Project 2: The Isoform repertoire and epigenome of Pediatric SLE

项目摘要 系统性红斑狼疮是一种发病率高的疾病，其治疗选择很差。确定相关目标， 由于该疾病的分子和临床异质性，治疗干预是困难的。通过 大量的工作，我们已经证明，基于血液的转录组学分析可以用来解析出 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个疾病亚组， 转录签名。最近，我们开发了一个复杂的集成分析和分析 我们使用iPAP来识别人类中有意义的表观遗传和转录组差异， 人群，包括SLE队列。建立在这一知识基础上将有助于我们确定潜在的驱动因素， 疾病，并最终新的生物标志物和新的SLE治疗靶点。我们假设SLE 由一系列疾病亚型组成，这些亚型可以根据相互不同的 表观基因组、同型和转录组特征。这个项目的目标是描述 免疫细胞亚型和表观基因组图谱与七种最常见的 儿科SLE亚组：SLE-浆母细胞（SLE-P），以浆母细胞转录特征为特征 以及循环浆母细胞增多; SLE-干扰素/髓样细胞（SLE-IM），这表明 IFN和嗜中性粒细胞标签;和显示IFN和嗜中性粒细胞标签的SLE-成浆细胞/干扰素/髓样细胞（SLE-PIM）。 三大签名我们将使用新的实验范式，将尖端的基因组 技术，并建立在我们在分子免疫分析方面的专业知识。我们的项目包含两个 具体目的：1）分析3例SLE患者血细胞的可变剪接转录组 患者亚组，其中我们将应用短读码（Illumina HiSeq2500）和长读码（PacBio RSII） 测序技术，以询问PBMC的同种型库并选择免疫细胞亚群;以及 2)确定三个SLE亚组血细胞的表观基因组，通过研究 在一个强大的和敏感的新的表观基因组测定称为ATAC-seq耦合到甲基-seq。健康的年龄- 匹配的样本将被用作所有实验的对照，我们还将检查儿童样本 活动性幼年皮肌炎和全身性幼年关节炎（每组n = 5），以确定这些亚型 以及SLE特有的与自身免疫相关的特征。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 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 数据，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 探针和单克隆抗体，用于适合于大患者群体的高通量筛选测定。 我们的项目的成功完成将揭示新的SLE生物标志物和/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.