孤独症谱系障碍患者胃肠功能紊乱相关的肠道菌群结构及其代谢产物研究

Studies showed that ASD patients were common affected with different level of Gastrointestinal (GI) problems, and the GI problems aggravated the abnormal behavior of ASD patients; Fecal microbiota transplantation showed promising in improving the GI problems and core phenotype in ASDs. The currently 16S rDNA sequencing based gut microbiota studies mainly focused on taxonomy and at high level, hardly associated ASDs or GI problems with specific species, microbe genes or functional pathways, and rarely focused on gut microbiota metabolites at the same time. Understand the differences in functional levels of gut microbiota in patients affected with ASDs will not only facilitate us to understand how gut microbiota participate in the occurrence of ASDs, but also provide a new perspective for the diagnosis and treatment of ASDs..In our project, we will used the the metagenome wide association study method which is established by applicant and successfully applied to the study gut microbiota of type 2 diabetes (Nature 2012). We will recruit 120 ASD patients, 120 gender and age matched normal children. We will simultaneously perform shotgun metagenome sequencing and Liquid chromatography-mass spectrometry (LC- MS) of gut microbiota to identify the correlation between gut microbiota composition, such as microbes, microbes genes or pathways, gut microbiota metabolites and ASDs or GI problems. We will also evaluated the potentiation of gut microbiota composition and metabolites been used as biomarkers of ASDs by qPCR and targeted metabolomics method. There was no report for ASDs gut microbiota metabolomic study. The joint use metagenomic and metabolomic reflected our innovation, at the same time, the applicant has also found that the ASDs patients with abnormal metabolic pathways, such as folic acid, laid a certain foundation for the project.

前人研究发现ASDs患者普遍存在胃肠功能紊乱，肠道功能紊乱会加重患者表型；进行粪便移植可以改善ASDs患者的胃肠功能紊乱与核心表型；目前肠道菌群研究较少涉及菌群功能水平，而了解ASDs患者肠道菌群功能水平的差异，找寻相关标志物，不但有助于深入认识肠道菌群如何参与ASDs的发生，还将为ASDs的诊断和治疗带来突破。本研究拟采样申请人前期参与建立的疾病宏基因组关联分析方法（Nature 2012），征集120例典型的ASDs患者和120例对照，首先采用肠道菌群宏基因组和代谢组联合研究方法来筛选肠道菌群标志物，然后基于qPCR和靶向代谢组技术来验证肠道菌群标志物。ASDs患者肠道菌群代谢组研究目前未有报道，宏基因组和代谢组联合研究更是首创，同时，申请人前期的组学研究也发现了ASDs患者存在叶酸等代谢通路的紊乱，为本项目奠定了一定的基础。

本研究拟采用宏基因组关联分析和LC-MS代谢组学分析方法，对同一批粪便样本同时进行宏基因组测序和代谢组检测，筛选和ASDs或者胃肠功能紊乱相关联的肠道菌群结构，包括细菌，基因和代谢小分子，并验证肠道菌群结构，如细菌，基因和代谢小分子的诊断价值，为ASDs的诊断和治疗提供新的思路。本研究的内容主要为四个方面，即：.第一部分，招募了43名ASD患者和31名性别和年龄匹配的典型发言（TD）儿童。通过酶联免疫吸附测定法测量粪便IgA含量，我们发现ASD患者较TD儿童肠道IgA含量显著升高（Zhou, et al., Res Dev Disabil 2018.通讯作者）。.第二部分，对43名ASD儿童（19名有GI和24名无GI患儿）和31例TD儿童（TD）的粪便样品进行鸟枪法宏基因组测序，鉴定出候选肠道微生物群相关表位（MEs），我们发现肠道IgA上升与肠道菌群抗原肽改变相关，并发现了34个在ASD和TD组差异显著的肠道菌群抗原表位肽（Wang, et al., Brain Behav Immun 2019.第一/通讯作者）。.第三部分，对来自ASD儿童（n = 43）和TD儿童（n = 31）的宏基因组数据集进行了宏基因组分析， 预测了毒性因子相关的肠道菌群（VFGM）基因组成，我们发现ASD患儿IgA上升与毒力因子相关的肠道菌群（VFGM）基因丰度改变相关，并基于机器学习发现肠道IgA水平和GBS基因是ASD早期诊断的潜在标志物（Wang, et al., Computational and Structural Biotechnology Journal 2021.第一/通讯作者）。.第四部分，共招募了92名ASD儿童和42名具有典型发育（TD）的年龄相匹配的儿童，完成了ASD患儿宏基因组-宏代谢组关联分析，发现ASD患儿肠道谷氨酸代谢改变与肠道菌群组成的变化相关，同时参与胆汁酸代谢的潜在有害菌（埃格特氏杆菌和肉毒梭菌）与肠道胆汁酸的改变与IgA升高相关（Wang, et al., Brain Behav Immun 2019.第一/通讯作者）。.本研究按计划顺利完成，以（共同）第一作者和（共同）通讯作者发表SCI论文11篇，累计影响因子>45，延续项目得到国家自然科学基金面上项目（82071733）的资助。