Molecular and Functional Analysis of Hirschsprung Defects in Humans and Mouse

先天性巨结肠症是一种先天性缺陷，其远端肠道缺乏肠神经系统。大多数HSCR患者RET活性降低，许多患者EDNRB（内皮素受体3）活性降低。尽管在80%的HSCR儿童中只有远端结肠是无神经节的（缺失神经元和神经胶质），但由肠神经元支配的近端肠区域（和胃肠道的其余部分）具有相同的突变基因。由于ENS发育的许多方面都需要RET和EDNRB，因此结肠神经节区域的肠神经元及其连接（以下称为ENS连接体）可能异常。与这一假设一致，HSCR手术切除无神经节肠并将“正常肠”重新连接到肛门边缘并不能缓解所有HSCR症状;高达50%的儿童在手术后存在持续问题，包括肠扩张、炎症、爆发性腹泻、便血、嗜睡和喂养不良。该研究项目的长期目标是确定HSCR相关基因的突变如何影响整个ENS并导致胃肠道功能障碍。短期目标是：1）鉴定HSCR患者和含有HSCR相关突变的小鼠中发生的RET和EDNRB突变下游的基因表达变化，和2）在HSCR小鼠模型中进行解剖学和功能研究以确定这些基因缺陷如何负面影响肠神经系统。指导这些实验的总体假设是，对于结肠的神经支配部分，不同的HSCR突变产生运动性缺陷，这是由独特的神经元子集（在肌间神经丛和自主神经系统（ANS）中）、ICC和神经胶质之间的通信的特定变化引起的。目标1：对野生型和HSCR小鼠模型的肠神经元进行合并和单细胞RNA-Seq分析;将鼠数据与来自HSCR患者和对照的RNA-Seq分析进行比较。目的2：确定HSCR相关突变对ENS/ICC/胶质细胞通讯的影响。目标3：检查HSCR小鼠模型中肌间神经元、ICC、神经胶质和相关平滑肌收缩的外在副交感神经和交感神经驱动。影响：转录组学表明，不同的HSCR突变将对ENS和ANS中不同细胞类型（神经元、神经胶质和ICC）的功能产生负面影响。涉及多种细胞类型的结果是，患者可能在病理学上看起来相似，但根本原因以及因此适当的治疗可能非常不同。该研究计划旨在确定疾病的突变特异性机制，作为开发患者特异性治疗的基础。

Hirschsprung’s disease (HSCR) is a birth defect where the enteric nervous system (ENS) is absent from the distal bowel. Most HSCR patients have reduced activity of RET and many have reduced EDNRB (endothelin receptor 3) activity. Although only the distal colon is aganglionic (missing neurons and glia) in 80% of children with HSCR, proximal bowel regions (and the rest of the GI tract) innervated by enteric neurons harbor the same mutated genes. Since RET and EDNRB are needed for many aspects of ENS development, it seems likely enteric neurons in ganglionic regions of the colon and their connections (hereafter referred to as the ENS connectome) are abnormal. Consistent with this hypothesis, HSCR surgery to remove aganglionic bowel and reconnect “normal bowel” to the anal verge does not alleviate all HSCR symptoms; up to 50% of children have ongoing issues post-surgery including bowel distension, inflammation, explosive diarrhea, blood in the stool, lethargy and poor feeding. The long-term goal of this research project is to determine how mutations in HSCR-related genes affect the entire ENS and contribute to GI tract dysfunction. The short-term goals are: 1) to identify the gene-expression changes downstream of mutations in RET and EDNRB that occur in HSCR patients and in mice in that contain HSCR-relevant mutations, and 2) Perform anatomical and functional studies in HSCR mouse models to determine how these gene defects negatively impact the enteric nervous system. The overall hypothesis guiding these experiments is that for the innervated portion of the colon, different HSCR mutations produce defects in motility resulting from specific changes in communication between unique subsets of neurons (in the myenteric plexus and autonomic nervous system (ANS)), ICC, and glia. Aim 1: Conduct pooled and single cell RNA-Seq analysis on enteric neurons of wild type and HSCR mice models; compare murine data with RNA-Seq analysis from HSCR patients and controls. Aim 2: Determine the effect of HSCR-associated mutations on ENS/ICC/glia communication. Aim 3: Examine extrinsic parasympathetic and sympathetic drive of myenteric neurons, ICC, glia and associated smooth muscle contractions in HSCR mouse models. Impact: Transcriptomics indicate that the different HSCR mutations will negatively affect the function of different cell types in the ENS and ANS (neurons, glia and ICC). The consequence of the involvement of multiple cell types is that patients may appear similar symptomatically, but the underlying cause, and hence appropriate treatment, may be very different. This research program is designed to identify mutation-specific mechanisms of disease as a basis for development of patient-specific treatments.