Cytosolic DNA, Telomeres/Subtelomeres, and Epigenetics: A Longitudinal Twin Study to Assess the Role of Genetics and Environment on their Frequency and Inter-relationships

在体细胞中获得的囊泡DNA（cyDNA）正在成为细胞凋亡的引发者/整合者。 与衰老相关的功能，但cyDNA的原因/后果知之甚少。人们要 具有产生cyDNA的遗传倾向或其频率受环境影响最大 因素？cyDNA是获得其他与年龄相关的生物标志物的早期触发因素吗？ 对这些特征的一个子集的干扰做出反应吗为了回答这些基本问题，我们将 完成100对双胞胎[70对同卵（MZ）和30对异卵（FZ）]的纵向研究（10至15年的时间范围 (DZ)cyDNA不一致（35 MZ; 15 DZ）或一致（35 MZ; 15 DZ）的双胞胎对; 200个个体 频率.这对双胞胎的年龄各不相同（目前为22岁至至少80岁），以便我们记录协会 衰老的标志和cyDNA的获得之间的联系。对于每个时间点，我们将确定：（a）cyDNA 水平，（B）染色体特异性端粒/亚端粒长度，（c）衰老标记，和（d）DNA 来自两种不同组织（血液和颊粘膜细胞）的细胞中的甲基化模式[以评估潜力 体细胞相关差异]）。将对cyDNA的两个指标进行定量：（1）微核（MN）频率;和（2） 染色体外环状DNA（eccDNA）频率。将为24个中的每个确定MN频率 使用我们开发的结合光谱核型分析和荧光分析的新方法， 原位杂交技术。eccDNA的遗传内容将使用我们的滚动来确定。 环扩增和测序方案。染色体特异性端粒和亚端粒长度将是 使用我们的Q-FISH方法和我们新开发的利用原子 力显微镜，CRISPR-Cas9和我们的新基因组序列算法，提供前所未有的 端粒/亚端粒测量的分辨率。我们还将使用我们开发/优化的“最先进”工具， 量化端粒功能障碍;衰老（SADS，经典和转录组研究），以及全基因组 DNA甲基化模式。使用鲁棒方差分量估计方法（在 FISHER定量遗传学软件包），这项研究将提供第一个衡量的程度， 确定cyDNA和亚端粒长度（与TERRA相关）的个体差异 加性遗传、共同环境和特定环境效应。我们还将使用“国家的 我们开发/优化了用于分析生物标志物模式的”统计建模和生物信息学工具 在个体内、双胞胎之间和双胞胎之间，以确定模式随年龄增长的稳定性， 确定cyDNA和其他衰老标志之间的时间关系以及驱动/中介关系 （端粒/亚端粒，DNA甲基化，衰老）。从这项研究中获得的信息也可以 导致健康筛查测试的发展和/或确定新的治疗靶点， 改变我们开发治疗方法的方法，以减轻与年龄有关的健康状况的症状。

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 the development of a health screening test(s) and/or identify new therapeutic targets that could transform our approach for developing treatments to alleviate symptoms of age-related health conditions.