Project 2: The Isoform repertoire and epigenome of Pediatric SLE

项目总结 系统性红斑狼疮是一种发病率很高的疾病，治疗方案很差。确定相关目标 由于疾病的分子和临床异质性，治疗干预是困难的。通过一个 大量的工作，我们已经证明了基于血液的转录图谱可以用来解析出 健康和疾病中免疫功能的明显转录差异。与当前应用程序相关 我们的研究是否表明SLE可以根据不同的血液分为七种疾病亚型 转录签名。最近，我们开发了一个用于集成分析和分析的复杂系统 我们用来确定人类有意义的表观遗传和转录差异的管道(IPAP) 人群，包括系统性红斑狼疮队列。在此知识库的基础上构建将帮助我们确定 疾病，最终是SLE的新生物标记物和新的治疗靶点。我们假设红斑狼疮 由一系列疾病亚型组成，这些亚型可以根据相互不同的情况进行分类 表观基因组学、异构体和转录体特征。这个项目的目标是描述剧目的特点 最常见的七种免疫细胞亚型和表观基因组图谱中的三种 儿童SLE亚型：以浆母细胞转录特征为特征的SLE-浆母细胞(SLE-P) 和循环中浆母细胞的增加；SLE-干扰素/髓样细胞(SLE-IM)，这表明 干扰素和中性粒细胞特征；SLE-浆母细胞/干扰素/髓系细胞(SLE-PIM)显示 三个主要签名。我们将使用包含尖端基因组的新的实验范例 技术，并以我们在分子免疫图谱方面的成熟专业知识为基础。我们的项目包含两个 具体目的：1)分析三种系统性红斑狼疮患者血细胞的选择性剪接转录组 患者分组，其中我们将同时应用短读(Illumina HiSeq2500)和长读(PacBio RSII) 测序技术，询问外周血单核细胞的异构体谱系并选择免疫细胞亚群； 2)通过研究确定三个SLE亚型的血细胞表观基因组 一种稳健而灵敏的新表观基因组分析，称为ATAC-SEQ，与甲基-SEQ偶联。健康的年龄- 所有实验中将使用匹配的样本作为对照，我们还将检查来自儿童的样本 活动期幼年皮肌炎和系统性幼年关节炎(每组5例)，以鉴定这些亚型 以及系统性红斑狼疮特有的签名，这些签名更普遍地与自身免疫有关。我们的团队包括 免疫学、基因组学、表观遗传学、疾病系统生物学、计算生物学、数据专家 管理和统计，他们带来了产生高质量基因组学所需的集体专业知识 并使用最先进的方法进行稳健的数据分析。我们还将创建新的工具，即NanoString 探针和单抗，适用于适合大患者队列的高通量筛查分析。 我们项目的成功完成将揭示SLE的新生物标记物和/或新的治疗靶点。

PROJECT SUMMARY SLE is a disease of high morbidity for which treatment options are poor. Identifying relevant targets for therapeutic intervention is difficult owing to the molecular and clinical heterogeneity of the disease. Through a large body of work, we have demonstrated that blood-based transcriptomic profiling can be used to parse out distinct transcriptional differences in immune function in health and disease. Relevant to the current application are our studies showing that SLE can be divided into seven disease subgroups based on distinct blood transcriptional signatures. More recently, we developed a sophisticated for integrated Profiling and Analysis Pipeline (iPAP) that we used to identify meaningful epigenetic and transcriptomic differences in human populations, including SLE cohorts. Building upon this knowledgebase will help us identify potential drivers of the disease, and ultimately novel biomarkers and novel therapeutic targets for SLE. We hypothesize that SLE is comprised of a spectrum of disease subtypes that can be categorized according to mutually distinct epigenomic, isoformic and transcriptomic features. The goal of this project is to characterize the repertoire of immune cell isoforms and the epigenome profiles associated with three of the seven most frequent pediatric SLE subgroups: SLE-Plasmablast (SLE-P), characterized by a plasmablast transcriptional signature and the presence of increased circulating plasmablasts; SLE-Interferon/Myeloid cells (SLE-IM), which shows an IFN and a neutrophil signature; and SLE-Plasmablast/ Interferon/Myeloid cells (SLE-PIM) displaying the three main signatures. We will use novel experimental paradigms that incorporate cutting-edge genomic technologies and build upon our established expertise in molecular immune profiling. Our project contains two Specific Aims: 1) To analyze the alternatively spliced transcriptome of blood cells from the three SLE patient subgroups, in which we will apply both short-read (Illumina HiSeq2500) and long-read (PacBio RSII) sequencing technologies to interrogate the isoform repertoire of PBMCs and select immune cell subsets; and 2) To determine the epigenome of blood cells from the three SLE subgroups, through studies capitalizing on a robust and sensitive new epigenomic assay called ATAC-seq coupled to Methyl-seq. Healthy age- matched samples will be used as controls in all experiments and, we will also examine samples from children with active juvenile dermatomyositis and systemic juvenile arthritis (n=5 per group), to identify those isoforms and signatures unique to SLE from those more generally associated with autoimmunity. Our team includes experts in immunology, genomics, epigenetics, systems biology of disease, computational biology, data management and statistics, who bring the collective expertise necessary for generating top quality genomics data and for robust data analysis using state-of-the-art methods. We will also create new tools, i.e., Nanostring probes and monoclonal antibodies, for high throughout screening assays suited to large patient cohorts. Successful completion of our project will reveal novel biomarkers of SLE and/or novel therapeutic targets.