Systematic Characterization of Tauopathy-Associated Genetic Variation using Multiplexed Reporter Assays

使用多重报告基因检测系统表征 Tau 病相关遗传变异

• 批准号: 10020162
• 负责人: Yonatan Cooper
• 金额: $ 3.73万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2023-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10020162
• 关键词: 17q21; ATAC-seq; Affect; Alzheimer' s Disease; Biological Assay; Biological Models; Biology; Brain; CRISPR/Cas technology; Cells; ChIP-seq; Chromatin; Clinical; Cognition Disorders; Complex; Data; Dementia; Dependovirus; Disease; Disease model; Disease susceptibility; Epidemic; Etiology; Follow-Up Studies; Frontotemporal Dementia; Future; Gene Expression; Gene Frequency; Genes; Genetic; Genetic Risk; Genetic Variation; Genetic study; Genome; Heritability; Human; In Vitro; Individual; Libraries; Linkage Disequilibrium; Methods; Modality; Modeling; Molecular; Molecular Conformation; Nerve Degeneration; Neurodegenerative Disorders; Neuroglia; Neurons; Nuclear Pore Complex; Nucleic Acid Regulatory Sequences; Parkinson Disease; Pathogenesis; Pathologic; Performance; Phenotype; Polygenic Traits; Progressive Supranuclear Palsy; Protocols documentation; Reporter; Risk; Role; Serotyping; Single Nucleotide Polymorphism; Structure; Susceptibility Gene; System; Tauopathies; Testing; Tissues; Transfection; Untranslated RNA; Variant; Viral; Virus Diseases; Work; aging population; causal variant; cell type; cohort; disorder risk; effective therapy; experimental study; functional genomics; genetic analysis; genetic architecture; genome sequencing; genome wide association study; genome-wide; genomic data; improved; in vitro Model; in vivo; innovation; insight; mortality; nerve stem cell; nervous system disorder; neurodegenerative dementia; new therapeutic target; novel; rare variant; risk variant; tau Proteins; tau aggregation; trait; whole genome

PROJECT SUMMARY/ABSTRACT Dementias, neurodegenerative cognitive disorders that include Alzheimer's disease (AD) and frontotemporal dementia (FTD), are slated to become a global epidemic due to a rapidly aging population. Disease etiology remains poorly understood and there are no effective treatments. Therefore, elucidating molecular mechanisms underlying the pathogenesis of neurodegeneration and identifying novel therapeutic targets is critically important. Progressive supranuclear palsy (PSP) is a neurodegenerative disease that has significant clinical, pathological, and genetic overlap with several dementias including AD, FTD, and Parkinson's disease. In contrast to these disorders, PSP has a relatively homogenous clinical and neuropathological phenotype, which motivates the study of PSP as a tractable yet generalizable model of neurodegenerative disease. One method that has proven useful for identifying casual disease mechanisms underlying PSP and neurodegeneration more broadly is genetic analysis. However, the majority of human variation is in non- coding regions of the genome and is difficult to functionally interpret. This includes rare and structural variants from whole genome sequencing (WGS) efforts and common susceptibility loci identified by genome-wide association studies (GWAS). Therefore, functional annotation of non-coding variation remains a major impediment to the elucidation of neurodegenerative disease. Massively Parallel Reporter Assays (MPRA) are a powerful approach for experimentally characterizing the regulatory effects of non-coding variation in a high- throughput manner, but have yet to be widely applied to neurologic disorders. The purpose of this study is to utilize MPRA to functionally characterize non-coding common and rare variation associated with PSP and identify causal genetic risk. To accomplish this, the experiments proposed in Aim 1 will first establish a novel protocol for implementing MPRA within differentiated human neural progenitor cells (hNPCs) – an in vitro model system germane to the study of neurological disease. A critical innovation will be to deliver the MPRA library into hNPCs using adeno-associated viruses (AAV). Aim 2 will use this approach to 1) identify causal variants underlying 9 PSP susceptibility loci identified by GWAS, and 2) model select functional variants in vitro using CRISPR-Cas9. This includes a systematic characterization of 17q21.31, an important risk locus associated with PSP, AD, and PD that has remained difficult to study. In Aim 3, variation from the largest PSP WGS study to date (1293 cases and 2000 controls) will be prioritized using multiple functional genomics datasets and characterized using MPRA, representing the first systematic analysis of PSP non-coding rare variation (allele frequency < 1%). Taken together, this work will elucidate the regulatory genetic architecture of PSP and provide mechanistic insights into variation underlying disease risk. More broadly, it will provide generalizable understanding of the genetics and fundamental biology of neurodegenerative disease and serve as a model for future studies seeking to comprehensively characterize non-coding variation in complex traits.

项目总结/摘要 痴呆，神经退行性认知障碍，包括阿尔茨海默病（AD）和额颞叶痴呆。 痴呆症（FTD）由于人口的快速老龄化而预定成为全球流行病。疾病病因 人们对此知之甚少，也没有有效的治疗方法。因此，阐明分子 神经退行性变发病机制的基础和识别新的治疗靶点， 至关重要。进行性核上性麻痹（PSP）是一种神经退行性疾病，具有显著的神经退行性病变。 在临床、病理和遗传学上与包括AD、FTD和帕金森病在内的几种痴呆重叠。 与这些疾病相反，PSP具有相对同质的临床和神经病理表型， 这激发了PSP作为神经退行性疾病的易处理但可推广的模型的研究。一 该方法已被证明可用于识别PSP的因果疾病机制， 神经退行性变更广泛地说是遗传分析。然而，大多数人类变异是在非- 基因组的编码区，很难从功能上解释。这包括罕见的和结构性的变异 从全基因组测序（WGS）的努力和常见的易感基因座确定的全基因组 关联研究（GWAS）。因此，非编码变异的功能注释仍然是一个主要的问题。 阻碍了神经退行性疾病的阐明。大规模平行报告基因测定（MPRA）是一种 一种强有力的方法，用于实验表征非编码变异在高 但是还没有广泛应用于神经系统疾病。本研究的目的是 利用MPRA在功能上表征与PSP相关的非编码常见和罕见变异， 确定因果遗传风险。为了实现这一点，目标1中提出的实验将首先建立一种新的 在分化的人神经祖细胞（hNPC）中实施MPRA的方案-体外 与神经系统疾病研究密切相关的模型系统。一个关键的创新将是提供MPRA 使用腺相关病毒（AAV）将文库导入hNPC。目标2将使用这种方法1）确定因果关系 GWAS鉴定的9个PSP易感基因座的基础变异，和2）模型选择功能性变异， 使用CRISPR-Cas9进行体外试验。这包括17q21.31的系统性特征，这是一个重要的风险位点， 与PSP，AD和PD相关的疾病，仍然难以研究。在目标3中，最大PSP的变化 迄今为止的WGS研究（1293例病例和2000例对照）将优先使用多功能基因组学 数据集和特点，使用MPRA，代表第一次系统分析PSP非编码罕见 变异（等位基因频率< 1%）。总之，这项工作将阐明调控遗传结构的 PSP和提供机制的变化，潜在的疾病风险的见解。更广泛地说，它将提供 对神经退行性疾病的遗传学和基础生物学有概括性的了解，并作为 一个模型，为未来的研究寻求全面表征非编码变异的复杂性状。