Deep Sequencing of Kawasaki Disease Tissues

描述（由申请人提供）： 川崎（KD）是一种儿童全身性炎症性疾病，在25%未经治疗的患者和5%接受静脉丙种球蛋白治疗的患者中引起冠状动脉瘤。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，包括季节性流行和抗生素治疗失败，强烈建议病毒的原因，但没有已知的病毒已被确定。我的实验室报告了急性KD中抗原驱动的伊加动脉免疫应答，并且这些抗体的合成版本在KD纤毛支气管上皮、肺、脾和淋巴结中的巨噬细胞亚群以及血管组织中鉴定了胞浆内包涵体（ICI）。石蜡包埋肺组织的光镜和透射电镜分析（TEM）显示ICI与病毒蛋白和RNA的聚集体一致。最近，来自三个病例的非包埋支气管组织的TEM显示病毒样颗粒（VLP）紧邻ICI。VLP在超微结构上不是任何已知病毒的典型。对其中一例病例的激光捕获显微切割支气管上皮细胞（400，000个序列）的cDNA进行高通量（454）测序，结果显示干扰素刺激的基因上调，与病毒感染一致。随后使用KD和对照肺样品的实时PCR阵列分析证实KD肺中α，β干扰素应答的刺激。这些发现使我们假设，一种“新的”，以前未经鉴定的RNA病毒，通过肺进入是KD的病原体，KD组织中的病毒序列可以通过高通量测序，使用生物信息学方法进行鉴定。由于与已知病毒的核苷酸或氨基酸同源性有限，和/或由于已检查的KD组织序列数量不足，该病毒迄今可能未被检测到。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，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，目标是确定与KD相关的病毒序列。我们为这项探索性赠款提出以下具体目标：1）对从KD组织扩增的cDNA进行超高通量测序，2）使用生物信息学分析来鉴定具有病毒同源性而没有匹配的序列，并对感兴趣的序列进行组装以产生最长的连续序列，和3）使用实时PCR分析确定另外的KD和对照组织中感兴趣的序列的存在。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，其长期目标是确定KD的病原体。KD病原体的鉴定将对该领域产生深远的影响，从而能够开发诊断测试，改进治疗方法，并最终预防这种可能致命的儿童疾病。

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.