Higher Order Chromatin and Genetic Risk for Alzheimer's Disease

项目摘要 阿尔茨海默病（AD）是最常见的痴呆形式，其特征在于认知障碍 和进行性神经退化AD的全基因组关联研究已经确定了30多种风险 然而，该领域的一个主要挑战是，这些风险因素中的大多数都存在于非基因组中。 编码区，其对AD发病机制的影响一直难以确定。因此，分子 AD发展和进展的基础仍然难以捉摸，到目前为止，还没有可靠的治疗方法。 found. AD相关的表观基因组调控变化可由原发性遗传和非遗传因素引起。 因果因素和附带现象，包括继发于疾病进展的变化。因此，表观基因组 是研究迟发性疾病（如AD）的一个强有力的代理标志物，其中可能存在多个 疾病进展开始和出现症状之间的数十年。在最初的R 01赠款中， 题为“高阶染色质和阿尔茨海默病的遗传风险”，我们扩大了分子组， 通过产生细胞类型特异性（神经元和非神经元）， AD病例和对照的内嗅皮层和上级颞回中的ATACseq。通过这样做，我们 确定了与临床痴呆症相关的共同和独特的调控基因组特征， 神经病理学病变和早期和晚期AD。与AD常见风险变体重叠 确定了由遗传变异驱动的初级染色质可及性扰动，与 没有明显遗传起源的继发性变化。 这项提案的总体目标是通过检查来解决我们以前研究的局限性。 以及在单细胞水平上验证染色质可及性和3D基因组的AD相关变化。 基于我们小组和其他人最近的数据，我们假设AD的基因型-表型相关性 是由基因表达调节机制中的细胞类型特异性改变引起的。到 为了检验我们的假设，我们提出以下具体目标：（1）执行多模式（即，细胞内）分析 染色质可及性和转录组在单细胞水平，以确定细胞类型特异性AD相关 3D基因组的变化;（2）精细绘制AD风险基因座，以识别致病变体、调控区域和基因; (3)使用新的方法在功能上验证推定的致病变体和调节序列， 联合收割机在神经元和小胶质细胞来源的细胞中进行大规模平行报告基因测定、CRISPR和单细胞测定 从诱导多能干细胞;和（4）开发和维护一个社区工作空间， 数据、分析和结果的快速传播和公开评估。总体而言，我们的多学科 计算和实验的方法将提供一个纲要的功能和因果验证的AD 风险位点，有可能导致新的见解和治疗发展的途径。

PROJECT SUMMARY Alzheimer's disease (AD) is the most common form of dementia and is characterized by cognitive impairment and progressive neurodegeneration. Genome-wide association studies of AD have identified more than 30 risk loci; however, a major challenge in the field is that the majority of these risk factors are harbored within non- coding regions where their impact on AD pathogenesis has been difficult to establish. Therefore, the molecular basis of AD development and progression remains elusive and, so far, reliable treatments have not been found. AD-associated changes in the regulation of the epigenome can result from primary genetic and non-genetic causal factors and epiphenomena, including changes secondary to disease progression. Thus, the epigenome is a strong proxy marker to study late-onset diseases such as AD, where there can be a gap of multiple decades between initiation of disease progression and the appearance of symptoms. In the original R01 grant titled “Higher Order Chromatin and Genetic Risk for Alzheimer's Disease”, we expanded the panel of molecular markers in the Mount Sinai Brain Bank AD cohort by generating cell type-specific (neurons and non-neurons) ATACseq in the entorhinal cortex and superior temporal gyrus of AD cases and controls. By doing so, we identified shared and distinct regulatory genomic signatures associated with clinical dementia and neuropathological lesions and with early and late stages of AD. Overlap with AD common risk variants identified primary chromatin accessibility perturbations that are driven by genetic variation, compared to secondary changes with no apparent genetic origin. The overarching goal of this proposal is to address the limitations of our previous research by examining and validating AD-related changes on chromatin accessibility and the 3D genome at the single cell level. Based on recent data from our group and others, we hypothesize that genotype-phenotype associations in AD are causally mediated by cell type-specific alterations in the regulatory mechanisms of gene expression. To test our hypothesis, we propose the following Specific Aims: (1) perform multimodal (i.e., within cell) profiling of the chromatin accessibility and transcriptome at the single cell level to identify cell type-specific AD-related changes on the 3D genome; (2) fine-map AD risk loci to identify causal variants, regulatory regions and genes; (3) functionally validate putative causal variants and regulatory sequences using novel approaches that combine massively parallel reporter assays, CRISPR and single cell assays in neurons and microglia derived from induced pluripotent stem cells; and (4) develop and maintain a community workspace that provides for the rapid dissemination and open evaluation of data, analyses, and outcomes. Overall, our multidisciplinary computational and experimental approach will provide a compendium of functionally and causally validated AD risk loci that has the potential to lead to new insights and avenues for therapeutic development.