increasing interindividual variability with age.
- Age-associated CpG sites were linked to nervous system development and disease pathways.
- Methylation variability at age-varying sites was associated with aging rates in eight organ systems.

## Abstract

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.

We longitudinally profiled DNA methylation in 135 relatively healthy older adults from the RLAS study, identifying 3145 CpG sites exhibiting increasing inter‐individual variability with age and 125,353 sites exhibiting significant changes in average methylation levels with age. These two CpG categories showed distinct functional enrichments with overlapping relevance to nervous system pathways. Furthermore, we identified significant associations between longitudinal change rates at these sites and the pace of aging across eight major organ systems, highlighting the importance of methylation variability in shaping aging heterogeneity.