Deep Sequencing of Kawasaki Disease Tissues

川崎病组织的深度测序

• 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
• 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

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）： 川崎疾病（KD）是一种儿童的全身性炎症性疾病，在25％的未治疗患者中引起冠状动脉动脉瘤，在接受静脉注射伽马卢蛋白治疗的患者中有5％。 KD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，包括季节性流行病和抗生素治疗的衰竭，强烈表明病毒原因，但尚未鉴定出已知的病毒。我的实验室报道了急性KD中由抗原驱动的IGA动脉免疫反应，这些抗体的合成版本在KD纤毛支气管上皮细胞中鉴定出胞内胞质内包含体（ICI），在肺部，spleen，spleen，spleen和lymphen，spleen和lymphen，spleen和lymphen，spleen和lympher nodes and in Ase in cy and in consue and spleen和lympher nodes and in in in consue and in con and in Aude and in consue and in in consue。石蜡包埋的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，来自三个病例的非嵌入支气管组织的TEM表现出与ICI紧邻的病毒样颗粒（VLP）。对于任何已知病毒而言，VLP并不是典型的超微结构。从这些病例之一（400,000个序列）中对激光捕获显微捕获的支气管上皮的cDNA的高通量（454）表明，干扰素刺激的基因被上调，与病毒感染一致。随后使用KD和对照肺样品的实时PCR阵列分析来确认刺激α肺中的β干扰素反应。这些发现使我们假设通过肺部进入的“新”，以前未识别的RNA病毒是KD的病因，并且可以使用生物信息学方法来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，该病毒可能已经逃脱了检测，并且/或因为已经检查了KD组织的序列数量不足。我们已经聚集了一个多学科团队，包括KD传染病和病理学专家，一名病毒学家，经验丰富的遗传学家，生物信息学家和生物统计学家，目的是识别与KD相关的病毒序列。 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. KD组织中ICI和VLP的鉴定提供了一个独特的机会，可以执行定向的高通量测序，以确定与KD相关的病毒序列，其长期目标是确定KD的病因学药物。鉴定KD的病因对该领域具有深远的影响，从而开发诊断测试，改善疗法，并最终预防这种潜在的致命童年疾病。