The Hallmarks of Aging: Assessing accumulation of DNA lesions with age using single cell DNA sequencing in GESTALT

National Institute On Aging

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Abstract

DNA is exposed to a large variety of potentially damaging challenges, including free radical species, ultraviolet light, ionizing radiation and environmental pollutants. These stresses activate the DNA damage response (DDR) that causes cell cycle arrest and triggers DNA repair mechanisms that specifically address different types of lesions, including base excision repair (BER), nucleotide excision repair (NER), non-homologous end joining (NHEJ) and homology-directed repair (HDR). If the DNA damage cannot be fixed, the DDR can induce apoptosis, push the cell into irreversible cell senescence or cause cancer. There is also evidence that DDR can cause severe mitochondrial dysfunction, perhaps by consuming NAD+ which is essential for mitochondrial energetic metabolism. At the organism level, there is evidence that response to DNA damage leads to the activation of the innate immune system as an attempt to eliminate damaged and senescent cells. The DDR activates many inflammatory pathways and by this mechanism suppresses the biological signaling of hormones and growth factors that are aimed at maintenance and repair of different tissues. Because of the widespread consequences of DNA damage accumulation, powerful and somewhat redundant DNA repair mechanisms were evolutionarily selected to cope with the thousand DNA lesions that occur daily in most cells. However, there is evidence that unrepaired DNA lesions tend to accumulate with age, and it has been suggested that accumulation of DNA damage is the primary mechanism for the development of most aging phenotypes. Multiple lines of evidence support this hypothesis: 1) Studies of comparative biology have suggested that DNA repair capacity is associated with longevity across species; 2) Genomic instability and accumulation of DNA damage trigger many of the biological mechanisms that have been associated with longevity, including senescence, mitochondrial dysfunction and inflammation; 3) studies in invertebrates and mammals have shown increased levels of DNA damage with aging in multiple tissues. However, robust evidence that DNA somatic mutations accumulate in human cells is still lacking and the magnitude of accumulation has not been quantified. More importantly, whether the frequency of these random lesions correlate with phenotypic manifestations of aging has not been established. Finally, it is not clear whether the accumulation of DNA somatic mutations is proportional to the DNA repair capacity in different individuals. To address these questions, we have designed an ancillary study nested in GESTALT. The primary aim of this study is to test the hypothesis that the number of random somatic mutations accumulated in the DNA extracted from circulating monocytes increases with aging in healthy individuals. To accomplish this goal, we used flow cytometry to isolate 119 single monocytes from PBMCs of 30 GESTALT donors, 4 cells/donor. We selected monocytes because they are involved in tissue maintenance/repair and because they are particularly susceptible to DNA damage. Participants are dispersed over a wide age-range (20-83 years). For the 30 donors, we performed whole genome sequencing (30X) on DNA extracted from buffy coats to be used as a reference. For the single cell, we amplified the DNA using single-cell multiple displacement amplification (SCMDA) method, performed DNA quality control and the high-quality amplified DNA was sequenced. For analysis we implemented the pipeline of SCcaller (Dong et al.) to identify somatic mutations of the nuclear DNA in each single cell while adjusting for sequencing coverage and sensitivity of the variant caller. Our preliminary analysis shows that the number of nuclear somatic mutations varies among the 4 single cells of the same individual and that the number of mutations does not increase significantly with age. These observations could be due to the unevenness of the SCMDA.