Statistical genetics of aging-related genomic and phenotypic change

为了帮助分析和理解受许多基因和环境因素影响的与衰老相关的“复杂”性状，我们遵循了开发用于全基因组基因分型和高通量测序研究分析的统计算法的道路。我们提出的新计算工具提供了分析其他类型数据的方法，从全基因组序列中有效地鉴定线粒体DNA（mtDNA）变体和估计mtDNA拷贝数。对于算法的实验测试，我们正在利用InCHIANTI项目（见年度报告AG 001050）和SardiNIA项目（见年度报告AG 000675）的特殊优势，以帮助在两个意大利队列中组装线粒体序列数据和多表型数据。 为了对大规模群体数据进行分析以研究mtDNA变异和拷贝数，我们开发了两个计算程序，为基于全基因组测序研究的mtDNA动力学分析提供了通用解决方案。一个程序（mitoCaller）专门用于识别mtDNA变异;另一个程序（mitoCalc）直接从基因组序列推断细胞中mtDNA的拷贝数。将这些程序应用于2，000名SardiNIA参与者和1，000名InChianti参与者的白细胞序列，我们已经表明异质性（一个位点上具有一个以上等位基因的mtDNA变体）随着年龄的增长而增加，并且拷贝数相对高度遗传，并且与代谢特征相关，特别是中央脂肪水平。在最近的工作中，我们将mitoCalc的速度提高了100倍（fastMitoCalc）。新程序正在应用于65，000个深度测序个体的白色细胞（TOPMed程序，NHLBI），用于拷贝数的GWAS。 凭借我们在研究mtDNA变异方面的专业知识，我们正在进行合作，研究DNA的一种特殊结构特征，G-四链体（G4）结构，作为干扰线粒体复制机制的潜在DNA路障。我们使用来自两个意大利队列的3,000个个体基因组的计算分析来证明G4和mtDNA变异之间的关联。使用G4 Hunter软件预测mtDNA中的G4形成区域，我们发现在稳定的G4区域中突变的统计学显著富集，优先发生在G4结构的环段中的变体。生物化学研究表明，G4结构在DNA合成中对人线粒体复制聚合酶有强有力的阻断作用，G4解旋酶Pif 1可以克服这种阻断作用。总之，计算和生物化学方法表明，mtDNA点突变富集在稳定的G4结构，与复制体停滞在G-四链体和依赖于易错的DNA合成一致。 扩展我们对mtDNA拷贝数（mtDNA Acn）分析的其他关注，我们正在着手研究巴尔的摩老龄化纵向研究（BLSA）参与者的mtDNA Acn和人格之间的关联。我们使用修订的NEO人格量表（NEO-PI-R）评估五大人格特质和方面，并从全基因组DNA序列中有效地估计mtDNA。我们的初步分析表明，mtDNAcn是显着相关的特定领域的人格清单。我们目前正在进行中介分析，以研究mtDNAcn对某些人格特质和死亡率之间关系的潜在影响。 在另一项研究中，我们创建了一个程序，使用机器学习方法来测量个人的有效衰老率。我们评估在何种程度上可以确定一个人的生理年龄作为一个综合评分推断出广泛的生化和生理特征的SardiNIA和InCHIANTI的纵向研究老化。从我们的框架推断的生理年龄与实足年龄高度相关（R2>0.8）。然后，我们定义了一个生理老化率（PAR）为每个主题，一个连续的特性测量的比例的主题预测的生理年龄，他/她的实足年龄。我们发现PAR在随访研究中是可重复的，可遗传的（h20.3），并可预测寿命和死亡率。对PAR的全基因组关联研究（GWAS）确定了先前建立的年龄相关基因座和几个新的遗传关联。我们的研究结果支持一种全身的、病理学无关的衰老效应，这种效应可以通过生理衰老率来总结，我们的方法可以用于评估针对衰老相关过程和疾病的治疗效果。

To help to analyze and understand aging-related "complex" traits that are affected by many genes and environmental factors, we have followed the path of developing statistical algorithms for the analyses of genome-wide genotyping and high-throughput sequencing studies. Our proposed new computational tools provide means to analyze additional types of data e.g., to identify mitochondrial DNA (mtDNA) variants and to estimate mtDNA copy number efficiently from whole-genome sequences. For experimental tests of the algorithms, we are capitalizing on the special advantages of the InCHIANTI project (see Annual Report AG001050) and SardiNIA project (see Annual Report AG000675) to help in the assembly of mitochondrial sequence data and multiple phenotypic data in the two Italian cohorts. In order to conduct analyses on large-scale consortium data to study mtDNA variation and copy number, we have developed two computational programs, providing a general solution for the analysis of mtDNA dynamics based on whole-genome sequencing studies. One program (mitoCaller) is designed specifically to identify mtDNA variants; the other (mitoCalc) infers mtDNA copy number in a cell directly from genome sequences. Applying the programs to leukocyte sequences of 2,000 SardiNIA participants and 1,000 InCHIANTI participants, we have shown that heteroplasmies (mtDNA variants with more than one allele at a site) increase with age, and that copy number is relatively highly heritable and is correlated with metabolic traits, particularly central fat levels. In more recent work, we have increased the speed of mitoCalc 100-fold (fastMitoCalc). The new program is being applied to white cells of 65,000 deeply sequenced individuals (TOPMed program, NHLBI), for GWAS on copy number. With our expertise in studying mtDNA variation, we have an ongoing collaborative effort to study a special structural feature of DNA, G-quadruplex (G4) structures, as potential DNA roadblocks that perturb mitochondrial replication machinery. We used computational analyses of 3,000 individual genomes from two Italian cohorts to demonstrate an association between G4s and mtDNA variation. Using the software G4Hunter to predict G4-forming regions in mtDNA, we found statistically significant enrichment of mutations in stable G4 regions, with preferential occurrence of variants in the loop segments of G4 structures. Biochemical studies demonstrated a potent block of human mitochondrial replicative polymerase in DNA synthesis by G4 structure, which could be overcome by the G4-resolving helicase Pif1. Altogether, the computational and biochemical approaches indicate that mtDNA point mutations are enriched at stable G4 structures, consistent with replisome stalling at G-quadruplexes and reliance on error-prone DNA synthesis. Expanding our other focus on the mtDNA copy number (mtDNAcn) analysis, we are setting out to examine the association between mtDNAcn and personality in participants of the Baltimore Longitudinal Study of Aging (BLSA). We assess the big five personality traits and facets using the Revised NEO Personality Inventory (NEO-PI-R) and estimate mtDNAcn efficiently from whole-genome DNA sequences. Our preliminary analyses show that mtDNAcn is significantly associated with specific domains of the personality inventory. We are currently performing mediation analysis to study the potential impact of mtDNAcn on the relationship between certain personality traits and mortality. In another study, we have created a program that uses machine learning methods to measure effective rates of aging for individuals. We assess the extent to which an individual's physiological age could be determined as a composite score inferred from a broad range of biochemical and physiological traits from the SardiNIA and InCHIANTI longitudinal studies of aging. Physiological age inferred from our framework was highly correlated with chronological age (R2>0.8). We then defined a physiological aging rate (PAR) for each subject, a continuous trait measured as the ratio of the subjects predicted physiological age to his/her chronological age. We found that PARs were reproducible across follow-up studies, heritable (h20.3), and predictive of lifespan and mortality. Genome-wide association studies (GWAS) on the PARs identified both previously established age-associated loci and several new genetic associations. Our findings support a whole-body, pathology-independent aging effect that can be summarized by the physiological aging rate and our method can be used to evaluate the efficacy of treatments that target aging-related processes and disease.