Elevated baseline DNA damage and hypomethylated circulating free DNA distinguish frailty from healthy aging

Aging is a primary risk factor for several diseases, including neurodegenerative disorders and cancer, with DNA damage accumulation being one of the key drivers. Frailty is a severe form of aging that affects many older adults and is characterized by dysfunction across multiple body systems, leading to increased vulnerability and late-life disability. The pathogenic mechanisms differentiating frailty from healthy aging, however, remain unclear. Here, we compared the DNA damage response (DDR) in skin fibroblasts and circulating free DNA (cfDNA) levels in the blood of frail individuals with those of healthy young and elderly controls. To infer a molecular fingerprint of frailty, we also sequenced the methylated cfDNA to identify potential tissue-specific dysfunction. Frail subjects exhibited significantly higher baseline levels of DNA damage, as evidenced by increased γH2AX and 53BP1 foci in fibroblasts under basal conditions. However, no significant differences in DDR capacity were observed post-irradiation between frail and age-matched controls. Elevated cfDNA levels in frail individuals correlated with higher TNF-α levels, suggesting a link between cfDNA, inflammation, and frailty. cfDNA methylation analysis revealed an abundance of hypomethylated fragments and identified a distinctive fingerprint of differentially methylated regions that discriminated frail subjects from age-matched controls. Moreover, deconvolution analysis identified the small intestine as a potential cfDNA tissue of origin, implicating a role of this anatomical site in frailty pathobiology. Our findings provide a molecular basis for understanding frailty and its links to aging-related diseases and may open avenues for biomarker development and targeted interventions. Introduction