Forward Genetic Analysis of Congenital Defects in Cortical Circuits and Structure

描述（由申请人提供）：哺乳动物的新皮层是一个巨大的细胞网络，每个细胞都有数千个连接，一系列神经系统疾病可能由不适当的皮层结构或连接引起。许多这些先天性脑缺陷都有遗传起源，但我们仍然缺乏对相关基因和机制的充分了解。本应用程序的总体目标是在小鼠和人类中使用正向遗传方法来识别和验证对皮层回路和整体结构发育重要的新等位基因。我们的中心假设是，在小鼠和人类中，协同和公正的正向遗传方法将导致皮层回路形成和结构发育遗传学的根本发现。这项研究的基本原理是，通过使用人类和小鼠遗传学，通过前向遗传方法识别正常皮质发育所需的新基因，我们将在随后的研究中利用这些信息和工具来研究人类皮质畸形的病因机制。我们将检验这一中心假设，并通过追求以下三个具体目标来实现本应用程序的目标：1)在小鼠中使用正向遗传学来有效地生成和捕获对皮质回路形成和结构发育重要的基因位点的基因突变；2)在皮质回路形成和结构脑缺陷的新型小鼠模型中识别和验证因果突变；3)应用下一代测序方法来识别导致人类运动障碍和结构脑缺陷的突变。目的是通过在小鼠中添加一种新的转基因报告基因，在皮层V层锥体神经元中特异性表达的ENU诱变方法来实现的。然后克隆这些突变，并通过一系列功能研究进行验证。人类遗传学研究是通过应用外显子组测序对精心选择的运动障碍和结构性脑畸形的家族病例进行的。这些研究将确定几个对哺乳动物前脑结构和功能至关重要的基因。这项工作的重要性体现在皮质电路和结构的具体应用上，而且这种无偏倚的方法有能力在神经系统疾病中揭示全新的途径。利用小鼠和人类遗传学的协同方法来具体查询神经发育的这些方面，可以从根本上了解发育和疾病的遗传学。这些知识不仅对进一步了解神经发育的基本机制至关重要，而且通过识别一些潜在的治疗靶点具有直接的临床意义。此外，这些小鼠模型提供了一种可重复使用的资源，可以直接表征突变基因在神经发育中的作用，并有可能作为测试未来治疗干预措施的工具。综上所述，这些发现适用于基础发育神经生物学、儿童和成人神经学、人类遗传学和遗传咨询。

DESCRIPTION (provided by applicant): The mammalian neocortex is an enormous network of cells, each making thousands of connections and an array of neurological conditions can result from inappropriate cortical structure or connectivity. Many of these congenital brain defects have a genetic origin but we still lack a full understanding of the genes and mechanisms involved. The overall objective of this application is to use forward genetic approaches in mouse and human to identify and validate novel alleles important for development of cortical circuitry and overall structure. Our central hypothesis is that a synergistic and unbiased forward genetic approach in mouse and human will lead to fundamental discoveries in the genetics of cortical circuit formation and structural development. The rationale of this proposed research is that by identifying novel genes through forward genetic approaches which are required for normal cortical development using both human and mouse genetics, we are then positioned to use this information and tools to study the etiological mechanisms of human cortical malformations in subsequent studies. We will test this central hypothesis and accomplish the goals of this application by pursuing the following three specific aims: 1) use forward genetics in the mouse to efficiently generate and capture genetic mutations in loci important for cortical circuit formation and structural development, 2) identify and validate causal mutations in novel mouse models of cortical circuit formation and structural brain defects, and 3) apply next-generation sequencing approaches to identify mutations leading to human movement disorders and structural brain defects. The aims are accomplished by an ENU mutagenesis approach in the mouse with the addition of a novel transgenic reporter which is expressed specifically in cortical layer V pyramidal neurons. The mutations are then cloned and validated through a number of functional studies. The human genetics studies are performed with the application of exome sequencing to carefully selected familial cases of movement disorders and structural brain malformations. These studies will identify several genes essential for mammalian forebrain structure and function. The significance of this work is found in the specific application to cortial circuitry and structure, and that an unbiased approach such as this has the capability to implicate entirely new pathways in neurological disease. A synergistic approach using both mouse and human genetics to specifically query these aspects of neural development allows fundamental insights into the genetics of development and disease. Such knowledge is not only critical to further understand the basic mechanisms of neurodevelopment, but also has immediate clinical relevance through identification of a number of potential therapeutic targets. Furthermore, these mouse models provide a reusable resource to directly characterize the role of the mutated gene in neurodevelopment, and potentially serve as a tool to test future therapeutic interventions. Taken together, these findings are therefore applicable to basic developmental neurobiology, pediatric and adult neurology, human genetics and genetic counseling.