Cystolic DNA (cyDNA), which is acquired in somatic cells, is emerging as an instigator/integrator of cellular functions associated with aging, yet the causes/consequences of cyDNA are poorly understood. Do individuals have a genetic predisposition to develop cyDNA or is its frequency most heavily influenced by environmental factors? Is cyDNA an early trigger for the acquisition of other age-related biomarker hallmarks, or does it arise in response to perturbations involving a subset of these hallmarks? To answer these primary questions, we will complete a longitudinal study (10 to 15 years timeframe) of 100 twin pairs [70 identical (MZ) and 30 fraternal (DZ) twin pairs; 200 individuals] who are discordant (35 MZ; 15 DZ) or concordant (35 MZ; 15 DZ) for cyDNA frequencies. The twin pairs will vary in age (currently 22 to at least 80 y.o) to allow us to chronicle associations between aging hallmarks and the acquisition of cyDNA. For each time point we will determine: (a) cyDNA levels, (b) chromosome specific-telomere/subtelomere lengths, (c) senescence markers, and (d) DNA methylation patterns in cells from two different tissues (blood and buccal mucosa cells [to assess potential soma-related differences]). Two measures of cyDNA will be quantified: (1) micronuclei (MN) frequency; and (2) extrachromosomal circular DNA (eccDNA) frequency. The MN frequencies will be identified for each of the 24 human chromosomes using a novel assay we developed that combines spectral karyotyping and fluorescence in situ hybridization technologies. The genetic contents of the eccDNA will be determined using our rolling circle amplification and sequencing protocol. Chromosome-specific telomere and subtelomere lengths will be determined using our Q-FISH method and our newly developed nanomapping method that exploits atomic force microscopy, CRISPR-Cas9, and our novel genome sequence algorithm to provide unprecedented resolution of telomere/subtelomere measures. We will also use “state of the art” tools we developed/optimized, to quantify telomere dysfunction; senescence (SADS, classical, and transcriptome studies), and genome-wide DNA methylation patterns. Using a method of robust variance component estimation (implemented in the FISHER quantitative genetics package), this study will provide the first measure of the extent to which individual differences in cyDNA and subtelomere lengths (which are associated with TERRA) are determined by additive genetic, common environmental, and specific environmental effects. We will also use “state of the art” statistical modeling and bioinformatic tools that we developed/optimized to analyze biomarker patterns within individuals, between co-twins, and among twin pairs to determine the stability of patterns with aging, and to identify temporal, as well as driver/mediator, relationships among cyDNA and other aging hallmarks (telomeres/subtelomeres, DNA methylation, senescence). The information gained from this study could also lead to...