Effect of Crohn's Disease Risk Alleles on Enteric Microbiota

描述：异常宿主-微生物相互作用与炎症性肠病（IBD）的发病机制有关。克罗恩病（CD）的风险等位基因，如NOD 2和ATG 16 L1，已与有缺陷的宿主遏制肠道细菌。我们假设这些CD风险等位基因有助于肠道微生物群的组成变化。这些微生物群的变化可能会减少保护性肠道微生物的比例，并增加引发肠道炎症的促炎微生物的数量。我们建议采取宏基因组学方法在三个具体目标中测试这一假设：1）。分析CD风险等位基因对从回肠CD、非回肠IBD和非IBD患者收集的GI组织中的粘膜相关微生物群的影响; 2.）开发和测试用于促炎性微生物的新型遗传探针; 3.）在四个主要IBD中心扩大GI组织的前瞻性采集。在UH 2试点年度，我们将重点研究已经在三个机构收集的364名受试者的755份现有GI组织。将在华盛顿大学对联合回肠组织进行高通量16 S rRNA序列分析，并对所有高度重现的CD风险等位基因（约30个，包括NOD 2和ATG 16 L1）进行高通量基因分型。将进行定量PCR以定量特定微生物亚组。宏基因组数据和基因分型数据的多变量分析将包括潜在的混杂变量，如年龄、疾病状态、药物和吸烟。粘附入侵E.大肠杆菌（AIEC）是一组新的细菌菌株，其被认为是引起肠道炎症的候选“致病”生物体。由于AIEC菌株的基因组信息有限，我们将产生从GI组织分离的AIEC菌株的基因组序列。在项目的UH 3阶段，将在四个主要IBD中心（华盛顿大学、北卡罗来纳州大学、克利夫兰诊所和西奈山医学中心）每年前瞻性采集700-800例回肠CD、非回肠IBD和非IBD患者的GI组织。将对选定的与回肠组织相关的粪便样本进行鸟枪测序，以鉴定由不可培养微生物引起的微生物组中的其他、辅助或协同致病因子或其他功能变化。通过挖掘细菌基因组数据和鸟枪测序数据开发的致病性细菌生物体的遗传探针将用于测试这些生物体数量增加与生态失调和/或CD风险等位基因相关的假设。我们在多个机构的多个学科的专业知识相结合，我们证明有能力收集大量的良好表型样本与纵向临床信息，将与主机响应分析和形态学研究，以及我们的联盟的高通量测序能力将被用来调查人类微生物组的改变如何与CD风险等位基因和CD发病机制。公共卫生相关性：我们结合了多个学科的专业知识和在六个机构收集大量良好表型的人类临床样本的能力（华盛顿圣路易斯大学、北卡罗来纳州大学、科罗拉多大学、新墨西哥州大学、西奈山医疗中心、和克利夫兰诊所）的能力，高-在华盛顿大学基因组中心进行的通量测序，研究人类肠道微生物组的改变与克罗恩病的关系。

DESCRIPTION: Abnormal host-microbial interactions are implicated in the pathogenesis of inflammatory Bowel disease (IBD). Crohn's disease (CD) risk alleles, such as the NOD2 and ATG16L1, have been associated with defective host containment of commensal bacteria. We hypothesize that these CD risk alleles contribute to compositional changes in intestinal microbiota. These changes in microbiota might reduce the proportion of protective commensal organisms and increase the numbers of proinflammatory organisms that incite intestinal inflammation. We propose to take a metagenomic approach towards testing this hypothesis in three Specific Aims: 1.) Analyze the effect of CD-risk alleles on mucosal associated microbiota in GI tissues collected from ileal CD, non-ileal IBD and non-IBD patients; 2. ) Develop and test novel genetic probes for pro-inflammatory commensal organisms; 3.) Expand prospective collection of GI tissues in four major IBD centers. During the UH2 pilot year, we will focus our study on 755 existing GI tissues from 364 subjects already collected at three institutions. High throughput 16S rRNA sequence analysis of the combined ileal tissues and high throughput genotyping for all highly reproducible CD risk alleles (~30, including NOD2 and ATG16L1) will be performed at Washington University. Quantitative PCR will be performed to quantify specific microbial subgroups. Multivariate analysis of the metagenomic data and the genotyping data will include potentially confounding variables such as age, disease state, medications and smoking. The adherent invasive E. coli (AIEC) are a novel group of bacterial strains that have been implicated as candidate "pathogenic" organisms that incite intestinal inflammation. Because there is limited genome information on AIEC strains, we will produce genome sequences of AIEC strains isolated from the GI tissues. During the UH3 phase of the project, prospective collection of GI tissues from 700-800 ileal CD, non-ileal IBD and non-IBD patients a year will be conducted at four major IBD centers: Washington University, University of North Carolina, Cleveland Clinic, and Mount Sinai Medical Center. Shotgun sequencing will be performed on selected fecal samples linked to the ileal tissues to identify additional, or auxiliary, or synergistic pathogenic factors or other functional changes in the microbiome that are contributed by uncultivatable organisms. Genetic probes for pathogenic commensal organisms developed by mining the bacterial genome data and shotgun sequencing data will be used to test the hypothesis that increased numbers of these organisms are associated with dysbiosis and/or CD risk alleles. Our combined expertise in multiple disciplines across multiple institutions, our demonstrated ability to collect a large number of well-phenotyped samples with longitudinal clinical information that will be linked to host response profiling and morphologic studies, and our consortium's capacity for high-throughput sequencing will be used to investigate how alterations in the human microbiome relate to CD risk alleles and CD pathogenesis. PUBLIC HEALTH RELEVANCE: We have combined expertise in multiple disciplines and the capability to collect a large number of well-phenotyped human clinical samples across six institutions (Washington University in St. Louis, University of North Carolina, University of Colorado, University of New Mexico, Mount Sinai Medical Center, and Cleveland Clinic) with the capacity for high-throughput sequencing at the Genome Center at Washington University to study how alterations in the human intestinal microbiome relate to Crohn's disease.