The impact of NOD2 variants on fecal microbiota in Crohn's disease and healthy controls

Background/AimsCurrent models of Crohn's disease (CD) describe an inappropriate immune response to gut microbiota in genetically susceptible individuals. NOD2 variants are strongly associated with development of CD, and NOD2 is part of the innate immune response to bacteria. This study aimed to identify differences in fecal microbiota in CD patients and healthy controls stratified by NOD2 genotype.MethodsPatients with CD and healthy controls of known NOD2 genotype were identified from patients in UK IBD genetics studies and the Cambridge bioresource (genotyped healthy individuals). Individuals with known CD-associated NOD2 mutations were matched to those with wild-type genotype. We obtained fecal samples from patients in clinical remission with low fecal calprotectin (<250µg/g) and controls without gastrointestinal disease. After extracting DNA, the V1-2 region of 16S rRNA genes were PCR-amplified and sequenced. Analysis was undertaken using the mothur package. Volatile organic compounds (VOC) were also measured.Results94 individuals were in the primary analysis (39 CD, 31 bioresource controls and 24 household controls). Comparing CD with healthy controls, there were reductions in bacterial diversity, Ruminococcaceae, Rikenellaceae and Christensenellaceae and an increase in Enterobacteriaceae. However, no significant differences could be identified in microbiota or VOC by NOD2 genotype either in the CD or healthy control cohorts.ConclusionsIn this well-controlled study of NOD2 genotype and fecal microbiota, we identified no significant genotype-microbiota associations. This suggests that the changes associated with NOD2 genotype might only be seen at the mucosal level, or that environmental factors and prior inflammation are the predominant determinant of the observed dysbiosis in gut microbiota.

背景与研究目的
目前关于克罗恩病（Crohn's disease, CD）的主流模型认为，遗传易感个体对肠道菌群存在不恰当的免疫应答。核苷酸结合寡聚化结构域2（NOD2）变异与克罗恩病的发病密切相关，且NOD2是机体对细菌产生固有免疫应答的组分之一。本研究旨在探究按NOD2基因型分层的克罗恩病患者与健康对照者的粪便菌群差异。

研究方法
本研究从英国炎症性肠病（Inflammatory Bowel Disease, IBD）遗传学研究队列以及剑桥生物样本库（已完成基因型检测的健康个体）中，招募了已知NOD2基因型的克罗恩病患者与健康对照者。将携带已知克罗恩病相关NOD2突变的个体与野生型基因型个体进行匹配。我们从处于临床缓解期且粪便钙卫蛋白水平较低（<250μg/g）的克罗恩病患者，以及无胃肠道疾病的健康对照者处获取粪便样本。提取DNA后，通过聚合酶链式反应（PCR）扩增16S rRNA基因的V1-V2区并进行测序，使用mothur软件包完成数据分析。同时检测了挥发性有机物（Volatile organic compounds, VOC）的含量。

研究结果
本研究的初步分析共纳入94名受试者，其中克罗恩病患者39名，生物样本库对照者31名，家庭对照者24名。与健康对照者相比，克罗恩病患者的细菌多样性、瘤胃球菌科（Ruminococcaceae）、理研菌科（Rikenellaceae）以及克里斯滕森菌科（Christensenellaceae）的相对丰度均有所降低，而肠杆菌科（Enterobacteriaceae）的相对丰度则升高。然而，无论在克罗恩病队列还是健康对照队列中，均未发现基于NOD2基因型的菌群或挥发性有机物含量存在显著差异。

研究结论
在这项针对NOD2基因型与粪便菌群的严格对照研究中，我们未发现基因型与菌群之间存在显著关联。这提示，与NOD2基因型相关的菌群变化可能仅存在于黏膜层面，或者环境因素与既往炎症反应是观察到的肠道菌群失调的主要驱动因素。