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

基本信息

批准号：
10597975
负责人：
Kathryn Marie Albers
金额：
$ 67.46万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-15 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10597975
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 Length Lethargies Mediating Modeling Molecular Motor Neurons Mus Muscle Contraction Mutant Strains Mice Mutate Mutation Myenteric Plexus Nervous System Neuroglia Neurons Operative Surgical Procedures Pathway interactions Patients Pattern Phenocopy Property Proto-Oncogenes Protocols documentation Recovery Regulation Research Research Project Grants Rest Signal Transduction Smooth Muscle Smooth Muscle Myocytes Submucosa 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 neural neurotransmission neurotrophic factor nodal myocyte novel prevent programs proto-oncogene protein c-ret single-cell RNA sequencing standard of care tool transcriptome sequencing transcriptomics

项目摘要

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.

Hirschsprung病（HSCR）是肠道肠道肠道（ENS）的出生缺陷，远端肠道不存在。大多数HSCR患者的RET活性降低，许多HSCR患者的EDNRB降低（内皮素受体3）活性。尽管在80％的HSCR儿童，近端肠道区域（以及GI段的其余部分）中，仅肠神经元与肠神经元相同的突变基因所支配。由于ENS开发的许多方面都需要RET和EDNRB，因此似乎可能在结肠的神经节区域中进入神经元及其连接（以下称为ENS Connectome）异常。与该假设一致，HSCR手术以去除阳式肠肠并重新连接到肛门的边缘并不能减轻所有HSCR症状；多达50％的儿童在手术后有持续的问题包括肠道延伸，感染，爆炸性腹泻，凳子上的血液，嗜睡和喂养不佳。该研究项目的长期目标是确定与HSCR相关基因中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）确定在HSCR患者中发生的RET和EDNRB突变下游的基因表达变化，而在HSCR患者中发生的RET和EDNRB的基因表达变化，其中包含与HSCR相关的突变的小鼠中，以及2）在HSCR小鼠模型中执行解剖学和功能性研究，以确定这些基因对这些基因的影响如何对肠道神经系统产生负面影响。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突变会导致运动性的缺陷，这是由于神经元（Myenteric Plexus和自主神经系统（ANS）），ICC和Glia之间通信的特定变化而导致的运动性缺陷。 AIM 1：对野生型和HSCR小鼠模型的进入神经元进行汇总和单细胞RNA-seq分析；将鼠数据与HSCR患者和对照组的RNA-seq分析进行比较。 AIM 2：确定与HSCR相关突变对ENS/ICC/GliA通信的影响。 AIM 3：检查HSCR小鼠模型中肌植物神经元，ICC，GliA和相关平滑肌收缩的外部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（神经元，GliA和ICC）中不同细胞类型的功能产生负面影响。多种细胞类型的参与的结果是，患者可能同样出现在症状上，但是基本原因及其因此可能会大不相同。该研究计划旨在识别疾病的突变特异性机制，作为患者特异性治疗的发展的基础。