PROJECT SUMMARY/ABSTRACT Due to advances in geroscience, aging must be considered a modifiable risk factor for the major causes of death. Interventions targeting human aging are ongoing, but there is a lack of predictive biomarkers to measure the effectiveness of these interventions. With age, somatically-derived mitochondrial DNA (mtDNA) deletions clonally accumulate within individual cells across a variety of tissues. Attaining high intracellular abundance, mtDNA deletions disrupt oxidative phosphorylation, cellular function, and result in cell death. We hypothesize that human mtDNA deletion frequency predicts the risk of morbidity and mortality and responds to interventions that alter healthspan. We have developed a digital PCR assay that quantifies mtDNA deletion frequency using total DNA samples from any tissue in rodents and humans. This assay provides absolute quantitation, is amenable to a 96-well format, correlates strongly with the subsequent cellular phenotypes including cell death, and has a detection limit below one part per million. MtDNA deletion mutation frequency increases exponentially with age and this increase parallels the age-induced accumulation of dysfunctional cells, tissue degeneration, and mortality. This project will further develop and validate mtDNA deletion frequency as a measure of cell death in human aging. We are validating the test in accordance with FDA guidelines for bioanalytical assays. We are measuring mtDNA deletion frequency in a number of human tissues and biofluids across the human lifespan and will establish the relationship between mtDNA deletion frequency, chronological age, clinical and physiological outcomes, and interventions targeting human aging.