Molecular and Functional Analysis of Hirschsprung Defects in Humans and Mouse

人类和小鼠先天性巨结肠缺陷的分子和功能分析

• 批准号: 10386806
• 负责人: Kathryn Marie Albers
• 金额: $ 68.57万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-15 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10386806
• 关键词: Adult; Affect; Afferent Neurons; Anal canal; Anatomy; Autonomic nervous system; Blood; Calcium; Cause of Death; Cell Count; Cells; Child; Childhood; Colon; Communication; Congenital Abnormality; Congenital Megacolon; DNA Sequence Alteration; Data; Defect; Development; Diarrhea; Disease; Distal; Endothelin Receptor; Engineering; Enteral; Enteric Nervous System; Excision; Family; Feces; Functional disorder; Ganglia; Gastrointestinal tract structure; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Human; Image; Inflammation; Interneurons; Interstitial Cell of Cajal; Intestinal Obstruction; Intestines; Lead; Length; Lethargies; Mediating; Modeling; Molecular; Motor Neurons; Mus; Muscle Contraction; Mutant Strains Mice; Mutate; Mutation; Myenteric Plexus; Neuroglia; Neurons; Operative Surgical Procedures; Pathway interactions; Patients; Pattern; Phenocopy; Piebaldism; Play; Property; Proto-Oncogenes; Protocols documentation; Recovery; Regulation; Research; Research Project Grants; Rest; Role; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Symptoms; Synapses; Transition zone of anal mucous membrane; Validation; Wild Type Mouse; calcium indicator; cell motility; cell type; common treatment; connectome; design; disabling symptom; endothelin-3 receptor; experimental study; feeding; glial cell-line derived neurotrophic factor; human data; human disease; individualized medicine; insight; motility disorder; mouse model; negative affect; nervous system development; neurotransmission; neurotrophic factor; nodal myocyte; novel; prevent; programs; proto-oncogene protein c-ret; relating to nervous system; single-cell RNA sequencing; standard of care; tool; transcriptome sequencing; transcriptomics

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.

先天性巨结肠症(HSCR)是一种先天缺陷，肠道神经系统(ENS)在远端肠道中缺失。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。虽然在80%的HSCR儿童中，只有远端结肠是无神经节细胞(缺失神经元和神经胶质细胞)，但由肠道神经元支配的近端肠道区域(和胃肠道的其余部分)拥有相同的突变基因。由于ENS发育的许多方面都需要RET和EDNRB，因此似乎结肠神经节区的肠神经元及其连接(以下称为ENS连接体)可能是异常的。与这一假设一致的是，HSCR手术切除无神经节段肠并将“正常肠”重新连接到肛缘并不能缓解所有HSCR症状；高达50%的儿童在手术后持续存在问题，包括肠胀、炎症、爆发性腹泻、大便出血、嗜睡和进食不良。这项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)确定HSCR患者和包含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；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对肠道神经系统造成负面影响。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突变会产生运动缺陷，这是由于独特的神经元亚群(肌间神经丛和自主神经系统(ANS))、ICC和胶质细胞之间的通信发生了特定的变化。目的：对野生型和HSCR小鼠模型的肠上皮神经元进行混合和单细胞RNA-Seq分析，并与HSCR患者和对照组的RNA-Seq分析结果进行比较。目的2：确定HSCR相关突变对ENS/ICC/GILA通讯的影响。目的3：检测HSCR小鼠模型肌间神经元、ICC、胶质细胞及相关的平滑肌收缩的外源性副交感神经和交感神经驱动。影响：转录学表明，不同的HSCR突变将对ENS和ANS中不同类型的细胞(神经元、胶质细胞和ICC)的功能产生负面影响。多种细胞类型参与的结果是，患者的症状可能相似，但潜在的原因以及相应的治疗可能会非常不同。这项研究计划旨在确定疾病的突变特异性机制，作为开发针对患者的治疗方法的基础。