BIOMARKER DISCOVERY AND VALIDATION IN PSP

摘要 进行性核上性瘫痪(PSP)是一种破坏性的非典型帕金森病，目前缺乏 有意义的对症治疗，缩短寿命，极大地损害日常功能和生活质量。它是 临床上常常难以与帕金森病(PD)相鉴别，这对恰当和及时地治疗帕金森病至关重要 管理、预后和临床试验招募。尽管迫切需要一种可靠的诊断标记物 帕金森病，目前还没有可用于常规临床实践的生物标记物 区分PSP和PD。这个项目的目的是发现脑脊液(CSF) 可靠区分PSP、PD和健康个体的生物标志物。辨认的困难 可靠的生物标志物可以归因于临床样本的变异性，低丰度的蛋白质 参与了PSP和PD的发病机制，以及在验证候选生物标记物时缺乏重复性。 为了克服这些限制，我们建议使用具有更大统计能力的大型脑脊液队列来进行TRUE 发现，并进行深入的蛋白质组分析，以揭示参与PSP发病机制的PSP生物标记物，但 以较低的丰度存在。此外，用等压串联质量标记法进行多路样品分析 (TMT)具有数据归一化的公共参考将确保可靠的定量分析精度 从更大的样本集合中发现蛋白质组学数据。此外，对大脑进行了额外的蛋白质组学分析 组织将被用来选择那些在脑脊液和球部显示差异表达的生物标记物 苍白球，一个具有代表性的大脑区域，用于从病理上定义PSP。这些发现平台将利用 一种生物信息学方法来选择最可信的候选对象进行靶向验证研究，然后 对已发现的生物标志物候选进行密集验证。为了实现这些目标，我们提出了四个目标： 具体目标1：前瞻性收集临床特征良好的PSP患者的脑脊液。特定目标 2：发现PSP患者与对照组和PD患者相比差异表达的蛋白质。我们 计划对PSP患者的脑脊液和苍白球样本进行定量蛋白质组学分析， PD和通过使用基于TMT的多路复用技术来自对照。通过这种方法，我们希望 更全面地覆盖分析样本中量化的更多蛋白质。 具体目标3：基于对脑脊液改变的综合分析，确定PSP生物标志物候选的优先顺序 和苍白球。通过将脑脊液和脑组织的表达变化与网络方法相结合 这利用了PSP中描述的已知生物途径，我们的建议将是 能够通过目标PRM实验选择可靠的PSP生物标记物候选进行验证。具体目标4： 使用靶向平行反应监测(PRM)验证较大队列中的候选蛋白质生物标记物 使用约翰·霍普金斯大学PSP队列中的脑脊液样本进行质谱分析 宾夕法尼亚州，加州大学旧金山分校和PDBP。由基于这些PRM实验的算法选择的生物标志物 最终将使用来自PSP的盲法PDBP脑脊液样本进行确认，并将其与脑脊液样本进行比较 从警局来的。通过以上概述的方法，我们期望发现和验证可靠的PSP生物标志物 以可重现的方式与PD区分。

ABSTRACT Progressive supranuclear palsy (PSP) is a devastating atypical parkinsonian disorder that currently lacks meaningful symptomatic therapies, reduces lifespan and greatly impairs daily function and quality of life. It is often difficult to distinguish from Parkinson disease (PD) clinically, which is crucial for appropriate and timely management, prognosis and clinical trial enrollment. Despite a critical need for a reliable diagnostic marker for parkinsonian disorders, there is currently no biomarker that can be used in routine clinical practice to distinguish between PSP and PD. The purpose of this project is to discover cerebrospinal fluid (CSF) biomarkers that reliably distinguish between PSP, PD and healthy individuals. The difficulty of identifying reliable biomarkers can be attributed to the variability of clinical samples, low abundance of proteins that are involved in the pathogenesis of PSP and PD, and the lack of reproducibility in validating biomarker candidates. To overcome these limitations, we propose use of a large CSF cohort with greater statistical power for true discovery, and deep proteome analysis to reveal PSP biomarkers that are involved in PSP pathogenesis, but are present at low abundance. In addition, multiplexed sample analysis by isobaric tandem mass tagging (TMT) with a common reference for data normalization will ensure robust analytical precision of quantitative proteomic data for discovery from a larger set of samples. Moreover, additional proteomic analysis of brain tissue will be used to select those biomarkers that show differential expression in CSF as well as the globus pallidus, a representative brain region used to pathologically define PSP. These discovery platforms will utilize a bioinformatics approach to select the most plausible candidates for targeted validation studies followed by an intensive validation of the discovered biomarker candidates. To achieve these goals, we propose four aims: Specific Aim 1: To prospectively collect CSF on patients with clinically well-characterized PSP. Specific Aim 2: To discover proteins that are differentially expressed in patients with PSP compared to controls and PD. We plan to carry out a quantitative proteomic analysis of CSF and globus pallidus samples from patients with PSP, PD and from controls by employing TMT-based multiplexing technology. With this approach, we expect to obtain a more comprehensive coverage of a larger number of proteins quantified across the analyzed samples. Specific Aim 3: To prioritize PSP biomarker candidates based on an integrative analysis of alterations in CSF and globus pallidus. By integrating the expression changes in CSF and brain tissue with a network approach that takes advantage of the known biological pathways that have been described in PSP, our proposal will be able to select reliable PSP biomarker candidates for validation by targeted PRM experiments. Specific Aim 4: To validate candidate protein biomarkers in a larger cohort using targeted parallel reaction monitoring (PRM) mass spectrometry using CSF samples from a PSP cohort at Johns Hopkins University, the University of Pennsylvania, UCSF and PDBP. Biomarkers that are selected by algorithms based on these PRM experiments will finally be confirmed using blinded PDBP CSF samples from PSP and will be compared to CSF samples from PD. Through the approaches outlined above, we expect to discover and validate reliable PSP biomarkers that are distinguishable from PD in a reproducible manner.