Multiparametric profiling of plasma cell-free nucleic acids in nonagenarians and centenarians highlights age-related...

Plasma circulating cell-free nucleic acids (ccfNAs) have emerged as promising non-invasive biomarkers of aging. While age-associated changes have been reported, data in relation to extreme aging and longevity remain scarce. Here, we assessed ccfNA levels and integrity, and ccfDNA methylation in a cohort of 86 individuals, analyzed both overall and stratified by sex, including nonagenarians (NON: 90-98 years, n = 29), centenarians (CEN: 100-109 years, n = 28), and a middle-aged control group (CG: 38-67 years, n = 29) of nonagenarians' and centenarians' offspring, using our previously optimized multiparametric analysis workflow targeting nuclear (ccfnDNA) and mitochondrial (ccfmtDNA) DNA, ribosomal RNA (ccfrRNA), messenger RNA (ccfmRNA), and microRNAs (ccfmiRNAs). ccfnDNA levels followed non-linear trajectories, decreasing from CG to nonagenarians (28%-64.5%, significant in nonagenarian men compared to CG), then slightly increasing in centenarians. ccfmRNA and ccfmtDNA followed the opposite pattern, with significantly lower ccfmRNA levels in centenarians (44.5%), and both were strongly correlated (r = 0.59-0.83, p < 0.0001), suggesting a shared regulatory mechanism. Additionally, ccfnDNA integrity significantly decreased from CG to CEN (20.7%), while ccfmtDNA and ccfrRNA integrities, and ccfDNA methylation, did not vary. Among the seven ccfmiRNAs analyzed, miR-93-5p, miR-126-3p, and liver-specific miR-122-5p, exhibited significant age-related decreases (40.5%-70.3%), reaching their lowest levels in centenarians. Our study thereby provides novel findings regarding age- and sex- related changes in ccfNAs, highlighting both dynamic and stable characteristics linked to longevity. It identified potential ccfNA-based longevity biomarkers, and supports ccfmiRNAs as the most promising and sensitive biomarkers of longevity.