Effect of Crohn's Disease Risk Alleles on Enteric Microbiota

描述：异常的宿主-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)的危险等位基因，如NOD2和ATG16L1，与宿主对共生菌的缺陷包容有关。我们假设这些CD风险等位基因对肠道微生物区系的组成变化有贡献。微生物区系的这些变化可能会减少保护性共生生物的比例，增加刺激肠道炎症的促炎微生物的数量。我们建议采用元基因组学的方法来检验这一假说，具体目标有三：1)分析CD易感等位基因对回肠CD、非回肠IBD和非IBD患者胃肠道黏膜相关微生物区系的影响；开发和测试致炎共生生物的新型遗传探针；在四个主要的IBD中心扩大预期的胃肠道组织收集。在UH2试点年，我们将重点研究现有的755个胃肠道组织，这些组织来自三个机构已经收集的364个受试者。华盛顿大学将对组合回肠组织进行高通量16S rRNA序列分析，并对所有高度可重复性的CD风险等位基因(~30，包括NOD2和ATG16L1)进行高通量基因分型。将进行定量聚合酶链式反应，以量化特定的微生物亚群。对元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。粘附性侵袭性大肠杆菌(AIEC)是一组新的细菌菌株，被认为是刺激肠道炎症的候选致病微生物。由于有关AIEC菌株的基因组信息有限，我们将从胃肠道组织中分离出AIEC菌株的基因组序列。在该项目的UH3阶段，将在四个主要的IBD中心：华盛顿大学、北卡罗来纳大学、克利夫兰诊所和西奈山医学中心，每年收集700-800名回肠CD、非回肠IBD和非IBD患者的胃肠道组织。将对与回肠组织相关的选定粪便样本进行鸟枪测序，以确定由不可培养生物造成的微生物群中的其他或辅助或协同致病因素或其他功能变化。通过挖掘细菌基因组数据和鸟枪测序数据开发的致病共生生物的遗传探针将被用来检验这样一个假设，即这些生物数量的增加与生态失调和/或CD风险等位基因有关。我们在多个机构的多学科专业知识的结合，我们证明有能力收集大量具有纵向临床信息的良好表型样本，这些信息将与宿主反应概况和形态研究联系起来，我们联盟的高通量测序能力将被用于调查人类微生物组的变化如何与CD风险等位基因和CD发病机制相关。与公共卫生相关：我们结合了多学科的专业知识和在六个机构(圣路易斯华盛顿大学、北卡罗来纳大学、科罗拉多大学、新墨西哥大学、西奈山医学中心和克利夫兰诊所)收集大量良好表型人类临床样本的能力，并在华盛顿大学基因组中心进行高通量测序，以研究人类肠道微生物组的变化与克罗恩病的关系。

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.