Dissecting the Genetic Basis of Congenital Diaphragmatic Hernias in Mice

剖析小鼠先天性膈疝的遗传基础

基本信息

批准号：
10569504
负责人：
Eric Bogenschutz
金额：
$ 6.95万
依托单位：
JACKSON LABORATORY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-03 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10569504
关键词：
Abdomen Alleles Animal Model Animals Appearance Automobile Driving Biological Assay Breeding Bypass Candidate Disease Gene Cell Lineage Cells Cessation of life Child Clustered Regularly Interspaced Short Palindromic Repeats Complement Complex Congenital Abnormality Congenital diaphragmatic hernia Consumption Data Set Defect Development Disease Dorsal Embryo Embryonic Development Engineering Etiology Fibroblasts Gene Expression Gene Expression Profiling Generations Genes Genetic Genetic Diseases Genetic Transcription Goals Hernia High-Throughput Nucleotide Sequencing Human In Vitro Invaded Knockout Mice Lead Live Birth Liver Location Lung Mediating Mesothelium Methods Modeling Molecular Morbidity - disease rate Morphogenesis Morphology Mus Muscle Muscular Atrophy Mutation Nature Organogenesis Pathway interactions Patients Penetrance Phenotype Population Process Protein-Serine-Threonine Kinases Rapid screening Reproductive Biology Respiratory Diaphragm Role Severities Skeletal Muscle Speed Structural Congenital Anomalies Structure Techniques Technology Tendon structure Testing Thoracic cavity structure Time Tissues Translating Validation Variant Ventral Hernia Work cell motility cell type cohort congenital heart disorder cost genetic pedigree genetic variant genome sequencing improved in vivo insertion/deletion mutation insight lung development migration model organism mortality mouse model muscular structure mutation screening novel pharmacologic programs renal agenesis repaired screening single-cell RNA sequencing validation studies

项目摘要

PROJECT SUMMARY Congenital diaphragm hernia (CDH) is a common and severe structural birth defect arising in 1 of every 3,000 live births and leading to mortality in an estimated 30-50% of patients. CDH is caused by either a weakening or partial loss of diaphragm skeletal muscle, allowing abdominal contents to herniate into the thoracic cavity, often leading to severe lung hypoplasia. The severity of CDH is directly related to the location in the diaphragm muscle sheet where the defect arises, with more dorsal hernias correlating with higher severity and morbidity. Though it is clear from human studies that CDH is largely a genetic disease, the genetics are complex and much remains unknown about the number and nature of the genes and pathways that underlie the defect. This is in part due to limitations in the number of functional validation studies as the diaphragm is a mammalian- specific structure, and thus only murine model organisms are available to study CDH. The goal of this proposal is to study both the genetics and cellular mechanisms underlying CDH using new gene-editing and transcriptional profiling techniques in the mouse model organism. From high-throughput sequencing of CDH patients, the number of novel variants predicted to underly CDH has grown exponentially. In the first aim of this proposal, I will optimize a method to rapidly create precise gene edits in the mouse embryo and screen embryos at the appropriate developmental stage without the need of further breeding. Using this discovery platform, I will then screen CDH-associated variants discovered from patient genome sequencing cohorts. To dissect the mechanism by which hernias arise in discrete regions of diaphragm, in aim 2 I will study the cellular dynamics leading to CDH within in mice homozygous null for the serine kinase encoding gene Cdc42bpb, a novel mouse model of CDH which develop unique ventral hernias. These studies will reveal key differences in the development of the symptomatic and asymptomatic forms of CDH. Finally, in aim 3 I will use single cell RNA sequencing to understand the cell types and lineages driving diaphragm development both temporally and spatially within the mouse. Using novel spatial gene expression techniques, I will specifically assay for two hypothesized cell populations in the developing diaphragm, a mesothelial layer surrounding the tissue and a fibroblast population driving the migration of the tissue across the liver through embryonic development. Overall, the work proposed will not only further our understanding of the mechanism by which CDH arises, it will create a novel transcriptional dataset to assist in prioritization of CDH-associated genes and provide a powerful method to screen disease-associated alleles rapidly in mouse, advancing our understanding of the genetic basis for CDH.

项目摘要先天性隔膜疝（CDH）是一种常见且严重的结构性先天性缺陷，每3,000次中有1个估计有30-50％的患者的活产和导致死亡率。 CDH是由弱化或 diaphragm骨骼肌的部分丧失，使腹部含量物疝通常造成胸腔腔，通常导致严重的肺发育不全。 CDH的严重程度与隔膜中的位置直接相关缺陷出现的肌肉片，更多的背疝与更高的严重程度和发病率相关。尽管从人类研究中可以明显看出，CDH在很大程度上是一种遗传疾病，但遗传学很复杂，并且关于缺陷构成的基因和途径的数量和性质，仍然未知。这部分原因是功能验证研究的局限性，因为膜片是哺乳动物 - 特定的结构，因此只有鼠模型生物可以研究CDH。该提议的目标是使用新的基因编辑和小鼠模型生物中的转录分析技术。来自CDH的高通量测序患者，预测基本CDH的新型变体数量已成倍增长。在第一个目的提案，我将优化一种在鼠标胚胎和屏幕上快速创建精确基因编辑的方法在适当的发育阶段的胚胎无需进一步繁殖。使用此发现平台，我将筛选从患者基因组测序队列中发现的与CDH相关的变体。到解剖疝气在隔膜离散区域中产生的机制，在AIM 2中，我将研究细胞导致CDH内部CDH的动力学，用于编码基因Cdc42bpb的丝氨酸激酶，A CDH的新型鼠标模型，形成独特的腹性疝。这些研究将揭示有关的关键差异 CDH有症状和无症状形式的发展。最后，在AIM 3中，我将使用单个单元格 RNA测序以了解细胞类型和谱系驱动diaphragm的发育均在时间上并在鼠标内空间。使用新型的空间基因表达技术，我将专门测定两个假设的隔膜中的细胞群，组织周围的间皮层和A 成纤维细胞种群通过胚胎发育推动组织迁移整个肝脏的迁移。总体而言，提出的工作不仅将进一步了解我们对CDH产生的机制的理解将创建一个新颖的转录数据集，以协助与CDH相关基因的优先次序并提供在小鼠中快速筛选疾病相关等位基因的强大方法，促进了我们对 CDH的遗传基础。