Deep Sequencing of Kawasaki Disease Tissues

川崎病组织的深度测序

基本信息

批准号：
8296545
负责人：
ANNE H ROWLEY
金额：
$ 18.99万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-07 至 2014-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8296545
关键词：
Acute Amino Acids Aneurysm Antibiotic Therapy Antibodies Antigens Bioinformatics Blood Vessels Child Childhood Clinical Communicable Diseases Complementary DNA Coronary artery DNA Sequence Data Set Detection Developed Countries Development Diagnostic tests Electron Microscope Electrons Epidemic Epidemiology Failure Future Genes Goals Grant Heart Diseases Human Immune response Immunoglobulin A Inclusion Bodies Infant Inflammatory Interferon-beta Interferons Intravenous Laboratories Lasers Length Lung Methods Microscopic Molecular Mucocutaneous Lymph Node Syndrome Nucleotides Paraffin Embedding Pathology Patients Prevention RNA RNA Viruses Reading Reporting Reverse Transcriptase Polymerase Chain Reaction Sampling Spleen Techniques Time Tissues Translating Viral Viral Proteins Virus Virus Diseases Virus-like particle bronchial epithelium cDNA Library disorder control experience improved interest light transmission lymph nodes macrophage microbial multidisciplinary novel response transmission process viral RNA

项目摘要

DESCRIPTION (provided by applicant): Kawasaki Disease (KD) is a systemic inflammatory illness of childhood that causes coronary artery aneurysms in 25% of untreated patients and in 5% of those treated with intravenous gammaglobulin therapy. KD is the most common cause of acquired heart disease in children in developed nations. Clinical and epidemiologic features including seasonal epidemics and failure of antibiotic therapy strongly suggest a viral cause, but no known virus has been identified. My laboratory reported an antigen-driven IgA arterial immune response in acute KD, and synthetic versions of these antibodies identified intracytoplasmic inclusion bodies (ICI) in KD ciliated bronchial epithelium, in a subset of macrophages in lung, spleen, and lymph nodes, and in vascular tissue. Light and transmission electron microscopic analysis (TEM) of paraffin-embedded lung showed that the ICI are consistent with aggregates of viral proteins and RNA. Most recently, TEM of non- embedded bronchial tissue from three cases demonstrated virus-like particles (VLP) in close proximity to ICI. The VLPs were not ultrastructurally typical for any known virus. High-throughput (454) sequencing of cDNA from laser-capture microdissected bronchial epithelium from one of these cases (400,000 sequences) showed that interferon-stimulated genes were upregulated, consistent with viral infection. Stimulation of the alpha, beta interferon response in KD lung was subsequently confirmed using real-time PCR array analysis of KD and control lung samples. These findings led us to hypothesize that a "new", previously unidentified RNA virus that enters through the lung is the causative agent of KD, and that viral sequences in KD tissues can be identified by high-throughput sequencing using a bioinformatics approach. This virus may have escaped detection to date because of limited nucleotide or amino acid homology with known viruses, and/or because an insufficient number of sequences from KD tissues have been examined. We have gathered a multidisciplinary team including KD infectious diseases and pathology experts, a virologist, an experienced geneticist, a bioinformatician, and a biostatistician, with the goal of identifying viral sequence(s) associated with KD. We propose the following specific aims for this exploratory grant: 1) Perform ultra high-throughput sequencing of cDNA amplified from KD tissues, 2) Use bioinformatics analysis to identify sequences with viral homology and without a match and perform assembly on sequences of interest to generate the longest contiguous sequences, and 3) Determine the presence of sequences of interest in additional KD and control tissues using real-time PCR analysis. The identification of ICI and VLP in KD tissues provides a unique opportunity to perform directed high-throughput sequencing to identify viral sequence(s) associated with KD, with the long-term goal of determining the etiologic agent of KD. Identification of the causative agent of KD would have profound implications for the field, enabling development of a diagnostic test, improved therapies, and ultimately, prevention of this potentially fatal illness of childhood.

描述（由申请人提供）：川崎病 (KD) 是一种儿童期全身性炎症性疾病，导致 25% 的未经治疗的患者和 5% 的接受静脉丙种球蛋白治疗的患者出现冠状动脉瘤。川崎病是发达国家儿童获得性心脏病的最常见原因。临床和流行病学特征，包括季节性流行和抗生素治疗失败，强烈提示病毒原因，但尚未发现已知病毒。我的实验室报告了急性川崎病中抗原驱动的 IgA 动脉免疫反应，这些抗体的合成版本在川崎病纤毛支气管上皮、肺、脾和淋巴结的巨噬细胞子集中以及维管组织。石蜡包埋肺的光和透射电子显微镜分析 (TEM) 表明 ICI 与病毒蛋白和 RNA 的聚集体一致。最近，来自三例病例的非包埋支气管组织的 TEM 显示病毒样颗粒 (VLP) 与 ICI 非常接近。对于任何已知病毒来说，VLP 的超微结构都不是典型的。对其中一例激光捕获显微切割的支气管上皮（400,000 个序列）进行 cDNA 高通量 (454) 测序表明，干扰素刺激的基因上调，与病毒感染一致。随后使用 KD 和对照肺样本的实时 PCR 阵列分析证实了 KD 肺中 α、β 干扰素反应的刺激。这些发现使我们推测，一种通过肺部进入的“新的”、先前未识别的 RNA 病毒是 KD 的病原体，并且可以使用生物信息学方法通过高通量测序来鉴定 KD 组织中的病毒序列。由于与已知病毒的核苷酸或氨基酸同源性有限，和/或因为已检查的来自 KD 组织的序列数量不足，该病毒迄今为止可能未能被检测到。我们聚集了一个多学科团队，包括川崎病传染病和病理学专家、病毒学家、经验丰富的遗传学家、生物信息学家和生物统计学家，目标是识别与川崎病相关的病毒序列。我们为这项探索性资助提出以下具体目标：1）对从 KD 组织中扩增的 cDNA 进行超高通量测序，2）使用生物信息学分析来识别具有病毒同源性和不匹配的序列，并对感兴趣的序列进行组装以生成最长的连续序列，以及 3) 使用实时 PCR 分析确定其他 KD 和对照组织中是否存在感兴趣的序列。 KD 组织中 ICI 和 VLP 的鉴定为进行定向高通量测序以鉴定与 KD 相关的病毒序列提供了独特的机会，长期目标是确定 KD 的病因。川崎病的致病因子的鉴定将对这一领域产生深远的影响，有助于开发诊断测试、改进治疗方法，并最终预防这种潜在的致命儿童疾病。