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

项目总结 我们研究的目标是识别和验证调节基因、蛋白质和生物途径 由突变的亨廷顿蛋白(MHTT)引起的神经变性，这种蛋白质导致亨廷顿病(HD)。这 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更容易下药的靶点。虽然mHTT是HD的主要原因，但一些研究已经 表明遗传修饰物与mHTT相互作用影响HD神经退行性变的进展。事实上，一个 对HD的重大遗传贡献并不仅仅由编码mHTT的基因或少数人来解释 其他研究小组已经确定的修饰物。我们假设罕见的基因变异 与全基因组关联遗漏的HD疾病的发生和发展有关 研究(Gwas)或以候选人为基础的方法。考虑到这一点，我们进行了全基因组测序 (WGS)对多个HD家系进行分析，并在先前未涉及HD的新基因中确定候选基因。他们 参与蛋白质清除和其他可能导致HD神经变性的细胞通路。 我们首次提供了直接证据，证明这些候选者的子集可以改变神经退行性变。 人纹状体类HD IPSC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调查这些潜力 遗传修饰物调节神经退行性变，并将我们的分析扩展到更多的变种及其细胞 导致HD神经退行性变的途径。人类神经元模型概括了几个关键特征 以及一种被称为机器人显微镜(RM)的细胞成像形式，使高通量(HT)、高内容、 这些模型的纵向单神经元分析。由RM生成的数据集揭示了 神经变性，包括存活，用强大的统计方法分析，或轴突长度的变化， 这是细胞压力的预测指标。我们的工具箱使用其他强大的方法来评估候选人的效果 神经退行性变，例如光脉冲标记(OPL)技术，可以测量 在单细胞内通过蛋白酶体活性或自噬清除蛋白质。我们有NIH X01拨款，这是 对19个新的HD家庭的104名额外成员进行测序，我们有大量的医疗记录和 临床病史记录。我们将把我们的WGS分析扩展到这些家庭，并结合数据来确定更多 一套完整的相互作用的基因伙伴和途径，并帮助我们集中目前的候选名单， 对HD的发病和轨迹有影响。新的假定变种将在我们的人类HD I-神经元模型中进行测试 确认它们是潜在的遗传修饰物，并更好地定义参与调控发病的细胞通路 高清。HD新的遗传修饰物的发现将进一步阐明HD的发病机制 确定新的方向，以开发疾病修正疗法，并对HD人群进行分层，以获得更多 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.