Rare Variant Whole Genome Analysis and iPSC Validation of Putative Genetic Modifiers of Huntington Disease

项目摘要 我们研究的目标是鉴定和验证基因、蛋白质和生物学途径， 由突变型亨廷顿蛋白（mHtt）诱导的神经变性，突变型亨廷顿蛋白是导致亨廷顿病（HD）的蛋白质。这 这些知识将为HD的潜在机制提供新的见解，并可能揭示新的治疗方法。 比mHtt更易被药物治疗的靶点虽然mHtt是HD的主要原因，但许多研究表明， 表明遗传修饰剂与mHtt相互作用以影响HD中神经变性的进展。其实一个 HD的实质性遗传贡献并不完全由编码mHtt的基因或由少数几个 其他研究小组已经确定的改性剂。我们假设罕见的基因变异 促进HD的疾病发作和进展，而全基因组关联性忽略了这一点 研究（GWAS）或基于候选人的方法。考虑到这一点，我们进行了全基因组测序， (WGS)对多个HD家族进行了研究，并在先前未涉及HD的新基因中确定了候选基因。他们 参与蛋白质清除和其他可能导致HD神经变性的细胞途径。 我们第一次提供了直接的证据，这些候选者的一个子集改变了神经变性。 人纹状体样HD iPSC衍生的神经元（HD纹状体i-神经元）。 在拟议的研究中，我们将进一步验证和研究这些潜在的机制， 遗传修饰剂调节神经变性，并将我们的分析扩展到其他变体及其细胞 导致HD神经退行性变的通路。人类神经元模型概括了几个关键特征 和一种称为机器人显微镜（RM）的细胞成像形式，能够实现高通量（HT），高内容， 这些模型的纵向单神经元分析。RM生成的数据集揭示了 神经变性，包括存活，通过强大的统计方法分析，或神经突长度的变化， 这是细胞压力的预测器。我们的工具箱使用其他强大的方法来评估候选人的影响 关于神经退行性疾病，例如光脉冲标记（OPL）技术，可以测量神经退行性疾病的发生率。 通过蛋白酶体活性或单细胞内的自噬清除蛋白质。我们有一个NIH X 01基金， 对19个新的HD家族的104名额外成员进行测序，我们有大量的医疗记录， 我们将把我们的WGS分析扩展到这些家庭，并结合联合收割机的数据，以确定一个更多的 一整套相互作用的基因伴侣和途径，并帮助我们集中目前的候选人名单， 有助于HD发作和轨迹。新的假定变体将在我们的人类HD i-神经元模型中进行测试， 验证它们作为潜在的遗传修饰剂，并更好地定义参与调节发病的细胞途径， HD的。HD的新遗传修饰物的发现将进一步阐明HD的疾病机制， 为开发疾病缓解疗法和对HD人群进行分层确定新的方向， 成功的临床试验。

PROJECT SUMMARY The goal of our studies is to identify and validate genes, proteins, and biological pathways that modulate neurodegeneration induced by mutant huntingtin (mHtt), the protein that causes Huntington's disease (HD). This knowledge will provide new insights into the underlying mechanisms of HD and may reveal novel therapeutic targets that are more druggable than mHtt. While mHtt is the major cause for HD, a number of studies have indicated that genetic modifiers interact with mHtt to affect progression of neurodegeneration in HD. In fact, a substantial genetic contribution to HD is not accounted for solely by the gene that encodes mHtt, or by the few modifiers that have been identified by other research groups. We hypothesize that rare genetic variants contribute to the disease onset and progression of HD that have been missed by genome-wide association studies (GWAS) or candidate-based approaches. With this in mind, we conducted whole-genome sequencing (WGS) on multiple HD families and identified candidates in novel genes not previously implicated in HD. They are involved in protein clearance and other cellular pathways that may contribute to neurodegeneration in HD. We provide direct evidence, for the first time, that a subset of these candidates modify neurodegeneration of human striatal-like HD iPSC-derived neurons (HD striatal i-neuron). In the proposed studies, we will further validate and investigate the mechanisms by which these potential genetic modifiers modulate neurodegeneration and expand our analysis to additional variants and their cellular pathways that contribute to neurodegeneration in HD. Human neuron models recapitulate several key features of HD, and a form of cellular imaging called robotic microscopy (RM) enables high-throughput (HT), high-content, longitudinal single-neuron analysis of these models. The data sets generated by RM reveal different aspects of neurodegeneration, including survival, analyzed by powerful statistical methods, or changes in neurite length, which is a predictor of cellular stress. Our toolbox uses other powerful approaches to assess a candidates' effects on neurodegeneration, such as an optical-pulse labeling (OPL) technology that can measure the rate of clearance of proteins by proteasome activity or autophagy within single cells. We have an NIH X01 grant that is sequencing 104 additional members of 19 new HD families for which we have extensive medical records and clinical history on. We will extend our WGS analysis to these families and combine the data to identify a more complete set of interacting gene partners and pathways and to help focus our list of current candidates that contribute to HD onset and trajectory. New putative variants will be tested in our human HD i-neuron model to validate them as potential genetic modifiers and to better define cellular pathways involved in modulating onset of HD. The discovery of novel genetic modifiers of HD will further elucidate the disease mechanisms in HD and identify new directions for developing disease-modifying therapeutics and for stratifying HD populations for more successful clinical trials.